Anti-pivka-ii antibodies and uses thereof

Abstract

The application provides a PIVKA-II antibody and application thereof, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof, and the light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 13 or a variant thereof. The PIVKA-II antibody of the application can be combined with PIVKA-II protein and used for detection of PIVKA-II.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202211736118.1, filed on December 30, 2022, entitled "Anti-PIVKA-II Antibody and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of antibody technology, and more specifically, to anti-PIVKA-II antibody and its applications. Background Technology

[0004] Des-gamma-carboxy prothrombin (DCP) is a protein induced by vitamin K deficiency or antagonist-II, also known as PIVKA-II, and is found in the serum of patients with vitamin K deficiency or hepatocellular carcinoma (HCC). It is a glycoprotein involved in blood clotting with a structure similar to prothrombin. Prothrombin is a protein with 579 residues, including γ-carboxyglutamate (Gla) residues resulting from the γ-carboxylation of 10 glutamate (Glu) residues near the N-terminus. The N-terminal region is called the Glu-Gla region. It is well known that during the production of prothrombin in the human body, vitamin K deficiency, liver dysfunction, use of vitamin K antagonists, and hepatocellular damage can lead to the un-γ-carboxylation of all or part of the 10 Glu residues in blood glycoproteins; these proteins are collectively referred to as PIVKA-II. The structures of prothrombin and PIVKA-II are identical except for the Glu-Gla region. Both proteins have two kringle domains in the middle region (prothrombin fragment 1 (F1) and prothrombin fragment 2 (F2) regions), and the thrombin region is located at the C-terminus.

[0005] In 1984, PIVKA-II was first recognized as a biomarker for primary liver cancer, a major global health problem and the leading cause of cancer-related death worldwide. Serum PIVKA-II levels in hepatocellular carcinoma patients are significantly higher than in patients with cirrhosis and metastatic liver cancer, and changes in serum PIVKA-II concentration are correlated with the dynamic changes in hepatocellular carcinoma (surgery, treatment, and recurrence, etc.). Currently, PIVKA-II is considered an extremely important indicator for liver cancer detection.

[0006] Currently, the detection method of PIVKA-II mainly has chemiluminescence method, which is a specific reaction based on antibody and antigen, and simultaneously uses a luminescent substance (such as peroxidase, isoluminol, etc.) to amplify and display the detected signal. Similar immunological detection methods include biochemical immunoturbidimetry, radioimmunoassay, fluorescent immunochromatography, etc. The above immunological detection methods all need antibodies specific to PIVKA-II. Therefore, there is a strong demand in the art for antibodies that effectively bind to PIVKA-II and detect the same. SUMMARY

[0007] The present application aims to provide anti-PIVKA-II antibodies, reagents and kits for detecting PIVKA-II.

[0008] Therefore, in a first aspect of the present application, the present application provides an antibody, comprising: a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof;

[0009] wherein the variant of SEQ ID NO: 1 comprises at least one of the following mutation sites: 35th, 44th, 45th, 46th and 50th, compared with the amino acid sequence shown in SEQ ID NO: 1;

[0010] the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13 or a variant thereof;

[0011] wherein the variant of SEQ ID NO: 13 comprises at least one of the following mutation sites: 32nd, 48th, 56th and 69th, compared with the amino acid sequence shown in SEQ ID NO: 13.

[0012] In a second aspect of the present application, the present application provides an antibody, comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprises / are the amino acid sequences identical to the HCDR1, HCDR2, HCDR3 of the heavy chain variable region defined by the antibody of the first aspect; the LCDR1, LCDR2, LCDR3 comprises / are the amino acid sequences identical to the LCDR1, LCDR2, LCDR3 of the light chain variable region defined by the antibody of the first aspect.

[0013] In a third aspect of the present application, the present application provides a nucleic acid molecule encoding the antibody of the first or second aspect.

[0014] In a fourth aspect of the present application, the present application provides a vector comprising the nucleic acid of the third aspect.

[0015] In a fifth aspect, the present application provides a cell comprising the nucleic acid molecule of the third aspect, the vector of the fourth aspect or expressing the antibody of the first or second aspect.

[0016] In a sixth aspect, the present application provides a method for preparing the antibody of the first or second aspect, the method comprising culturing the cell of the fifth aspect.

[0017] In a seventh aspect, the present application provides an antibody conjugate comprising the antibody of the first or second aspect and a conjugating moiety conjugated thereto.

[0018] In an eighth aspect, the present application provides a reagent or kit comprising the antibody of the first or second aspect or the antibody conjugate of the seventh aspect.

[0019] In a ninth aspect, the present application provides the use of the aforementioned antibody, antibody conjugate, reagent or kit for detecting PIVKA-II, preparing a product for detecting PIVKA-II or diagnosing a PIVKA-II related disease.

[0020] In a tenth aspect, the present application provides a method for detecting PIVKA-II in a test sample, the method comprising: contacting the aforementioned antibody, antibody conjugate or reagent or kit with PIVKA-II antigen in the sample to be detected to form an immune complex.

[0021] In an eleventh aspect, the present application provides a method for screening a PIVKA-II antibody, the method comprising: a) designing primers for performing amino acid substitution at the defined mutation site in the antibody or original binding fragment of the first aspect, at least one of the defined X1, X2, X3, X4 sites in the antibody or original binding fragment of the second aspect; b) constructing a mutation library using the primers of a) as templates with the nucleic acid of the third aspect, the vector of the fourth aspect or the cell of the fifth aspect; c) screening a PIVKA-II antibody from the mutation library.

[0022] In a twelfth aspect, the present application provides a mutation library comprising the antibody of the first or second aspect.

[0023] The present application has at least the following advantages:

[0024] The present application provides antibodies effectively binding PIVKA-II, and the anti-PIVKA-II antibodies with higher PIVKA-II binding activity or affinity after mutation modification, which better meet the requirements of scientific research and clinical screening detection. DETAILED DESCRIPTION

[0025] The embodiments of the present application are described in detail below, which are exemplary and intended to explain the present application, and cannot be understood as a limitation of the present application.

[0026] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. These endpoints and any values between them are included in the range. For ranges comprising values "between" and between individual points, the range is intended to include the values of and between the endpoints. For ranges having endpoints of "about" a value and between individual points, the range is intended to include the value about the endpoint and between the individual points.

[0027] In order to facilitate the understanding of the present application, some technical and scientific terms are defined specifically below. Unless otherwise explicitly defined herein elsewhere, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the present application belongs. The abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids.

[0028] In the process of describing the present application, the explanations and descriptions of the terms related herein are only for the convenience of understanding the scheme, and cannot be regarded as a limitation of the protection scheme of the present application.

[0029] Herein, the term "antibody" is used in the broadest sense, and can include full-length monoclonal antibodies, bispecific, multispecific antibodies, chimeric antibodies or antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0030] The term "antigen binding fragment" is a moiety comprising a portion or all of the CDRs of an antibody that lacks at least some of the amino acids present in a full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to an antigen and can compete with other antigen binding molecules, including intact antibodies, for binding to a given epitope. Such fragments include at least one of an Fv fragment, a disulfide stabilized Fv fragment (dsFv), an F(ab')2 fragment, a Fab' fragment, a Fab fragment, an F(ab)2 fragment, a scFv fragment, a scFv-Fc fusion protein, a scFv-Fv fusion protein, a Fv-Fc fusion protein, a multispecific antibody formed from antigen binding fragments, a single domain antibody, a VHH nanobody, a domain antibody, a bivalent domain antibody, or a minimal recognition unit. Such fragments can be produced by recombinant nucleic acid techniques, or can be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

[0031] As used herein, the term "complementarity determining region," "CDR," or "CDRs" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, referring to the regions that comprise one or more, or even all, of the primary amino acid residues that affect the binding affinity of an antibody to its recognized antigen or epitope. In the DETAILED DESCRIPTION of the disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of the antibody.

[0032] As used herein, heavy chain complementarity determining regions (heavy chain variable region CDRs) are denoted "HCDR," which includes HCDR1, HCDR2, and HCDR3; light chain complementarity determining regions (light chain variable region CDRs) are denoted "LCDR," which includes LCDR1, LCDR2, and LCDR3. Commonly used CDR numbering schemes include: Kabat numbering, Chothia numbering, IMGT numbering, Martin numbering, and AHo numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition. As described herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). See Chothia et al., J Mol Biol 196:901-917 (1987) for "Chothia definition." Exemplary defined CDRs are listed in Table 1 below. One of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of a given antibody.

[0033] Table 1: CDR Definitions 1

[0034] CDR Kabat AbM 2 ]] IMGT HCDR1 31-35 26-35 26-35 HCDR2 50-65 50-58 51-56 HCDR3 95-102 95-102 93-102 LCDR1 24-34 24-34 27-32 LCDR2 50-56 50-56 50-51 LCDR3 89-97 89-97 89-97

[0035] 1 The numbering of all CDRs defined in Table 1 is according to the Kabat numbering system (see below).

[0036] 2 “AbM” as used in Table 1, with a lower case “b”, refers to CDRs defined by the “AbM” antibody modeling software of Oxford Molecular.

[0037] The present disclosure employs the Kabat definition of CDRs, but CDRs defined by other methods are within the scope of the present disclosure.

[0038] As used herein, “framework region” or “FR” region, which includes heavy chain framework regions and light chain framework regions, refers to the regions of an antibody heavy chain variable region (which can be denoted as VH) and light chain variable region (which can be denoted as VL) other than the CDRs; wherein the heavy chain framework regions are denoted as “HFR” and can be further subdivided into mutually separated adjacent regions by CDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; and the light chain framework regions are denoted as “LFR” and can be further subdivided into mutually separated adjacent regions by CDRs, comprising LFR1, LFR2, LFR3, and LFR4 framework regions.

[0039] As used herein, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; and the light chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combination: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0040] In the present context, the terms "identity" or "homology" when used in reference to an amino acid sequence or a nucleic acid sequence relative to a reference sequence, are determined by the percent of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences, as determined by conventional methods, see, e.g., Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978) Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, D.C.). There are a number of algorithms that can be used to align sequences and determine sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the search for similarity method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and the BLAST family of algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs are available that use these algorithms to compare sequences, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program from Intelligenetics, Mountain View, California.

[0041] Without materially affecting the activity of the antibody (retaining at least 95% activity), one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids can be substituted, added and / or deleted from the sequences of the present application to obtain variants of the sequences of the antibody by one skilled in the art. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences of the present application can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequences. The sequence identity of the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences referred to in the present application are all shown in the manner of N-terminal to C-terminal. In addition, "having at least 80% identity or homology" herein means having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0042] It should be noted that in the claims and specification herein, the variants of the antibody sequences are obtained by adding and / or deleting one or more amino acids based on the heavy chain, light chain, variable region of the heavy chain or variable region of the light chain, and the position or number of the mutation sites defined in the specification and claims of the present application also need to be adjusted according to the number and position of the added and / or deleted amino acids. For example, the variant of SEQ ID NO: 1 is obtained by adding one amino acid before the 35th amino acid (e.g. 10th, 27th), and one skilled in the art can understand that S35V should be adjusted to S36V.

[0043] In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule comprising one or more nucleotide or amino acid mutations.

[0044] In this document, the term "Fab antibody" or "Fab" is generally composed of VH and CH1 of the heavy chain and the complete light chain, and the light chain and the heavy chain are connected by one disulfide bond.

[0045] In this document, the term "F(ab')2 antibody" or "F(ab')2" has two antigen-binding F(ab') parts connected together by disulfide bonds.

[0046] In this document, the term "Fab'" fragment contains VH and CH1 of the heavy chain, the complete light chain, and the hinge region, and the light chain and the heavy chain are connected by one disulfide bond.

[0047] In the present document, the term "Fv antibody" generally refers to an antibody formed by a non-covalent association of a light chain variable region (VL) and a heavy chain variable region (VH) and is the smallest functional fragment of an antibody that retains the intact antigen binding site.

[0048] In the present document, the term "single chain antibody" or "scFv" refers to a fragment formed by a short peptide connecting an antibody heavy chain variable region and a light chain variable region.

[0049] Antibody

[0050] In one aspect of the present application, the present application provides an antibody. According to an embodiment of the present application, the antibody comprises: the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1 or a variant thereof;

[0051] wherein the variant of SEQ ID NO: 1 comprises at least one of the following mutation sites: 35th, 44th, 45th, 46th and 50th, compared with the amino acid sequence shown in SEQ ID NO: 1;

[0052] the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 13 or a variant thereof;

[0053] wherein the variant of SEQ ID NO: 13 comprises at least one of the following mutation sites: 32nd, 48th, 56th and 69th, compared with the amino acid sequence shown in SEQ ID NO: 13.

[0054] According to an embodiment of the present application, the above-mentioned antibody can further comprise at least one of the following additional technical features:

[0055] According to an embodiment of the present application, the variant of SEQ ID NO: 1 comprises a mutation of at least one of S35V, R44P, L45A, L45F, L45I, L45M, L45S, L45R, L45T, E46H, E46T, T50V, T50L, T50P, compared with the amino acid sequence shown in SEQ ID NO: 1.

[0056] According to an embodiment of the present application, the 35th is mutated to S35V.

[0057] According to an embodiment of the present application, the 44th is mutated to R44P.

[0058] According to an embodiment of the present application, the 45th is mutated to L45A, L45F, L45I, L45M, L45S, L45R or L45T.

[0059] According to embodiments of the present application, the 46th position is mutated to E46H or E46T.

[0060] According to embodiments of the present application, the 50th position is mutated to T50V, T50L or T50P.

[0061] According to embodiments of the present application, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-12, 25-41 and 79-84.

[0062] According to embodiments of the present application, the variant of SEQ ID NO: 13 comprises a mutation at at least one of S32R, S32K, S48Q, S48G, V56H, V56K, V56L, V56N, V56Q, V56R, V56T, V56A, V56M, V56S, V56Y, G69S, G69T, G69V, as compared to the amino acid sequence of SEQ ID NO: 13.

[0063] According to embodiments of the present application, the 32nd position is mutated to S32R or S32K.

[0064] According to embodiments of the present application, the 48th position is mutated to S48Q or S48G.

[0065] According to embodiments of the present application, the 56th position is mutated to V56H, V56K, V56L, V56N, V56Q, V56R, V56T, V56A, V56M, V56S or V56Y.

[0066] According to embodiments of the present application, the 69th position is mutated to G69S, G69T or G69V.

[0067] According to embodiments of the present application, the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 13-24, 42-66 and 85-105.

[0068] According to embodiments of the present application, the heavy chain variable region is not the same as the light chain variable region as set forth in SEQ ID NO: 1 and SEQ ID NO: 13, respectively.

[0069] According to embodiments of the present application, the antibody comprises a heavy chain variable region and a light chain variable region as set forth in the following table:

[0070]

[0071]

[0072] It should be noted that the above-mentioned positions are numbered by sequentially numbering the amino acid sequences shown in SEQ ID NO: 1 and 13 from N-terminus to C-terminus. For example, the 35th position refers to the 35th amino acid from N-terminus in the amino acid sequence shown in SEQ ID NO: 1; and the "S35V" refers to the substitution of the 35th amino acid in the amino acid sequence shown in SEQ ID NO: 1 from serine to valine.

[0073] In yet another aspect of the present application, the present application provides an antibody. According to embodiments of the present application, the antibody comprises HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprises / consists of an amino acid sequence identical to the HCDR1, HCDR2, HCDR3 of the heavy chain variable region as previously described; and the LCDR1, LCDR2, LCDR3 comprises / consists of an amino acid sequence identical to the LCDR1, LCDR2, LCDR3 of the light chain variable region as previously described.

[0074] According to embodiments of the present application, the above-mentioned antibody can comprise at least one of the following additional technical features:

[0075] According to embodiments of the present application, the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by any one of Kabat, Chothia, IMGT, AbM or Contact system or a combination of multiple systems.

[0076] According to embodiments of the present application, the HCDRs and LCDRs comprise or are the amino acid sequences shown in the following:

[0077] HCDR1: SYGMX1, wherein X1 is S or V;

[0078] HCDR2: X2ISRGGSSTYYPDSVKG, wherein X2 is T, L, P or V;

[0079] HCDR3: LNYGNFFDY;

[0080] LCDR1: RSSQSLVHX3NGNTYLH, wherein X3 is S, R or K;

[0081] LCDR2: KX4SNRFS, wherein X4 is V, H, K, L, N, Q, R, T, A, M, S or Y; and LCDR3: SQNRHVPPT.

[0082] and X1 / X2 / X3 / X4 are not simultaneously S / T / S / V combination;

[0083] According to embodiments of the present application, said X1 is S.

[0084] According to embodiments of the present application, said X1 is V.

[0085] According to embodiments of the present application, said X2 is T.

[0086] According to embodiments of the present application, said X2 is L.

[0087] According to embodiments of the present application, said X2 is P.

[0088] According to embodiments of the present application, said X2 is V.

[0089] According to embodiments of the present application, said X3 is S.

[0090] According to embodiments of the present application, said X3 is R.

[0091] According to embodiments of the present application, said X3 is K.

[0092] According to embodiments of the present application, said X4 is V.

[0093] According to embodiments of the present application, said X4 is H.

[0094] According to embodiments of the present application, said X4 is K.

[0095] According to embodiments of the present application, said X4 is L.

[0096] According to embodiments of the present application, said X4 is N.

[0097] According to embodiments of the present application, said X4 is Q.

[0098] According to embodiments of the present application, said X4 is R.

[0099] According to embodiments of the present application, said X4 is T.

[0100] According to embodiments of the present application, said X4 is A.

[0101] According to embodiments of the present application, said X4 is M.

[0102] According to embodiments of the present application, said X4 is S.

[0103] According to embodiments of the present application, said X4 is Y.

[0104] According to embodiments of the present application, said X1 / X2 / X3 / X4 is selected from any one of the following combinations 1-52:

[0105]

[0106]

[0107] According to an embodiment of the present application, when the antibody has the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 described above, the antibody has higher binding activity to the PIVKA-II protein.

[0108] According to an embodiment of the present application, the antibody further includes at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4.

[0109] At least a part of at least one of the HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR4 is derived from at least one of a murine antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cattle antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0110] According to an embodiment of the present application, the HFR1 amino acid sequence is as shown in SEQ ID NO: 71 or has at least 80% homology thereto.

[0111] The HFR2 amino acid sequence is as shown in SEQ ID NO: 72 or has at least 80% homology thereto.

[0112] The HFR3 amino acid sequence is as shown in SEQ ID NO: 73 or has at least 80% homology thereto.

[0113] The HFR4 amino acid sequence is as shown in SEQ ID NO: 74 or has at least 80% homology thereto.

[0114] The LFR1 amino acid sequence is as shown in SEQ ID NO: 75 or has at least 80% homology thereto.

[0115] The LFR2 amino acid sequence is as shown in SEQ ID NO: 76 or has at least 80% homology thereto.

[0116] The LFR3 amino acid sequence is as shown in SEQ ID NO: 77 or has at least 80% homology thereto.

[0117] The LFR4 amino acid sequence is as shown in SEQ ID NO: 78 or has at least 80% homology thereto.

[0118] According to embodiments of the present application, the HFR2 amino acid sequence is WVRQTPDKX5X6X7WVA, wherein X5 is R or P, X6 is L, A, F, I, M, R, S or T, and X7 is E, H or T.

[0119] According to embodiments of the present application, the LFR2 amino acid sequence is WYLQKPGQX8PKLLIY, wherein X8 is S, Q or G.

[0120] According to embodiments of the present application, the LFR3 amino acid sequence is GVPDRFSX9SGSGTDFTLKISRVEAEDLGVYFC, wherein X9 is G, S, T or V.

[0121] According to embodiments of the present application, X5 is R.

[0122] According to embodiments of the present application, X5 is P.

[0123] According to embodiments of the present application, X6 is L.

[0124] According to embodiments of the present application, X6 is A.

[0125] According to embodiments of the present application, X6 is F.

[0126] According to embodiments of the present application, X6 is I.

[0127] According to embodiments of the present application, X6 is M.

[0128] According to embodiments of the present application, X6 is R.

[0129] According to embodiments of the present application, X6 is S.

[0130] According to embodiments of the present application, X6 is T.

[0131] According to embodiments of the present application, X7 is E.

[0132] According to embodiments of the present application, X7 is H.

[0133] According to embodiments of the present application, X7 is T.

[0134] According to embodiments of the present application, X8 is S.

[0135] According to embodiments of the present application, X8 is Q.

[0136] According to embodiments of the present application, X8 is G.

[0137] According to embodiments of the present application, X9 is G.

[0138] According to embodiments of the present application, the X9 is S.

[0139] According to embodiments of the present application, the X9 is T.

[0140] According to embodiments of the present application, the X9 is V.

[0141] According to embodiments of the present application, the X5 / X6 / X7 / X8 / X9 is selected from any one of the following numbers 1-58:

[0142]

[0143]

[0144] According to embodiments of the present application, the antibody comprises a heavy chain variable region identical to the amino acid sequence of the heavy chain variable region according to any one of the preceding embodiments, and / or a light chain variable region identical to the amino acid sequence of the light chain variable region according to any one of the preceding embodiments.

[0145] According to embodiments of the present application, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 1 or a variant thereof;

[0146] wherein the variant of SEQ ID NO: 1 comprises at least one of the following mutation sites: 35th, 44th, 45th, 46th and 50th, as compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0147] the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 13 or a variant thereof;

[0148] wherein the variant of SEQ ID NO: 13 comprises at least one of the following mutation sites: 32nd, 48th, 56th and 69th, as compared to the amino acid sequence set forth in SEQ ID NO: 13.

[0149] According to embodiments of the present application, the antibody described above can further comprise at least one of the following additional technical features:

[0150] According to embodiments of the present application, the variant of SEQ ID NO: 1 comprises a mutation of at least one of the following sites: S35V, R44P, L45A, L45F, L45I, L45M, L45S, L45R, L45T, E46H, E46T, T50V, T50L, T50P, as compared to the amino acid sequence set forth in SEQ ID NO: 1.

[0151] According to embodiments of the present application, the 35th is mutated to S35V.

[0152] According to embodiments of the present application, the 44th position is mutated to R44P.

[0153] According to embodiments of the present application, the 45th position is mutated to L45A, L45F, L45I, L45M, L45S, L45R or L45T.

[0154] According to embodiments of the present application, the 46th position is mutated to E46H or E46T.

[0155] According to embodiments of the present application, the 50th position is mutated to T50V, T50L or T50P.

[0156] According to embodiments of the present application, the heavy chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 1-12, 25-41 and 79-84.

[0157] According to embodiments of the present application, the variant of SEQ ID NO: 13 comprises a mutation at at least one of S32R, S32K, S48Q, S48G, V56H, V56K, V56L, V56N, V56Q, V56R, V56T, V56A, V56M, V56S, V56Y, G69S, G69T, G69V, as compared to the amino acid sequence of SEQ ID NO: 13.

[0158] According to embodiments of the present application, the 32nd position is mutated to S32R or S32K.

[0159] According to embodiments of the present application, the 48th position is mutated to S48Q or S48G.

[0160] According to embodiments of the present application, the 56th position is mutated to V56H, V56K, V56L, V56N, V56Q, V56R, V56T, V56A, V56M, V56S or V56Y.

[0161] According to embodiments of the present application, the 69th position is mutated to G69S, G69T or G69V.

[0162] According to embodiments of the present application, the light chain variable region comprises an amino acid sequence selected from any one of SEQ ID NOs: 13-24, 42-66 and 85-105.

[0163] According to embodiments of the present application, the heavy chain variable region is not the same as the light chain variable region as set forth in SEQ ID NO: 1 and SEQ ID NO: 13, respectively.

[0164] According to an embodiment of the present application, the antibody comprises a heavy chain variable region and a light chain variable region as shown in the following table:

[0165]

[0166]

[0167] It should be noted that the above-mentioned site numbers are obtained by sequentially numbering the amino acid sequences shown in SEQ ID NO: 1 and 13 from N-terminus to C-terminus. For example, the 35th site refers to the 35th amino acid from N-terminus in the amino acid sequence shown in SEQ ID NO: 1; and the "S35V" refers to the substitution of the 35th amino acid in the amino acid sequence shown in SEQ ID NO: 1 with valine.

[0168] According to an embodiment of the present application, the antibody comprises at least one of a heavy chain constant region and a light chain constant region.

[0169] According to an embodiment of the present application, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the light chain constant region of kappa type or lambda type.

[0170] According to an embodiment of the present application, the species origin of the constant region is bovine, equine, bovine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, cock or human.

[0171] Alternatively, the species origin of the constant region is mouse.

[0172] According to an embodiment of the present application, the heavy chain constant region has the amino acid sequence shown in SEQ ID NO: 67 or an amino acid sequence having 80% or more homology with SEQ ID NO: 67; and the light chain constant region has the amino acid sequence shown in SEQ ID NO: 68 or an amino acid sequence having 80% or more homology with SEQ ID NO: 68.

[0173] According to an embodiment of the present application, the antibody comprises a light chain and / or a heavy chain, the heavy chain comprising the heavy chain variable region and the heavy chain constant region according to any one of the preceding embodiments; and the light chain comprising the light chain variable region and the light chain constant region according to any one of the preceding embodiments.

[0174] According to an embodiment of the present application, the N-terminus of the heavy chain constant region is connected to the C-terminus of the heavy chain variable region; and the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region.

[0175] Nucleic acid molecule, vector, recombinant cell

[0176] In the process of preparing or obtaining these antibodies, nucleic acid molecules expressing these antibodies can be used, linked with different vectors, and then expressed in different cells to obtain the corresponding antibodies.

[0177] In another aspect of the present application, the present application provides a nucleic acid molecule encoding the aforementioned antibody. The antibody obtained from the nucleic acid molecule according to some embodiments of the present application has higher PIVKA-II binding activity.

[0178] According to some embodiments of the present application, the aforementioned nucleic acid molecule can further comprise at least one of the following additional technical features:

[0179] According to some embodiments of the present application, the nucleic acid molecule comprises DNA or RNA.

[0180] It should be noted that, for the nucleic acid mentioned in the specification and claims of the present application, a person skilled in the art should understand that, actually, either one of the complementary double strands or both are included. For the convenience, in the specification and claims, although only one strand is given in most cases, actually, the other complementary strand is also disclosed. In addition, the nucleic acid sequence in the present application includes DNA form or RNA form, and disclosing one means that the other is also disclosed.

[0181] In some embodiments, the present application provides a vector carrying the aforementioned nucleic acid molecule. In the process of linking the aforementioned nucleic acid molecule to the vector, the nucleic acid molecule can be directly or indirectly linked with the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid can be operably linked with the control elements. "Operably linked" herein means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc. can play their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the nucleic acid molecule encoding the antibody can be inserted into different vectors respectively and independently, and commonly inserted into the same vector. Commonly used vectors can be plasmids, bacteriophages, etc. After the expression vector according to some embodiments of the present application is introduced into a suitable recipient cell, the aforementioned antibody expression can be effectively realized under the mediation of the regulation system, and then the antibody can be obtained in large quantities in vitro.

[0182] According to some embodiments of the present application, the aforementioned vector can further comprise at least one of the following additional technical features:

[0183] According to some embodiments of the present application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0184] In some further aspects of the present application, the present application provides a cell, which carries the nucleic acid molecule as described above, or expresses the antibody as described above. The cell according to some embodiments of the present application can be used for in vitro expression and mass production of the antibody as described above under suitable conditions.

[0185] According to some embodiments of the present application, the cell as described above can further include at least one of the following additional technical features:

[0186] According to some embodiments of the present application, the cell is obtained by introducing the expression vector as described above into a host cell.

[0187] According to some embodiments of the present application, the expression vector is introduced into the host cell by electroporation.

[0188] According to some embodiments of the present application, the cell is a mammalian cell.

[0189] It should be noted that the cell according to the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Giardia lamblia, etc. The eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cell such as Spodoptera exigua, plant cell such as tobacco, mammalian cell such as BHK cell, CHO cell, COS cell, myeloma cell, etc. In some embodiments, the cell according to the present application is preferably a mammalian cell, including BHK cell, CHO cell, NSO cell or COS cell, and does not include animal reproductive cell, fertilized egg or embryonic stem cell.

[0190] It should be noted that the "suitable conditions" as described in the present application means conditions suitable for expression of the antibody according to the present application. It is easily understood by those skilled in the art that the conditions suitable for the antibody include but are not limited to suitable transformation or transfection method, suitable transformation or transfection condition, healthy state of host cell, suitable density of host cell, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions suitable for expression of the antibody according to the specific environment of the laboratory.

[0191] In one aspect of the present application, the present application provides a method for preparing the antibody as described above. The method includes culturing the cell as described above. The method according to some embodiments of the present application can effectively mass produce the antibody.

[0192] Antibody conjugate, reagent or kit and use in the preparation of a detection or diagnostic product

[0193] In yet another aspect of the present application, there is provided an antibody conjugate comprising the antibody as described above and a conjugate moiety conjugated thereto.

[0194] According to some embodiments of the present application, the antibody conjugate as described above can further comprise at least one of the following additional technical features:

[0195] According to some embodiments of the present application, the conjugate moiety is selected from a purification tag or a detectable label, including one or more of colloidal gold, a radioactive label, a luminescent substance, a colored substance, an enzyme, such as a fluorescent label, a chromophore label, an electron-dense label, such as a radioisotope, a fluorophore, rhodamine and its derivatives, luciferase, fluorescein, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, a carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / avidin, and a spin label.

[0196] According to embodiments of the present application, the conjugate moiety is selected from a magnetic microsphere, a plastic microsphere, a plastic microparticle, a microwell plate, glass, a capillary tube, nylon, and a nitrocellulose membrane.

[0197] In yet another aspect of the present application, there is provided a reagent or a kit comprising the antibody or the antibody conjugate as described above. As described above, the antibody according to some embodiments of the present application is capable of effectively binding to PIVKA-II protein, and thus the reagent or the kit comprising the antibody is capable of effectively detecting PIVKA-II protein qualitatively or quantitatively. The reagent or the kit according to the present application can be used in, for example, immunoblotting, immunoprecipitation, and other tests involving the specific binding of PIVKA-II and its antibody. As described above, the antibody according to some embodiments of the present application has a higher binding activity to PIVKA-II, and thus the reagent or the kit comprising the antibody has a higher detection sensitivity or specificity.

[0198] The kits can comprise any one or more of the following: a treatment solution, an anti-PIVKA-II antibody, a PIVKA-II control, an anti-IgG antibody, instructions or literature, etc. The anti-PIVKA-II antibody can be used in various types of diagnostic tests, such as tests for detecting the presence of various diseases or drugs, or other proteins, in vitro or in vivo. For example, the presence of a disease can be tested by detecting serum or blood from a subject.

[0199] In another aspect of the present application, the present application provides use of the antibody, the antibody conjugate, the reagent or the kit as described above in detecting PIVKA-II, preparing a product for detecting PIVKA-II or preparing a product for diagnosing a PIVKA-II related disease.

[0200] As described above, the antibody of some embodiments of the present application can effectively bind to PIVKA-II protein, and thus the antibody has the use as described above.

[0201] According to some embodiments of the present application, the use as described above can further include at least one of the following additional technical features:

[0202] According to some embodiments of the present application, the PIVKA-II related disease includes at least one of vitamin K deficiency and hepatocellular carcinoma.

[0203] In one aspect of the present application, the present application provides a method for detecting PIVKA-II in a test sample, which includes contacting the antibody, the antibody conjugate or the reagent or the kit as described above with PIVKA-II antigen in the sample to be detected to form an immune complex.

[0204] According to embodiments of the present application, the immune complex further includes a second antibody, which binds to the antibody.

[0205] According to embodiments of the present application, the immune complex further includes a second antibody, which binds to the PIVKA-II antigen.

[0206] It should be noted that the "test sample" as described above can be a sample to be detected from a patient, such as a serum sample or the like; or can be a non-patient sample that can contain PIVKA-II, for example, in scientific research, the above method is only used to detect the presence of PIVKA-II or the content of PIVKA-II in the sample, and does not involve disease diagnosis.

[0207] In another aspect of the present application, the present application provides a method for screening a PIVKA-II antibody, which includes: a) designing primers for amino acid substitution at one, two, three or four of the mutation sites or X1, X2, X3, X4 sites as described above; b) constructing a mutation library using the primers of a) as a template of the nucleic acid, the vector or the cell as described above; and c) screening a PIVKA-II antibody from the mutation library.

[0208] According to embodiments of the present application, the primers are designed for amino acid substitution at one, two, three or four of the mutation sites or X1, X2, X3, X 4， X 5、 X 6、 X7、 X 8、 1, 2, 3, 4, 5, 6, 7, 8 or 9 positions in the X9 position are substituted with an amino acid substitution;

[0209] According to embodiments of the present application, the mutant library is a single point saturation mutant library.

[0210] According to embodiments of the present application, the mutant library is a combinatorial mutant library.

[0211] According to embodiments of the present application, the PIVKA-II antibody comprises or is the antibody as previously described.

[0212] In another aspect of the present application, the present application provides a mutant library comprising the antibody as previously described; according to embodiments of the present application, the mutant library is constructed using the step of constructing a mutant library in the methods as previously described. The amino acid sequences involved herein are shown in Table 2:

[0213] Table 2

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely. In the embodiments, the specific conditions not noted are carried out according to the conventional conditions or the conditions suggested by the manufacturers. The reagents or instruments not noted the manufacturers are all the conventional products that can be obtained by the market purchase.

[0221] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

[0222] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (J. E. Coligan et al., eds., 2011), each of which is incorporated herein by reference in its entirety.

[0223] Example 1: Construction and screening of a mutation library

[0224] 1.1 Construction of wild-type (WT) PIVKA-II antibody template plasmid

[0225] (1) WT PIVKA-II antibody gene synthesis

[0226] The sequences encoding the heavy chain variable region of wild-type PIVKA-II antibody and the light chain variable region were E. coli codon-optimized, and then the antibody gene sequence was submitted to a company for gene synthesis. The heavy chain and light chain amino acid sequences of WT PIVKA-II antibody are shown in SEQ ID NOs: 69, 70, respectively.

[0227] (2) WT PIVKA-Ⅱ antibody gene fragment amplification

[0228] The antibody coding sequence synthesized in step (1) is subjected to PCR amplification using a DNA polymerase, and the antibody band is separated by agarose gel electrophoresis, and the antibody gene fragment is purified by a gel recovery kit.

[0229] (3) WT PIVKA-Ⅱ antibody gene fragment digestion and ligation

[0230] The antibody gene fragment obtained in step (2) and the V01 carrier plasmid are simultaneously subjected to double digestion with a restriction endonuclease, and the antibody gene fragment and the V01 carrier with sticky ends are purified by a gel recovery kit. Then, the antibody gene fragment and the V01 carrier are ligated at 22°C for 4 hours using a T4 DNA ligase, and the ligation reaction product is recovered and purified, and the DNA concentration is measured. Finally, 100 ng of the plasmid is transformed into 100 μL of TG1 E. coli competent cells, and the bacterial solution is completely coated on a plate containing ampicillin resistance, and cultured at 37°C overnight.

[0231] (4) Extraction and sequencing verification of WT PIVKA-Ⅱ template plasmid

[0232] Ten single colonies cultured overnight in step (3) are selected, colony PCR and gel electrophoresis are performed using Taq DNA polymerase, and the correct insertion of the antibody gene sequence is selected for culture and amplification. The WT template plasmid is extracted by a plasmid extraction kit, and the gene sequencing verification is performed by a sequencing company.

[0233] 1.2 Construction of single-point mutation library

[0234] In this part of the experiment, the VH and VL full variable regions of the WT PIVKA antibody are subjected to single-point saturation mutation to construct a single-point mutation library.

[0235] (1) Primer design and synthesis

[0236] First, the single-point saturation mutation upstream and downstream primers 230 pairs of the VH and VL full variable regions (230 amino acid sites) of the WT PIVKA antibody are designed using the annexed base codon, and the primer synthesis is outsourced to a company.

[0237] (2) Single-point saturation mutation plasmid PCR amplification

[0238] The primer obtained in step (1) is used to amplify the single point saturation mutation plasmid by PCR. The reaction system is configured according to Table 3, and then the PCR reaction conditions in Table 3 are used to amplify the single point saturation mutation library plasmid. Finally, the WT template plasmid is digested with a restriction enzyme at 37°C for 1 hour to obtain 230 mutant library plasmids.

[0239] Table 3

[0240] Component Addition WT PIVKA antibody template plasmid 50 ng DNA polymerase 1 μL DNA polymerase buffer 10 μL dNTP (2.5 mM) 4 μL Upstream primer (10 uM) 1 μL Downstream primer (10 uM) 1 μL ddH2O q.s. to 50 μL

[0241] Table 4

[0242] Item Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Temperature 95℃ 95℃ 55-60℃ 72℃ 72℃ 4℃ Time 5 min 30s 30s 2 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min 5 min ∞

[0243] Note: Step 2-Step 4 for 22 cycles

[0244] (3) Single point saturation mutation plasmid transformation

[0245] Take 10 μL of each of the 230 mutant library plasmids obtained in step (2) and transform them into 100 μL of TG1 E. coli competent cells. Spread the bacterial solution on a plate containing ampicillin resistance, and incubate at 37°C overnight.

[0246] 1.3 Screening of single point mutation library

[0247] (1) Mutant library antibody expression

[0248] In the 96-well culture plate, 500 μL of culture medium is added in advance. For each single point mutation library, 92 single colonies of the single point saturation mutation plasmid transformed by the above overnight culture are selected, and WT, negative, and blank control colonies are set. After incubation at 37°C for 5-6 hours, the bacterial solution is transferred to a new 96-well culture plate, and then incubated at 37°C for 1-2 hours. Finally, the induction medium is added, and the antibody is expressed at 37°C overnight to obtain the antibody expression supernatant of the 230 mutant libraries.

[0249] (2) Mutant library screening and sequencing

[0250] PIVKA-Ⅱ was added to 230 ELISA plates at an amount of 0.032 μg / mL, 100 μL / well, and reacted for coating at 4°C overnight. The next day, 1% to 2% skim milk was used for blocking. The antibody expression supernatant of the 230 mutant libraries obtained in step (1) was added to the ELISA plate wells at an amount of 100 μL / well, and WT, negative, and blank controls were set. Incubation was performed at room temperature for 2 hours. Subsequent plate washing, color development, and reading were performed using the conventional ELISA detection method. Finally, the data results were sorted and analyzed, and the Ratio value was calculated. The Ratio value represents the degree of affinity improvement. When the Ratio value is equal to 1, it indicates that the affinity of the mutant clone is the same as that of WT. When the Ratio value is greater than 1, it indicates that the affinity has improved. Then, the clones with improved affinity compared to the WT group were sequenced. Finally, the sequencing results were analyzed, and the mutation sites of the 22 unique mutant candidate clones shown in Table 5 were combined to construct a mutant library.

[0251] Table 5

[0252]

[0253] 1.4 Construction of a combined mutant library

[0254] (1) Library primer design and synthesis

[0255] According to the mutation sites on the WT PIVKA-Ⅱ antibody VH and VL obtained by screening in Section 1.3, the amplification primers for the combined mutant library were designed, and the primers were synthesized by an outsourcing company. It should be noted that:

[0256] a. Since there are many mutation amino acids (A / F / R / T) at L45 of VH, a compatible base was used when designing the primers, so the mutation amino acids at this site are not only A / F / R / T.

[0257] b. Since there are many mutation amino acids (Q / N / R / L) at V56 of VL, a compatible base was used when designing the primers, so the mutation amino acids at this site are not only Q / N / R / L.

[0258] (2) Amplification and ligation of combined mutant antibody fragments

[0259] The combined mutant antibody fragments obtained in step (1) were amplified according to the PCR system shown in Table 3 and the PCR reaction conditions shown in Table 4, and then the antibody mutant fragments were gel recovered. The antibody mutant fragments were spliced into complete antibody fragments using the method of Overlap PCR.

[0260] Finally, the antibody fragments were inserted into the VOl vector by enzyme digestion and ligation to form the complete antibody expression plasmid, and the operation method of this part was referred to the step (3) "WT PIVKA-Ⅱ antibody gene fragment enzyme digestion and ligation" in section 1.1. 100 ng of plasmid was transformed into 100 μL of TG1 E. coli competent cells, and the bacterial solution was completely coated on a plate containing ampicillin resistance and cultured at 37°C overnight.

[0261] 1.5 Screening of combined mutation library

[0262] Randomly selected 70 combined mutant antibodies were subjected to supernatant expression, ELISA screening detection and positive clone sequencing analysis.

[0263] The ratio value determination is shown in Table 6, and the position of the mutant amino acid is obtained by sequentially numbering the amino acid sequence of the WT PIVKA-Ⅱ antibody heavy chain or light chain from N-terminus to C-terminus.

[0264] Table 6

[0265]

[0266]

[0267] Example 2: Expression of mutant PIVKA-Ⅱ antibody

[0268] This example is for the expression of the mutant PIVKA-Ⅱ antibody obtained by screening in Example 1, and the specific experimental operation is as follows:

[0269] 2.1 Construction of eukaryotic recombinant expression plasmid

[0270] A recombinant antibody eukaryotic expression vector pcDNA TM 3.4 vector was constructed, and the expression vector has introduced multiple cloning enzyme digestion sites such as HindIII, BamHI and EcoRI, and is named pcDNA3.4A expression vector, which is hereinafter referred to as 3.4A expression vector; according to the variable region gene sequence of the antibody in Example 1, the corresponding antibody VL and VH gene specific amplification primers and constant region overlap primers were designed, the two end primers were respectively provided with HindIII, EcoRI enzyme digestion sites and protection bases, and the 0.73 KB light chain (Light Chain) gene fragment and the 1.40 kb heavy chain (Heavy Chain) gene fragment were amplified by PCR amplification method.

[0271] The gene fragments encoding the heavy chain and the light chain are respectively subjected to HindIII / EcoRI double enzyme digestion, the 3.4A vector is subjected to HindIII / EcoRI double enzyme digestion, the antibody light chain and heavy chain gene fragments and the vector after enzyme digestion are purified and recovered, then the antibody light chain and heavy chain gene fragments are respectively connected into the 3.4A expression vector and transformed into the DH5α E. coli competent cells, after the colonies are grown, single colonies are respectively picked for PCR identification of positive clones, the positive clones are picked for sequencing to determine the correctness of the sequences. The correct sequencing clones are selected for plasmid extraction for standby use.

[0272] 2.2 Sample preparation of mutant antibodies

[0273] The HEK293 cells are recovered in advance, subcultured to 200 mL system, and the cell density is adjusted to 3-5 x 10 6 cells / mL, and the cell viability is greater than 95%; then the cells are centrifuged and washed, resuspended with the culture medium, and the cell density is adjusted to 2.9 x 10 6 cells / mL as a cell diluent. The plasmid DNA and the transfection reagent diluent are respectively prepared with the culture medium. The transfection reagent diluent is added to the plasmid DNA diluent, mixed, and then placed at room temperature for 15 min; the mixture is slowly added to the above-mentioned cell diluent within 1 min, mixed, sampled, counted, and the cell viability after transfection is recorded and observed, and the mixture is placed in a 35℃ constant temperature incubator for culture at a rotation speed of 120 rpm and a CO2 content of 8% for 13 days, and then centrifuged to collect the sample. The collected liquid is subjected to affinity purification with a protein A affinity chromatography column to obtain the mutant PIVKA-Ⅱ antibody.

[0274] 2.3 Affinity analysis

[0275] The dissociation curve of the antigen PIVKA-Ⅱ and the mutant PIVKA-Ⅱ antibody is tested on a Biacore 8K+ device, and the affinity constant, the association rate and the dissociation rate are obtained by automatic fitting of the instrument. In the affinity detection result, KD represents the equilibrium dissociation constant, that is, the affinity constant; ka represents the association rate; and kd represents the dissociation rate. The result shows that the mutant PIVKA-Ⅱ antibody screened in Example 1 has improved affinity with PIVKA-Ⅱ compared with WT. The affinity detection result of the exemplary antibody is shown in Table 7 as follows:

[0276] Table 7: Antibody affinity detection result

[0277]

[0278]

[0279] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0280] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. An anti-PIVKA-II antibody, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region as shown in any combination of the following table:

2. An anti-PIVKA-II antibody, characterized in that, comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 are identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 defined in any combination of the antibody of claim 1; The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by any one of Kabat, Chothia, IMGT, AbM or Contact system.

3. An anti-PIVKA-II antibody comprising HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, characterized in that, The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are amino acid sequences as shown in the following: HCDR1: SYGMX1, wherein X1 is S or V; HCDR2: X2ISRGGSSTYYPDSVKG, wherein X2 is T, L, P or V; HCDR3: LNYGNFFDY; LCDR1: RSSQSLVHX3NGNTYLH, wherein X3 is S, R or K; LCDR2: KX4SNRFS, wherein X4 is V, H, K, L, N, Q, R, T, A, M, S or Y; LCDR3: SQNRHVPPT; The X1 / X2 / X3 / X4 is selected from any one of the following combinations 1-52:

4. The antibody according to any one of claims 1 to 3, characterized in that, The antibody comprises HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4; The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are from at least one of murine antibody, primate antibody, bovine antibody, equine antibody, porcine antibody, ovine antibody, caprine antibody, canine antibody, feline antibody, rabbit antibody, camelid antibody, donkey antibody, cervine antibody, mink antibody, chicken antibody, duck antibody, goose antibody.

5. The antibody of claim 4, wherein The HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are from at least one of human antibody, bovine antibody, turkey antibody, cockfighting chicken antibody.

6. The antibody of claim 4, wherein The HFR1 amino acid sequence is an amino acid sequence having at least 80% homology with SEQ ID NO: 71; The HFR2 amino acid sequence is an amino acid sequence having at least 80% homology with SEQ ID NO: 72; The HFR3 amino acid sequence is an amino acid sequence having at least 80% homology with SEQ ID NO: 73; The HFR4 amino acid sequence is an amino acid sequence having at least 80% homology with SEQ ID NO: 74; The LFR1 amino acid sequence is an amino acid sequence having at least 80% homology with SEQ ID NO: 75; the LFR2 amino acid sequence is an amino acid sequence having at least 80% homology to SEQ ID NO: 76; the LFR3 amino acid sequence is an amino acid sequence having at least 80% homology to SEQ ID NO: 77; the LFR4 amino acid sequence is an amino acid sequence having at least 80% homology to SEQ ID NO:

78.

7. The antibody of claim 6, wherein the HFR2 amino acid sequence is WVRQTPDKX5X6X7WVA, wherein X5 is R or P, X6 is L, A, F, I, M, R, S or T, and X7 is E, H or T.

8. The antibody of claim 6, wherein the LFR2 amino acid sequence is WYLQKPGQX8PKLLIY, wherein X8 is S, Q or G.

9. The antibody of claim 6, wherein the LFR3 amino acid sequence is GVPDRFSX9SGSGTDFTLKISRVEAEDLGVYFC, wherein X9 is G, S, T or V.

10. The antibody of any one of claims 7-9, wherein, the X5 / X6 / X7 / X8 / X9 is selected from any one of the following numbers 1-58: 。 11. The antibody of any one of claims 1-3, 5-9, wherein, the antibody comprises a heavy chain constant region and a light chain constant region.

12. The antibody of claim 11, wherein the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the kappa type or lambda type light chain constant region.

13. The antibody of claim 11, wherein the species origin of the constant region is bovine, equine, porcine, ovine, caprine, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose or human.

14. The antibody of claim 11, wherein the species origin of the constant region is bovine, turkey or cock.

15. The antibody of claim 11, wherein the species origin of the constant region is mouse.

16. The antibody of claim 11, wherein the heavy chain constant region is an amino acid sequence having more than 80% homology to SEQ ID NO: 67, and the light chain constant region is an amino acid sequence having more than 80% homology to SEQ ID NO:

68.

17. A nucleic acid molecule, characterized in that, the nucleic acid molecule encodes the antibody of any one of claims 1-16.

18. A vector, characterized in that, comprising the nucleic acid molecule of claim 17.

19. A cell, comprising: the cell comprises the nucleic acid molecule of claim 17, the vector of claim 18 or expresses the antibody of any one of claims 1-16.

20. A method of producing an antibody according to any one of claims 1 to 16, characterized in that, the method comprises culturing the cell of claim 19.

21. An antibody conjugate, characterized in that, the antibody conjugate consists of the antibody of any one of claims 1-16 and a purification tag or label coupled thereto, the purification tag or label being selected from the group consisting of a luminescent substance, an enzyme or biotin / avidin.

22. An antibody conjugate, characterized in that, the antibody conjugate consists of the antibody of any one of claims 1-16 and a purification tag or label coupled thereto, the label being selected from a radioactive label, an electron-dense label or a spin label.

23. An antibody conjugate, characterized in that, the antibody conjugate consists of the antibody of any one of claims 1-16 and a purification tag or label coupled thereto, the purification tag or label being selected from one or more of colloidal gold, magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

24. The antibody conjugate of claim 21, wherein, the luminescent substance is selected from a fluorescent label or a chromophore label.

25. The antibody conjugate of claim 24, wherein, the fluorescent label is selected from a fluorophore, rhodamine or fluorescein.

26. The antibody conjugate of claim 21, wherein, The enzyme is selected from one or more of luciferase, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase.

27. A reagent or kit, characterized in that, The reagent or kit comprises the antibody of any one of claims 1 to 16 or the antibody conjugate of any one of claims 21 to 26.

28. Use of the antibody of any one of claims 1 to 16, the antibody conjugate of any one of claims 21 to 26, or the reagent or kit of claim 27 for the manufacture of a product for detecting PIVKA-II or for detecting PIVKA-II for non-disease diagnostic purposes.

29. Use of an antibody according to any one of claims 1 to 16, an antibody conjugate according to any one of claims 21 to 26 or a reagent or kit according to claim 27 for the manufacture of a product for the detection of PIVKA-II, characterized in that, comprising: contacting the antibody of any one of claims 1 to 16, the antibody conjugate of any one of claims 21 to 26, or the reagent or kit of claim 27 with PIVKA-II antigen in a sample to be tested to form an immunocomplex.

30. Use according to claim 29, characterized in that, The immunocomplex further comprises a second antibody that binds to the antibody.

31. The use according to claim 29, characterized in that, The immunocomplex further comprises a second antibody that binds to the PIVKA-II antigen.

32. A method of screening for antibodies to PIVKA-II, comprising: contacting a sample with a PIVKA-II antigen; and detecting the presence of antibodies to PIVKA-II in the sample. The method comprises: a) designing primers for amino acid substitution at X1, X2, X3, X4 sites defined by any one of the combination of antibodies of claim 3; b) constructing a mutation library using the primers of a) as templates with the nucleic acid of claim 17, the vector of claim 18, or the cell of claim 19; c) screening PIVKA-II antibodies from the mutation library.

33. The method of claim 32, wherein, The mutation library is a single point saturation mutation library.

34. The method of claim 32, wherein, The PIVKA-II antibody comprises or is the antibody of any one of claims 1 to 16.

35. A library of mutations, wherein, The mutation library comprises the antibody of any one of claims 1 to 16.

36. The mutation library of claim 35, which is obtained using the method of any one of claims 32 to 34.

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