High affinity antibodies that specifically bind to α-1,6-core-fucosylated alpha-fetoprotein

Abstract

The present invention relates to monoclonal antibodies and antigen-binding fragments that specifically bind to α-1,6-core-fucosylated alpha-fetoprotein (AFP), which is the core component of AFP-L3. Thus, the antibodies and antigen-binding fragments provided herein may also be referred to as AFP-L3 antibodies. Also provided are polynucleotides encoding the antibodies or antigen-binding fragments of the present invention, host cells expressing the antibodies and antigen-binding fragments of the present invention, methods for producing the antibodies and antigen-binding fragments of the present invention, and uses of the antibodies and antigen-binding fragments of the present invention. Also provided herein are pretreatment agents that facilitate the binding of the antibodies and antigen-binding fragments of the present invention to α-1,6-core-fucosylated AFP. The present invention further relates to kits comprising the antibodies and antigen-binding fragments of the present invention, optionally together with the pretreatment agents of the present invention.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies and antigen-binding fragments that specifically bind to α-1,6-core-fucosylated alpha-fetoprotein (AFP), which is the core component of AFP-L3. Accordingly, the antibodies and antigen-binding fragments provided herein may also be referred to as AFP-L3 antibodies. Also provided are polynucleotides encoding the antibodies or antigen-binding fragments of the present invention, host cells expressing the antibodies and antigen-binding fragments of the present invention, methods for producing the antibodies and antigen-binding fragments of the present invention, and uses of the antibodies and antigen-binding fragments of the present invention. Also provided herein are pretreatment agents that facilitate the binding of the antibodies and antigen-binding fragments of the present invention to α-1,6-core-fucosylated AFP. The present invention further relates to kits comprising the antibodies and antigen-binding fragments of the present invention and optionally the pretreatment agents of the present invention.

Background Art

[0002] Liver cancer is the seventh most common cancer in the world and the second leading cause of cancer death.

[0003] Hepatocellular carcinoma (HCC) is the major histological type among primary liver cancers occurring worldwide and accounts for 70% to 85% of the total burden. It is known that underlying liver diseases such as hepatic fibrosis and cirrhosis are the main risk factors for the development of HCC. HCC can be treated by resection, liver transplantation, or local ablation by high frequency for patients diagnosed early. Therefore, detection at the early stage of HCC and minimally invasive screening methods are very important.

[0004] The most common methods for the diagnosis of HCC are imaging techniques such as ultrasound detection, computed tomography (CAT scan), or magnetic resonance imaging (MRI), and serological biomarkers. However, ultrasound detection requires a tumor mass of at least 2 cm, resulting in a poor prognostic value. Imaging techniques have low sensitivity for each lesion and are costly. On the other hand, in recent years, much focus has been placed on the discovery of new blood biomarkers that can be used in surveillance programs for the early detection of HCC in high-risk patients (Yang JD. Detect or not to detect very early stage hepatocellular carcinoma? The western perspective. Clin Mol Hepatol. 2019;25(4):335-43).

[0005] Some current medical guidelines recommend monitoring high-risk patients every six months using ultrasound or other imaging modalities. However, limitations of the imaging approach include low sensitivity for early tumors, operator dependence, and poor quality in patients with obesity or non-alcoholic steatohepatitis.

[0006] Alpha-fetoprotein (AFP) is the most well-established current biomarker for hepatocellular carcinoma (HCC). However, there is a need to further improve the sensitivity and specificity of AFP for HCC diagnosis, especially for early HCC diagnosis.

[0007] AFP is a glycoprotein, and various glycosylated forms of AFP have been described. In particular, AFP is N-glycosylated at asparagine 251. Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the sugar chain structure of glycoproteins, it is possible to separate and concentrate marker glycoprotein fractions (plural possible) having a specific sugar chain structure. In the case of AFP, lectins derived from Lens culinaris agglutinin-A (LCA) are widely used. Using LCA, AFP can be fractionated into three variants L1, L2, and L3, and AFP-L3 has the highest affinity for LCA. The AFP-L3 fraction is composed of AFP, and AFP is N-glycosylated at Asn-251 using an N-glycan containing α-1,6-core-fucosylated (i.e., AFP in which a fucose sugar is bound to N-acetylglucosamine (GlcNAc) located at the reducing end of the N-type sugar chain via an α-1,6 bond). Therefore, since AFP-L3 is composed of α-1,6-core-fucosylated AFP, the terms AFP-L3 and α-1,6-core-fucosylated AFP are used interchangeably herein. The Lens culinaris agglutinin (LCA) reactive fraction of alpha-fetoprotein (AFP-L3) specifically increases in HCC patients (Khien VV et al., The International Journal of Biological Markers. 2001;16(2):105-111).

[0008] Recent publications have clearly pointed out that scores including the gender, age, AFP, and des-carboxyprothrombin (DCP = PIVKA-II) (= GAAD score) or gender, age, AFP, AFP-L3, and des-carboxyprothrombin (DCP = PIVKA-II) (= GALAD score) of input variable factors can also improve the results of HCC screening efforts, especially the detection of early HCC, and will improve these (Zhou, J-M., Wang, T., Zhang, K-H. AFP-L3 for the diagnosis of early hepatocellular carcinoma, Medicine 2021;100(43):p e27673).

[0009] Therefore, there is a high need to reliably detect and quantify AFP-L3.

[0010] The μTASWako AFP-L3 assay (Fujifilm Wako) is currently the most widely used method for detecting and quantifying AFP-L3. This assay is based on affinity capillary electrophoresis using LCA to separate AFP-L3. However, this method has several drawbacks. First, N-glycan branching can result in the detection of incorrect AFP-L3 levels. Second, this method is cumbersome and requires special equipment. Therefore, it is highly desirable to design an antibody-based method (Egashira Y et al., Scientific Reports, 2019, 9:12359). Third, lectins are less specific than antibodies and typically show low binding affinity.

[0011] Japanese Patent Laid-Open No. 63-307900 discloses an antibody that binds to LCA-binding AFP (AFP-LCA-R) but does not bind to LCA-non-binding AFP (AFP-LCA-NR). However, the epitope of the antibody is unknown, and it is also unknown whether the antibody recognizes proteins other than AFP having the same sugar moiety.

[0012] European Patent No. 3252073, US Patent Application Publication No. 2018 / 0110889, and Egashira Y et al. (Scientific Reports, 2019, 9:12359) disclose monoclonal antibodies that bind in a core-fucosylation-dependent manner and also bind to a part of the AFP peptide backbone, namely, core-fucosylated AFP (i.e., AFP-L3). However, as is well known in the art and as described in Egashira Y et al. (Scientific Reports, 2019, 9:12359), most anti-carbohydrate antibodies have low affinity for their antigens. The affinity of the antibody FasMab is described as stronger than that of typical anti-carbohydrate antibodies, but the K D value is only 6.5×10 -7 M. Typically, antibodies used in fully automated immunoassays such as the Elecsys® assay (Roche) have significantly lower K D values (e.g., in the low nanomolar concentration range), i.e., higher affinity for AFP-L3.

[0013] Therefore, there is a need to provide a novel antibody that specifically binds to α-1,6-core-fucosylated alpha-fetoprotein (i.e., AFP-L3) and has improved kinetic properties, particularly higher affinity (i.e., lower K D ). Furthermore, there is a need to provide an improved immunoassay for detecting AFP-L3.

Summary of the Invention

[0014] The above need is solved by the monoclonal antibodies and antigen-binding fragments thereof provided in the present invention, as well as their use.

[0015] In a first aspect of the present invention, there is provided a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation.

[0016] The monoclonal antibodies and antigen-binding fragments thereof provided herein distinguish α-1,6-core-fucosylated AFP (also referred to herein as core-fucosylated AFP and 1,6fucAFP) from related structures with high specificity. First, the monoclonal antibodies or antigen-binding fragments thereof provided herein can distinguish α-1,6-core-fucosylated AFP from AFP lacking an α-1,6-core-fucose residue (i.e., aglycosylated AFP or AFP having an N-glycan lacking an α-1,6-core-fucose residue). Second, the monoclonal antibodies or antigen-binding fragments provided herein can distinguish α-1,6-core-fucosylated AFP from free N-glycans having an α-1,6-core-fucose residue. Third, the monoclonal antibodies or antigen-binding fragments provided herein distinguish α-1,6-core-fucosylated AFP from an aglycosylated AFP peptide containing the N-glycosylation site N-251. Based on this binding behavior, it can be concluded that the antibodies and antigen-binding fragments thereof bind to α-1,6-core-fucosylated AFP depending on the presence of both α-1,6-core-fucosylation and, for example, the AFP peptide sequence of SEQ ID NO: 2. Thus, the antibodies and antigen-binding fragments provided herein are specific for α-1,6-core-fucosylated AFP and do not significantly recognize (i) other proteins having a similar α-1,6-core-fucosylated N-glycan structure and (ii) AFP lacking an α-1,6-core-fucose residue or a partial sequence thereof.

[0017] Due to this specificity, the antibodies and antigen-binding provided herein are valuable tools for use in immunoassays for detecting the level of α-1,6-core-fucosylated AFP (clinically equivalent to AFP-L3) in a sample. Thus, the antibodies and antigen-binding fragments and immunoassays using them can assist in the detection of HCC, particularly early HCC.

[0018] The antibodies and antigen-binding fragments provided herein exhibit surprisingly high affinity for α-1,6-core-fucosylated AFP, as exemplified herein by analyzing binding to α-1,6-core-fucosylated AFP peptides. The K D measured for such interactions is in the single-digit nanomolar range, i.e., lower than the K D that could be predicted based on prior art teachings for such AFP glycopeptide antibodies (Egashira Y et al., Scientific Reports, 2019, 9:12359). Egashira Y et al. reported an affinity of 650 nM as surprisingly low K D (i.e., high binding affinity) for antibodies against α-1,6-core-fucosylated AFP, emphasizing the unexpected finding that antibodies with the affinities described herein can be identified.

[0019] Interestingly, the two best monoclonal antibodies identified herein, namely clone 19B12 and 3C5, also show very high sequence homology, especially at the CDR residues. There are a total of three amino acid differences at the CDR residues, one insertion and two amino acid substitutions.

[0020] Thus, in an embodiment, a monoclonal antibody or antigen-binding fragment according to a first aspect of the disclosure comprises (i) a heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution; CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions; CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution, and (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (the CDR-L1 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 7 or 8 modified by at most two amino acid substitutions; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (the CDR-L2 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (the CDR-L3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution.

[0021] In a second aspect, the present invention (i) a heavy chain or heavy chain variable domain of the monoclonal antibody or antigen-binding fragment of the first aspect, and / or (ii) a light chain or light chain variable domain of the monoclonal antibody or antigen-binding fragment of the first aspect, and provides a polynucleotide encoding the same.

[0022] In a third aspect, a vector comprising the polynucleotide according to the second aspect of the present invention is disclosed.

[0023] According to a fourth aspect of the present invention, a host cell comprising the polynucleotide of the second aspect or the vector of the third aspect is provided herein.

[0024] In a fifth aspect, the present invention relates to a method for producing the monoclonal antibody or antigen-binding fragment of the first aspect, the method comprising culturing the host cell of the fourth aspect and isolating the antibody or antigen-binding fragment. Antibodies obtainable or obtainable by such methods are also disclosed.

[0025] According to a sixth aspect, a composition comprising the monoclonal antibody or antigen-binding fragment of the first aspect, the polynucleotide of the second aspect, the vector of the third aspect, or the host cell of the fourth aspect is provided.

[0026] In an embodiment of the sixth aspect, a diagnostic composition comprising the antibody according to the first aspect of the present invention is provided.

[0027] Using the antibody or antigen-binding fragment of the present invention, the inventors have developed an immunoassay for determining the level of α-1,6-core-fucosylated AFP. The level of α-1,6-core-fucosylated AFP measured in this assay correlates very well with the level measured by the μTASWako AFP-L3 assay, which is currently the most widely used method for detecting and quantifying AFP-L3. Thus, the immunoassay using the antibody of the present invention is a reliable tool that overcomes the drawbacks of μTASWako AFP-L3 and, for example, aids in HCC detection.

[0028] Thus, in a seventh aspect, the present invention relates to the use of the antibody or antigen-binding fragment of the first aspect for an in vitro immunoassay, particularly for an in vitro immunoassay for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP) or AFP-L3.

[0029] Similarly, an eighth aspect of the present invention relates to an in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence containing such α-1,6-core-fucosylation in a sample using the monoclonal antibody of the first aspect or an antigen-binding fragment thereof.

[0030] The inventors have also found that pretreatment (e.g., using a reducing agent) can improve the signal obtained in such immunoassays, suggesting that the epitopes recognized by the antibodies and antigen-binding fragments provided herein become more readily accessible upon such pretreatment.

[0031] Thus, in a ninth aspect of the present specification, a pretreatment agent or pretreatment composition containing a reducing agent (e.g., DTT) is also provided.

[0032] In a tenth aspect, the present invention provides a parts kit comprising the monoclonal antibody or antigen-binding fragment of the first aspect of the present invention. In an embodiment, the kit is for an immunoassay for the detection and / or quantification of α-1,6-core-fucosylated AFP. In an embodiment, the kit further comprises the pretreatment agent or composition of the ninth aspect of the present invention.

Brief Description of the Drawings

[0033] The following figures are provided to assist in the understanding of the present invention.

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4

Figure 5-1

Figure 5-2

Figure 5-3

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0034] Hereinafter, the elements of the present invention will be described. Although these elements are listed with specific embodiments and implementations, it should be understood that they can be combined in any way and in any number to create additional embodiments and implementations.

[0035] In a first aspect, the present disclosure relates to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing such α-1,6-core-fucosylation.

[0036] As is known in the art, human AFP is a glycoprotein having the amino acid sequence of SEQ ID NO: 1 (or its natural variants described in Uniprot ID P02771 (version 209)), and contains a single N-glycosylation site corresponding to Asn-251 of Uniprot ID P02771. The N-glycan of Asn-251 can contain a core fucose residue (see Figure 1). As further understood in the art, the term "core fucosylation" within a glycan indicates that a fucose residue is α-1,6-linked to a core GlcNac residue bound to Asn-251 of the AFP protein or to a subsequence thereof containing Asn corresponding to Asn-251. The terms "core-fucosylation" and "α-1,6-core-fucosylation" are recognized as interchangeable. Thus, the term "specific (or specifically binding) to core-fucosylated AFP and / or to subsequences of AFP containing core-fucosylation" is interchangeable with the term "specific (or specifically binding) to α-1,6-core-fucosylated AFP and to subsequences thereof containing α-1,6-core-fucosylation". The antibodies and antibody-antigen binding fragments of the present invention are also herein referred to interchangeably as 1,6fucAFP antibodies and their antigen-binding fragments.

[0037] As noted above, the α-1,6-core-fucose referred to in connection with a 1,6fucAFP antibody or antigen-binding fragment is part of the N-glycan bound to residue N251 of human AFP (see SEQ ID NO: 1).

[0038] The position of the α-1,6-core-fucose in the N-glycan is shown in Figure 1.

[0039] The subsequence of AFP can include SEQ ID NO: 2. In certain embodiments, the peptide portion of the subsequence of AFP consists of SEQ ID NO: 2.

[0040] As demonstrated in the attached examples, the inventors have identified two monoclonal antibodies (19B12 and 3C5) that are characterized by high specificity for α-1,6-core-fucosylated AFP (i.e., the component that characterizes AFP-L3). The monoclonal antibodies bind in an α-1,6-core-fucose-dependent manner, but the peptide portion of AFP also contributes to the binding of α-1,6-core-fucosylated AFP. Thus, the monoclonal antibodies and antigen-binding fragments thereof identified by the inventors distinguish α-1,6-core-fucosylated AFP from different AFP species lacking α-1,6-core-fucosylation at Asn-251, but also from other proteins modified with α-1,6-core-fucosylated N-glycans.

[0041] The monoclonal antibodies and antigen fragments of the present invention exhibit particularly high affinity (i.e., low K D ). This was somewhat unexpected and surprising based on previously reported antibodies against α-1,6-core-fucosylated AFP (Egashira Y et al., Scientific Reports, 2019, 9:12359). Furthermore, the monoclonal antibodies of the present invention have favorable kinetic parameters for use in fully automated high-throughput immunoassays, such as high association rate constant k a and low dissociation rate constant k d .

[0042] As stated above, the monoclonal 1,6fucAFP antibodies or antigen-binding fragments provided herein specifically bind to α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation. The α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation may comprise or consist of a glycopeptide of formula I.

Chemical formula

[0043] The glycopeptide of formula I represents an AFP peptide having the amino acid sequence of SEQ ID NO: 2 containing an Asn amino acid residue corresponding to Asn-251 of AFP at position 3, and the AFP peptide is modified with an N-glycan containing α-1,6-core-fucosylation.

[0044] Accordingly, the 1,6fucAFP antibodies of the present invention and their antigen-binding fragments can specifically bind to the glycopeptide of formula I or a glycoprotein containing the glycopeptide of formula I.

[0045] In an embodiment, the α-1,6-core-fucosylated AFP or a partial sequence of AFP containing the α-1,6-core-fucosylation specifically bound by the monoclonal 1,6fucAFP antibody or antigen-binding fragment provided herein may comprise or consist of a glycopeptide of formula II.

Chemical formula

[0046] The glycopeptide of formula II represents an AFP peptide having the amino acid sequence of SEQ ID NO: 2 containing an Asn amino acid residue corresponding to Asn-251 of AFP at position 3, and the AFP peptide is modified with an N-glycan containing α-1,6-core-fucosylation.

[0047] In an embodiment where the α-1,6-core-fucosylated AFP or a partial sequence of AFP containing the α-1,6-core-fucosylation comprises a glycopeptide of formula II, for example, additional amino acids may be present in the peptide sequence or additional sugar moieties may be present in the glycan (preferably bound to GlcNAc).

[0048] Accordingly, the 1,6fucAFP antibodies of the present invention and their antigen-binding fragments can specifically bind to the glycopeptide of formula II or a glycoprotein containing the glycopeptide of formula II.

[0049] In embodiments, the 1,6fucAFP antibodies of the invention and antigen-binding fragments thereof can specifically bind to a glycopeptide of formula I or II, or a glycoprotein comprising a glycopeptide of formula I or II.

[0050] As demonstrated in the appended examples, the 1,6fucAFP antibodies and antigen-binding fragments of the present disclosure have high affinity (i.e., low K D ) for binding to both the glycopeptide of formula I and the glycopeptide of formula II. Surprisingly, the binding affinity for the glycopeptide of formula I is slightly higher than the binding to the glycopeptide of formula II (i.e., K D is lower). This indicates that the added sugar moiety in the N-glycan of the glycopeptide of formula I somewhat facilitates the binding. In nature, N-glycan structures are typically more complex, such as those shown for the glycopeptide of formula I, so this binding behavior can be advantageous in increasing specificity for more complex N-glycosylated AFP species.

[0051] Accordingly, in embodiments, the 1,6fucAFP antibodies of the invention and antigen-binding fragments thereof can bind to the glycopeptide of formula I with a higher affinity (i.e., a lower K D ) than the glycopeptide of formula II. In embodiments, the ratio of K D for the binding of the glycopeptide of formula II to the glycopeptide of formula I can be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 10. In certain embodiments, the ratio of K D for the binding of the glycopeptide of formula II to the glycopeptide of formula I can be at least 7. In embodiments, the ratio of K D for the binding of the glycopeptide of formula II to the glycopeptide of formula I can be at most 11, at most 12, or at most 15. In embodiments, the ratio of K D for the binding of the glycopeptide of formula II to the glycopeptide of formula I can be at most 12.

[0052] As discussed above, the 1,6fucAFP antibodies of the present invention and their antigen-binding fragments distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., Formula I), and (ii) AFP or a partial sequence thereof lacking an α-1,6-core-fucose residue.

[0053] AFP or a partial sequence thereof lacking an α-1,6-core-fucose residue includes (i) AFP or a partial sequence thereof that is N-glycosylated at Asn-251 or a corresponding position but lacks core fucose in the N-glycan, and (ii) aglycosylated AFP or a partial sequence thereof.

[0054] Furthermore, the 1,6fucAFP antibodies of the present invention and their antigen-binding fragments distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., Formula I), and (ii) α-1,6-core-fucosylated N-glycans in the isolation or presence of proteins other than AFP.

[0055] Also, as defined hereinbelow, the term "distinguish" means that the 1,6fucAFP antibody and antigen-binding fragment bind to a specific antigen target (i.e., core-fucosylated AFP and its core-fucosylated partial sequences) with higher affinity and / or specificity than they bind to other antigens ("non-target antigens"), e.g., in isolated form, or in the context of other glycosylated proteins or peptides, to an AFP / AFP partial sequence lacking a core fucose residue and / or a core-fucosylated glycan. For example, the characteristic of distinguishing a target antigen from / against a non-target antigen can be a 1,6fucAFP antibody or antibody antigen-binding fragment having an affinity for the target antigen that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold or at least 245-fold superior to its affinity for the non-target antigen. (I.e., the K of binding to the target antigen D is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold or at least 245-fold lower than the KD of binding to the non-target antigen). The formulation is at least "XX" is K for the non-target antigen D Embodiments are also included where it is very high and cannot be detected by the methods used. Thus, whether an antibody or antigen-binding fragment can distinguish a target structure from a non-target structure depends on the same method (e.g., K as described below herein D (a preferred method for determining) to determine the K for each binding D value.

[0056] In an embodiment, the ability of an antibody to distinguish a target antigen from a non-target antigen can be evaluated using an immunoassay in which the binding of the antibody or antigen-binding fragment being tested to the target structure is detected. Using such an immunoassay, the immunoassay signal obtained in a first sample containing a defined concentration of the target antigen can be compared with the immunoassay signal obtained in a second sample containing the same concentration of the non-target antigen. That the immunoassay signal of the first sample is higher than the immunoassay signal from the second sample indicates discrimination between the target antigen and the non-target antigen. In an embodiment, the antibody or antigen-binding fragment being tested can distinguish the target structure from the non-target structure if the immunoassay signal of the first sample is at least 5-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold or at least 100-fold higher than that of the second sample. Exemplary but non-limiting immunoassays that can be used for such analysis are provided in Example 7. Exemplary concentrations of the target antigen and the non-target antigen can be 12 nM.

[0057] In an embodiment, AFP lacking an α-1,6-core-fucose residue or a partial sequence thereof can comprise or consist of the glycopeptide of Formula III.

[0058] [Chemical formula] (SEQ ID NO: 2) (Formula III) Since Formula III lacks a core-fucose, a 1,6fucAFP antibody or an antigen-binding fragment thereof can distinguish this from the glycopeptide of Formula I or a glycoprotein containing the same and the glycopeptide of Formula III.

[0059] Accordingly, provided herein are 1,6fucAFP antibodies and antigen-binding fragments thereof that distinguish between the glycopeptide of Formula I (or an AFP sequence containing the same) and the glycopeptide of Formula III (or an AFP sequence containing the same).

[0060] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the present invention distinguish between the glycopeptide of Formula II (or an AFP sequence containing the same) and the glycopeptide of Formula III (or an AFP sequence containing the same).

[0061] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the present invention distinguish between the glycopeptide of Formula I (or an AFP sequence containing the same) and the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence containing the same).

[0062] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the present invention distinguish between the glycopeptide of Formula II (or an AFP sequence containing the same) and the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence containing the same).

[0063] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment provided herein distinguish between the glycopeptide of Formula I (or an AFP sequence containing the same) and both the glycopeptide of Formula III (or an AFP sequence containing the same) and the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence containing the same).

[0064] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the present invention distinguish between the glycopeptide of formula II (or an AFP sequence containing the same), the glycopeptide of formula III (or an AFP sequence containing the same), and the peptide of SEQ ID NO: 25 (or an AFP sequence containing the same).

[0065] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the present invention have an equilibrium dissociation constant K for binding to (i) α-1,6-core-fucosylated AFP, or a partial AFP sequence containing α-1,6-core-fucosylation (e.g., the glycopeptide of formula I), and (ii) AFP lacking an α-1,6-core-fucose residue, or a partial sequence of AFP lacking an α-1,6-core-fucose residue (e.g., the glycopeptide of formula III or SEQ ID NO: 2 or 25). D at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, at least 183-fold, or at least 245-fold lower K D for binding. In a specific embodiment, the difference in K D is at least 100-fold. In another specific embodiment, the difference in the dissociation constant is at least 180-fold. In yet another specific embodiment, the difference in dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0066] In an embodiment, the AFP lacking an α-1,6-core-fucose residue or a partial sequence of AFP lacking an α-1,6-core-fucose residue is N-glycosylated AFP or a partial sequence thereof lacking a core fucose residue (e.g., the glycopeptide of formula III), and the 1,6fucAFP antibody and antigen-binding fragment of the present invention have an equilibrium dissociation constant (K) for binding to (i) α-1,6-core-fucosylated AFP, or a partial AFP sequence containing α-1,6-core-fucosylation (e.g., SEQ ID NO: 2), and (ii) AFP lacking an α-1,6-core-fucose residue, or a partial sequence of AFP lacking an α-1,6-core-fucose residue. D at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, at least 183-fold or at least 245-fold lower K than D and binds. In a specific embodiment, K D for said difference is at least 100-fold. In another specific embodiment, said difference in dissociation constant is at least 180-fold. In yet another specific embodiment, said difference in dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0067] In an embodiment, the 1,6fucAFP antibody and antigen-binding fragment of the invention have an equilibrium dissociation constant (K D for the binding of an antibody or antigen-binding fragment to (i) a glycopeptide of formula I and (ii) a glycopeptide of formula III) that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, at least 183-fold or at least 245-fold lower than K D and binds. In a specific embodiment, K D for said difference is at least 100-fold. In another specific embodiment, said difference in dissociation constant is at least 180-fold. In yet another specific embodiment, said difference in dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0068] In an embodiment, AFP lacking an α-1,6-core-fucose residue or a partial sequence of AFP lacking an α-1,6-core-fucose residue is an aglycosylated AFP or an aglycosylated partial AFP sequence (e.g., SEQ ID NO: 2 or 25), and the 1,6fucAFP antibody and antigen-binding fragment of the invention have an equilibrium dissociation constant (K D at least 10-fold, at least 20-fold, at least 50-fold or at least 100-fold, at least 150-fold, at least 180-fold, at least 183-fold or at least 245-fold, at least 500-fold, at least 1000-fold, at least 3000-fold or at least 5000-fold lower K D binds. In some embodiments, K D of said difference is at least 1000-fold. In some embodiments, K D of said difference is at least 5000-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0069] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments of the invention have a K D that is at least 10-fold, at least 20-fold, at least 25-fold or at least 100-fold, at least 150-fold, at least 180-fold, at least 183-fold or at least 245-fold, at least 500-fold, at least 1000-fold, at least 3000-fold or at least 5000-fold lower than the K D for binding to the (i) glycopeptide of formula I and (ii) the AFP peptide of SEQ ID NO: 50. In some embodiments, K D of said difference is at least 1000-fold. In some embodiments, K D of said difference is at least 4000-fold. In some embodiments, K D of said difference is at least 5000-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0070] As demonstrated using the respective glycopeptides and glycans in the appended examples and figures, the monoclonal antibodies or antigen-binding fragments of the invention distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., the glycopeptide of formula I), and (ii) an isolated α-1,6-core-fucosylated glycan (e.g., of formula (IV)) and other glycoproteins having an α-1,6-core-fucosylated glycan (i.e., proteins containing the glycopeptide of formula IV but not the glycopeptide of formula I).

[0071] The sugar chain of formula IV has the following structure.

Chemical formula

[0072] Accordingly, in an embodiment, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention distinguishes between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., the glycopeptide of formula I), and (ii) an isolated α-1,6-core-fucosylated glycan (e.g., of formula (IV)) and other glycoproteins having an α-1,6-core-fucosylated glycan (i.e., proteins containing the glycopeptide of formula IV but not the glycopeptide of formula I).

[0073] In certain embodiments, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention has an equilibrium dissociation constant (K D At least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, at least 150-fold, at least 180-fold, at least 245-fold lower K than D binds. The binding affinities for (i) and (ii) are determined under the same conditions. In a specific embodiment, K D of this difference is at least 100-fold. In another specific embodiment, this difference in dissociation constant is at least 180-fold. In yet another specific embodiment, this difference in dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0074] In an even more specific embodiment, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has an equilibrium dissociation constant (K D ) that is at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold, at least 150-fold, at least 180-fold, at least 245-fold lower than the binding of the antibody or antigen-binding fragment to (i) the glycopeptide of formula I and (ii) the isolated α-1,6-core-fucosylated glycan of formula (IV). D binds. The binding affinities for (i) and (ii) are determined under the same conditions. In a specific embodiment, K D of this difference is at least 100-fold. In another specific embodiment, this difference in dissociation constant is at least 180-fold. In yet another specific embodiment, this difference in dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0075] In an embodiment, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention distinguishes between the glycopeptide of formula I and both the glycopeptide of formula III and the core-fucosylated glycan of formula IV. What has been said regarding the fold difference in K D values between the individual structural pairs referred to elsewhere in this specification (e.g., above) applies with the necessary modifications.

[0076] In an embodiment, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention distinguishes the glycopeptide of Formula I from all of the following three: the glycopeptide of Formula III, the core-fucosylated glycan of Formula IV, and the AFP peptide of SEQ ID NO: 25 or 2 (e.g., SEQ ID NO: 25).

[0077] The monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has surprisingly high affinity (i.e., low K D ) for the glycopeptide of Formula I (see above), i.e., for the AFP sequence containing α-1,6-core-fucosylation. A low K D is typically advantageous for immunoassays.

[0078] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K D of 100 nM or less, 20 nM or less, preferably 10 nM or less, more preferably 3.1 nM or less or 2.5 nM or less. When K D is determined by the affinity in solution, K D can also be 0.9 nM or less or 0.4 nM or less. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K D of 20 nM or less. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K D of 10 nM or less. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K D of 5 nM or less.

[0079] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K of less than 10-fold, less than 8-fold, less than 6-fold, less than 4-fold, or less than 2-fold of the K of a rabbit IgG antibody (e.g., antibody 19B12) comprising the heavy-chain variable domain of SEQ ID NO: 11 and the light-chain variable domain of SEQ ID NO: 13 against the glycopeptide of formula I D and binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K D equal to or less than that of a rabbit IgG antibody (e.g., antibody 19B12) comprising the heavy-chain variable domain of SEQ ID NO: 11 and the light-chain variable domain of SEQ ID NO: 13 against the glycopeptide of formula I D and binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. D

[0080] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K of less than 10-fold, less than 8-fold, less than 6-fold, less than 4-fold, or less than 2-fold of the K of a rabbit IgG antibody (e.g., antibody 3C5) comprising the heavy-chain variable domain of SEQ ID NO: 12 and the light-chain variable domain of SEQ ID NO: 14 against the glycopeptide of formula I D and binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K D equal to or less than that of a rabbit IgG antibody (e.g., antibody 3C5) comprising the heavy-chain variable domain of SEQ ID NO: 12 and the light-chain variable domain of SEQ ID NO: 14 against the glycopeptide of formula I D and binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. D

[0081] ​​The monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a surprisingly high association rate (k a ) constant for binding to the glycopeptide of formula I (see above), i.e., the AFP sequence containing α-1,6-core-fucosylation. A high k a is important for the antibody to bind rapidly to the antigen at equilibrium. Thus, a high k a is important for low incubation times in immunoassays.

[0082] The monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has at least 2.0×10 4 M -1 s -1 , in embodiments at least 2.1×10 4 M -1 s -1 , in embodiments at least 10 5 M -1 s -1 , in embodiments at least 2.5×10 5 M -1 s -1 , in embodiments at least 6.0×10 5 M -1 s -1 , and in embodiments at least 1.0×10 6 M -1 s -1 and may have an association rate constant (k a ) for binding to the glycopeptide of formula I.

[0083] In embodiments, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of formula I with a k a that is 3.4%, 5%, 10%, 20%, 50%, 80% or 90% higher than the k a of a rabbit IgG antibody (e.g., antibody 19B12) comprising the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 13 against the glycopeptide of formula I, and k a The values are measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k of a rabbit IgG antibody (e.g., antibody 19B12) containing the heavy-chain variable domain of SEQ ID NO: 11 and the light-chain variable domain of SEQ ID NO: 13 with respect to the glycopeptide of formula I a equal to or higher than k a and binds to the glycopeptide of formula I, and the k a values are measured using the same method under the same conditions.

[0084] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k that is 2.0%, 3.4%, 5%, 10%, 20%, 50%, 80% or 90% higher than the k of a rabbit IgG antibody (e.g., antibody 3C5) containing the heavy-chain variable domain of SEQ ID NO: 12 and the light-chain variable domain of SEQ ID NO: 14 with respect to the glycopeptide of formula I a and binds to the glycopeptide of formula I, and the k a values are measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k of a rabbit IgG antibody (e.g., antibody 3C5) containing the heavy-chain variable domain of SEQ ID NO: 12 and the light-chain variable domain of SEQ ID NO: 14 with respect to the glycopeptide of formula I a equal to or higher than k a and binds to the glycopeptide of formula I, and the k a values are measured using the same method under the same conditions. a The monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has been further found to have a surprisingly low dissociation rate (k

[0085] (dissociation constant) for binding to the glycopeptide of formula I (see above), i.e., an AFP sequence containing α-1,6-core-fucosylation. d )

[0086] The monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a maximum of 1.2×10 -2 s -1 , up to 1.3×10 -2 s -1 , up to ×10 -2 s -1 , up to 8.0×10 -3 s -1 , up to 7.3×10 -3 s -1 , up to 3.2×10 -3 s -1 or up to 1.5×10 -3 s -1 and may have a dissociation rate constant (k d ) for the glycopeptide of Formula I.

[0087] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K d that is 1.5, 2, 2.5, 3, 3.5, or 3.75 times lower than the K d of a rabbit IgG antibody (e.g., antibody 19B12) comprising the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 13 against the glycopeptide of Formula I, and the K d value is measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a k d that is equal to or lower than the k d of a rabbit IgG antibody (e.g., antibody 19B12) comprising the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 13 against the glycopeptide of Formula I, and the k d value is measured using the same method under the same conditions.

[0088] In an embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binds to the glycopeptide of Formula I with a K d that is 1.5, 2, 2.5, 3, 3.5, 3.75, 5, 6, 7, or 8 times lower than the K d of a rabbit IgG antibody (e.g., antibody 3C5) comprising the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 14 against the glycopeptide of Formula I, and the K d Values are measured using the same method under the same conditions. In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k of a rabbit IgG antibody (e.g., antibody 3C5) containing the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 14 against the glycopeptide of formula I d equal to or lower than the k d for binding to the glycopeptide of formula I, and the k d value is measured using the same method under the same conditions.

[0089] In an embodiment, all K D , k a and k d values may be the K D , k a and k d values at 37°C.

[0090] As understood in the art, the K D , k a and k d values can be determined by any conventional means known to those skilled in the art or as described herein.

[0091] In the context of the present invention, the K D , k a and k d values herein, particularly the K D values, the two k a values and the two k d values, preferably are determined by surface plasmon resonance spectroscopy (e.g., BIAcore®). The fold difference (i.e., ratio) between two K D values, two k a values and two k d values can be calculated based on the respective two values measured by plasmon resonance spectroscopy. Preferred peptides and methods for performing the analysis are disclosed in the accompanying examples and figures.

[0092] Herein, surface plasmon resonance spectroscopy described in Example 3 is used to determine K D , k a and k d It is particularly preferable to determine the value.

[0093] Therefore, K D , k a and / or k d Surface plasmon resonance spectroscopy for determining the value of K, k, and / or k may include capturing a monoclonal antibody or antigen-binding fragment on a C1 sensor chip (e.g., a series S C1 sensor chip) and injecting the glycopeptide to be analyzed as an analyte, and the determination is performed at a temperature of 37°C. HBS-ET pH 7.4 (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w / v Tween 20 (registered trademark)) may be used as the system buffer, and the system buffer supplemented with 1 mg / ml carboxymethyl dextran may be used as the sample buffer.

[0094] In an embodiment, the binding parameter K D and / or k a and / or k d can be determined, for example, according to BIAcore (trademark) T200 Evaluation SW 3.2, by a Langmuir fit 1:1 fitting model. Preferably, the fitting is global. Alternatively or additionally, Langmuir 1:1 fitting Scrubber-SW V2.0c may be applied. max

[0095] Surface plasmon resonance spectroscopy can be performed using different devices known in the art. In a particularly preferred embodiment, a BIAcore (registered trademark) T200 or 8K device can be used.

[0096] The concentration of the antibody or antigen-binding fragment used for surface plasmon resonance measurement may be 150 nM, and for example, it may be injected at 10 μl / min for 30 seconds. For the analyte (e.g., peptide or glycopeptide), a concentration series of 3.3 to 270 nM may be used. The injection of the analyte may be at 60 μl / min.

[0097] ​ In surface plasmon resonance spectroscopy, the association phase can be monitored for, for example, 3 minutes. The dissociation phase can be monitored for, for example, 10 minutes. Regeneration may be performed by injecting 10 mM glycine pH 2.0 at 20 μL / min for 30 seconds, followed by injecting 10 mM glycine pH 2.25 twice for 60 seconds each.

[0098] In a preferred embodiment, K D and / or k a and / or k d can be determined as follows.

[0099] A BIAcore™ T200 instrument manufactured by GE Healthcare at 37 °C using a Series S C1 sensor can be used. A rabbit antibody capture system (e.g., as described in the examples) can be immobilized in the flow cell at, for example, 700 RU to 800 RU. One flow cell can be used as a control, or three flow cells can be used for measurement. Then, 150 nM of the antibody or antigen-binding fragment of interest can be injected at 10 μL / min for 30 seconds. The capture level (CL) in resonance units RU can be monitored. A series of analyte concentrations from 3.3 to 270 nM can be injected at 60 μl / min. The association phase can be monitored for 3 minutes, and the dissociation phase can be monitored for 10 minutes. Regeneration may be performed by injecting 10 mM glycine pH 2.0 at 20 μL / min for 30 seconds, followed by injecting 10 mM glycine pH 2.25 twice for 60 seconds each. The kinetic rate constant and dissociation equilibrium constant K D can be determined using the Langmuir 1:1 fitting model according to BIAcore™ T200 Evaluation SW 3.2. Next, the Langmuir 1:1 fitting Scrubber-SW V2.0c may be applied.

[0100] Unless expressly stated to the contrary herein, the K provided herein D The value (and rate constant) is determined by surface plasmon resonance spectroscopy which uses capturing a monoclonal antibody or antigen-binding fragment on a chip (e.g., C1 sensor chip) and injecting the analyte to be analyzed. As is well known in the art, K D There are also alternative methods known in the art for determining the value and / or rate constant. Such alternative methods may also be used as long as the results obtained for antibodies 3C5 and 19B12 using these methods are consistent with the results reported herein for the antibodies in Example 3.

[0101] In the attached examples, K D Alternative methods for determining the value: Affinity in solution analysis is also described. As shown in the attached examples, the K D value determined using the affinity in solution analysis is very similar to, but may be somewhat different from, the capture-based method described above. The K D value referred to herein relates only to the K D measured by the affinity in solution when such techniques are explicitly referred to. In case of inconsistency, the antibody capture-based surface plasmon resonance spectroscopy dataset described above shall be used.

[0102] Affinity in solution measurements can be performed as described in the vendor's instructions for the CAP-Kit (Cytiva). For example, a biotinylated analyte (e.g., a glycopeptide or peptide) can be captured on the surface of a CAP chip sensor. A mixture of a 10 nM antibody or antigen-binding fragment being tested and various concentrations of non-biotinylated AFP(243-261)-G0F from 120 nM to 0.01 nM, and various concentrations of non-biotinylated AFP(248-256) from 200 μM to -0.1 nM can be incubated until equilibrium is achieved. Binding events of the mixture to the analyte displayed on the surface may be monitored. As the concentration of the peptide as a competitor increases, the "free" antibody in solution decreases. Then, the free antibody concentration determined for the competition experiment may be plotted against the peptide competitor concentration. The data can be evaluated using an affinity model in solution from the BIAcore® Evaluation software to determine K D and may be determined.

[0103] In some embodiments, the method for determining K D is (1) a rabbit IgG antibody comprising a heavy chain variable domain having the sequence of SEQ ID NO: 11 and a light chain variable domain having the sequence of SEQ ID NO: 13, and (2) a glycopeptide of Formula I, is selected such that the binding constant KD between them is determined to be 2.5 (± error of the method, e.g., 0.08%) nM.

[0104] In some embodiments, the method for determining K D is (1) a rabbit IgG antibody comprising a heavy chain variable domain having the sequence of SEQ ID NO: 12 and a light chain variable domain having the sequence of SEQ ID NO: 14, and (2) a glycopeptide of Formula I, is selected such that the binding constant KD between them is determined to be 3.1 (± error of the method, e.g., 0.13%) nM.

[0105] As demonstrated in the attached examples and as further discussed below in this specification, the 1,6fucAFP antibody or antigen-binding fragment can detect α-1,6-core-fucosylated alpha-fetoprotein (AFP) (e.g., natural α-1,6-core-fucosylated occurring in a blood sample (e.g., plasma or serum) obtained from a human subject) pretreated with a pretreatment agent according to the invention (see other places in this specification) much better than without such pretreatment. Thus, in embodiments, the monoclonal antibody or antigen-binding fragment of the present disclosure binds to α-1,6-core-fucosylated alpha-fetoprotein (AFP) better than α-1,6-core-fucosylated alpha-fetoprotein (AFP) without such pretreatment. A measure for detecting such "better" binding can be to transmit a signal using one of the immunoassay settings disclosed in the attached examples. A reducing agent such as DTT is preferred as the pretreatment agent.

[0106] Surprisingly, the two best monoclonal 1,6fucAFP antibodies (i.e., 19B12 and 3C5) identified herein show very high sequence homology, but there are certain differences not only in the framework regions but also in the CDR residues. This demonstrates that the CDR sequences as well as the VH and VL sequences underlying the identified antibodies define very valuable components for making 1,6fucAFP antibodies and their antigen-binding fragments. However, the sequence variability seen in VH and VL also demonstrates that specific amino acid exchanges can be made in both the CDR and framework regions of VH and VL without significantly adversely affecting antibody properties. As will be appreciated, one of ordinary skill in the art can make facile variants of the antibody or antigen-binding fragment sequences of the present invention and determine based on the disclosure herein whether such antibodies maintain the advantageous properties disclosed herein. For example, affinity maturation can be applied to obtain variant monoclonal antibodies or antigen-binding fragments that include VH and VL of 19B12 or 3C5 with amino acid substitutions obtained by such affinity maturation that have the same or improved properties as 19B12 or 3C5.

[0107] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein are (i) a heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5) or a variant thereof modified by one amino acid substitution, insertion or deletion; CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions, insertions or deletions; and CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution, insertion or deletion, and (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5 respectively), or a variant of SEQ ID NO: 7 or 8 modified by substitution, insertion or deletion of up to two amino acids; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5) or a variant thereof modified by substitution, insertion or deletion of one amino acid; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5) or a variant thereof modified by substitution, insertion or deletion of one amino acid, comprising a light chain variable domain (VL).

[0108] In a specific embodiment, the monoclonal antibody or antigen-binding fragment provided herein (i) A heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5); a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5 respectively), or a variant of SEQ ID NO: 4 or 5 modified by substitution of one amino acid and / or insertion or deletion of one amino acid, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5 respectively), or a variant of SEQ ID NO: 7 or 8 modified by substitution, insertion or deletion of one amino acid; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5); and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5), comprising a light chain variable domain (VL).

[0109] In a specific embodiment, the monoclonal antibody or antigen-binding fragment provided herein (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5); a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by one amino acid substitution and / or one amino acid insertion or deletion, and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution, insertion or deletion; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5); and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5), and a light chain variable domain (VL).

[0110] In an embodiment, the monoclonal antibody or antigen-binding fragment provided herein (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5) or a variant thereof modified by one amino acid substitution, insertion or deletion; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions, insertions or deletions; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution, insertion or deletion, (ii) a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5 respectively), or a variant of SEQ ID NO: 7 or 8 modified by substitution, insertion or deletion of at most two amino acids; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5) or a variant thereof modified by substitution, insertion or deletion of one amino acid; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5) or a variant thereof modified by substitution, insertion or deletion of one amino acid, and a light chain variable domain (VL).

[0111] Mutations in the CDR sequences of monoclonal antibodies or antigenic fragments can be such that the total of substitutions, insertions and deletions in all CDR sequences is at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2.

[0112] In certain embodiments, the total of amino acid substitutions, insertions and deletions in all CDR sequences can be at most 3. In embodiments, the at most three amino acid substitution, insertion and deletion events can be at most two amino acid substitutions and one insertion or deletion.

[0113] In another particular embodiment, the total of amino acid substitutions, insertions and deletions in all CDR sequences can be at most 2.

[0114] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein are (i) A heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution, (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 7 or 8 modified by up to two amino acid substitutions; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5), or a variant thereof modified by one amino acid substitution.

[0115] As used herein, the expression "a variant thereof modified by one amino acid substitution" means that there is exactly one (one or less) amino acid substitution in the variant sequence relative to the indicated parental sequence.

[0116] In certain embodiments, the monoclonal antibody or antigen-binding fragment (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution, (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by at most two amino acid substitutions; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution, and a light chain variable domain (VL).

[0117] The mutations in the CDR sequences of the monoclonal antibody or antigen fragment can be such that the total number of amino acid substitutions in all CDR sequences is at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2.

[0118] In certain embodiments, the total number of amino acid substitutions in all CDR sequences can be at most 3.

[0119] In another certain embodiment, the total number of amino acid substitutions in all CDR sequences can be at most 2.

[0120] In embodiments, the monoclonal antibody or antigen-binding fragment provided herein (i) A CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5), or a variant thereof modified by one conservative amino acid substitution; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by at most two conservative amino acid substitutions; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one conservative amino acid substitution, and a heavy chain variable domain (VH). (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 7 or 8 modified by at most two conservative amino acid substitutions; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5), or a variant thereof modified by one conservative amino acid substitution; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5), or a variant thereof modified by one conservative amino acid substitution, and a light chain variable domain (VL).

[0121] In certain embodiments, the monoclonal antibody or antigen-binding fragment (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one conservative amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by at most two conservative amino acid substitutions; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one conservative amino acid substitution, and a heavy chain variable domain (VH). (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by at most two conservative amino acid substitutions; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one conservative amino acid substitution; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one conservative amino acid substitution, and a light chain variable domain (VL).

[0122] In embodiments, the monoclonal antibody or antigen-binding fragment provided herein (i) A heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3 (CDR-H1 of 19B12 and 3C5), or a variant thereof modified by one highly conserved amino acid substitution; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 (CDR-H2 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 4 or 5 modified by up to two highly conserved amino acid substitutions; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5), or a variant thereof modified by one highly conserved amino acid substitution, (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 (CDR-L1 of 19B12 and 3C5, respectively), or a variant of SEQ ID NO: 7 or 8 modified by up to two highly conserved amino acid substitutions; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9 (CDR-L2 of 19B12 and 3C5), or a variant thereof modified by one highly conserved amino acid substitution; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10 (CDR-L3 of 19B12 and 3C5), or a variant thereof modified by one highly conserved amino acid substitution.

[0123] In certain embodiments, the monoclonal antibody or antigen-binding fragment (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one highly conserved amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by up to two highly conserved amino acid substitutions; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one highly conserved amino acid substitution, (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by at most two highly conserved amino acid substitutions; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one highly conserved amino acid substitution; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one highly conserved amino acid substitution, and a light chain variable domain (VL).

[0124] The amino acid sequences in the CDRs of the 1,6fucAFP antibodies 19B12 and 3C5 differ by three amino acids. Specifically, there are amino acid insertions and amino acid substitutions in the CDR-H2 of 19B12 (i.e., SEQ ID NO: 4) compared to the CDR-H2 of 3C5 (i.e., SEQ ID NO: 5). Furthermore, the CDR-L1 of 19B12 (i.e., SEQ ID NO: 7) and the CDR-L1 of 3C5 (i.e., SEQ ID NO: 8) differ by one amino acid.

[0125] Therefore, the 1,6fucAFP antibodies or antigen-binding fragments provided herein (i) A heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one amino acid substitution; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10.

[0126] In certain embodiments, the monoclonal antibody or antigen-binding fragment (i) A heavy chain variable domain (VH) comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one amino acid substitution; and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution; CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, may be included.

[0127] In an embodiment, the antibody or antigen-binding fragment (i) A heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 3; CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one conservative amino acid substitution; and CDR-H3 having the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one conservative amino acid substitution; CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and CDR-L3 having the amino acid sequence of SEQ ID NO: 10, may be included.

[0128] In certain embodiments, the monoclonal antibody or antigen-binding fragment (i) A heavy chain variable domain (VH) comprising CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one conservative amino acid substitution; and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one conservative amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain (VL) comprising a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, may be included.

[0129] In an embodiment, the antibody or antigen-binding fragment is (i) A heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one conservative or highly conserved amino acid substitution; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one conservative or highly conserved amino acid substitution; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, may be included.

[0130] In certain embodiments, the monoclonal antibody or antigen-binding fragment is (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one highly conserved amino acid substitution; and a heavy chain variable domain (VH) comprising a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one highly conserved amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, may be included.

[0131] The amino acid sequences in the CDRs of the 1,6fucAFP antibodies 19B12 and 3C5 differ by three amino acids. Specifically, there is an amino acid insertion at position 3 of the CDR-H2 of 19B12 (i.e., SEQ ID NO: 4) relative to that of 3C5 (i.e., SEQ ID NO: 5). Also, the amino acid at position 5 of the CDR-H2 of 19B12 (i.e., SEQ ID NO: 4) is different from the amino acid at position 4 of the CDR-H2 of 3C5 (i.e., SEQ ID NO: 5). These positions correspond to each other considering the aforementioned insertion. Finally, the CDR-L1 of 19B12 (i.e., SEQ ID NO: 7) and the CDR-L1 of 3C5 (i.e., SEQ ID NO: 8) differ in the amino acid at position 7.

[0132] Therefore, the monoclonal 1,6fucAFP antibodies or antigen-binding fragments of the present disclosure (i) a heavy chain variable domain (VH) comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one amino acid substitution at the 5th amino acid, or a variant of SEQ ID NO: 5 modified by one amino acid substitution at the 4th amino acid; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, and (ii) a light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution at the 7th amino acid; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10.

[0133] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one amino acid substitution at the 5th amino acid, or a variant of SEQ ID NO: 5 modified by one amino acid substitution at the 4th amino acid; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and a heavy chain variable domain (VH), (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 in which the amino acid at position 7 is modified by one amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL).

[0134] In an embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one conservative amino acid substitution at the amino acid at position 5, or a variant of SEQ ID NO: 5 modified by one conservative amino acid substitution at the amino acid at position 4; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, and a heavy chain variable domain (VH). (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 in which the amino acid at position 7 is modified by one conservative amino acid substitution; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL).

[0135] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one conservative amino acid substitution at the amino acid at position 5, or a variant of SEQ ID NO: 5 modified by one conservative amino acid substitution at the amino acid at position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and a heavy chain variable domain (VH). (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 in which the amino acid at position 7 is modified by one conservative amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain (VL) comprising a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10.

[0136] In an embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one highly conservative amino acid substitution at the amino acid at position 5, or a variant of SEQ ID NO: 5 modified by one highly conservative amino acid substitution at the amino acid at position 4; and a heavy chain variable domain (VH) comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 in which the amino acid at position 7 is modified by one highly conservative amino acid substitution; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain (VL) comprising a CDR-L3 having the amino acid sequence of SEQ ID NO: 10.

[0137] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one highly conservative amino acid substitution at the amino acid at position 5, or a variant of SEQ ID NO: 5 modified by one highly conservative amino acid substitution at the amino acid at position 4; and a heavy chain variable domain (VH) comprising a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 in which the amino acid at position 7 is modified by one highly conserved amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain (VL) comprising a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10.

[0138] Unless otherwise specified, any amino acid substitution(s) in any one of the variants of SEQ ID NOs: 3-10 (e.g., as a whole) can be any amino acid exchange. In certain embodiments, any amino acid substitution(s) in any one of the variants of SEQ ID NOs: 3-10 (e.g., as a whole) can be a conservative amino acid substitution(s). In even more specific embodiments, any amino acid substitution(s) in any one of the variants of SEQ ID NOs: 3-10 (e.g., as a whole) can be a highly conserved amino acid substitution(s).

[0139] In embodiments, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 in which the amino acid at position 5 is modified by one amino acid substitution, or a variant of SEQ ID NO: 5 in which the amino acid at position 4 is modified by one amino acid substitution; and a heavy chain variable domain (VH) comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, (ii) A light chain variable domain (VL) comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, The amino acid at position 5 of the variant of SEQ ID NO: 4 is modified to serine, and the amino acid at position 4 of the variant of SEQ ID NO: 5 is modified to asparagine.

[0140] In more specific embodiments, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one amino acid substitution at the 5th amino acid, or a variant of SEQ ID NO: 5 modified by one amino acid substitution at the 4th amino acid; and a heavy chain variable domain (VH) containing a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a light chain variable domain (VL) containing a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, The 5th amino acid of the variant of SEQ ID NO: 4 is modified to serine, and the 4th amino acid of the variant of SEQ ID NO: 5 is modified to asparagine.

[0141] The monoclonal antibody or antigen-binding fragment according to the present invention may contain the CDR sequences of the monoclonal antibody 19B12 identified herein.

[0142] Therefore, the monoclonal antibody or antigen-binding fragment of the present invention (i) A heavy chain variable domain (VH) containing a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) containing a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10,

[0143] The monoclonal antibody or antigen-binding fragment according to the present invention may contain the CDR sequences of the monoclonal antibody 3C5 identified herein.

[0144] Therefore, the monoclonal antibody or antigen-binding fragment of the present disclosure (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 5; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 8; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, may be included.

[0145] In the present specification above, several embodiments listing the amino acid sequences as shown in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9 and 10 and optionally their variants are disclosed. Corresponding embodiments are also provided herein where the reference to "SEQ ID NO: 3" is replaced by the "CDR-H1 sequence contained in SEQ ID NO: 11 or 12", the reference to "SEQ ID NO: 4" is replaced by the "CDR-H2 sequence contained in SEQ ID NO: 11", the reference to "SEQ ID NO: 5" is replaced by the "CDR-H2 sequence contained in SEQ ID NO: 12", the reference to "SEQ ID NO: 6" is replaced by the "CDR-H3 sequence contained in SEQ ID NO: 11 or 12", the reference to "SEQ ID NO: 7" is replaced by the "CDR-L1 sequence contained in SEQ ID NO: 11", the reference to "SEQ ID NO: 8" is replaced by the "CDR-L1 sequence contained in SEQ ID NO: 12", the reference to "SEQ ID NO: 9" is replaced by the "CDR-L2 sequence contained in SEQ ID NO: 11 or 12", and / or the reference to "SEQ ID NO: 10" is replaced by the "CDR-L1 sequence contained in SEQ ID NO: 11 or 12".

[0146] In an embodiment, the monoclonal antibody or its antigen-binding fragment is (i) A heavy chain variable domain (VH) having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.5% sequence identity with SEQ ID NO: 11 or 12, and (ii) a light chain variable domain (VL) having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.3% sequence identity with SEQ ID NO: 13 or 14.

[0147] Accordingly, a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., the glycopeptide of formula I), (i) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 11 or 12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.5% sequence identity with SEQ ID NO: 11 or 12, (ii) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 13 or 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.3% sequence identity with SEQ ID NO: 13 or 14, and a monoclonal antibody or an antigen-binding fragment thereof are provided herein.

[0148] As is apparent from the accompanying examples and figures, the two antibodies 19B12 and 3C5 identified herein show significant sequence similarity in the CDRs as well as the framework regions of VH and VL, but also have some amino acid substitutions. Based on the VH and VL sequences of these antibodies, additional antibodies can be generated that share similar characteristics (e.g., specificity, affinity, and other kinetic parameters for α-1,6-core-fucosylated AFP) with 19B12 and 3C5. For example, such variant antibodies can be generated by affinity maturation.

[0149] Accordingly, in an embodiment, the antibody or antigen-binding fragment of the present invention is (i) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 11 or 12, or an affinity matured variant of SEQ ID NO: 11 or 12, and (ii) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 13 or 14, or an affinity matured variant of SEQ ID NO: 13 or 14.

[0150] In an embodiment, the antibody or antigen-binding fragment of the present invention (i) CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 modified by one amino acid substitution at the 5th amino acid, or a variant of SEQ ID NO: 5 modified by one amino acid substitution at the 4th amino acid; and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; or a heavy chain variable domain (VH) comprising an affinity matured variant of said VH, and (ii) CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8; CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10; or a light chain variable domain (VL) comprising an affinity matured variant of said VL.

[0151] By definition, an affinity matured variant has at least the same affinity as 19B12 or 3C5 and exhibits at least the same binding preference as 19B12 or 3C5 (i.e., discrimination of binding of different structures). An affinity matured variant may include amino acid substitutions, insertions and / or deletions. In an embodiment, the affinity matured variant may include amino acid substitutions. In an embodiment, the affinity matured variant exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with each respective parental VH or VL sequence (e.g., any one of SEQ ID NOs: 11-14).

[0152] It is known in the art that the heavy chain variable domain or the light chain variable domain of an antibody contains four framework domains in addition to three CDRs. Specifically, it is known that framework region 1 (FW1) represents most of the N-terminal portion of the variable chain domain, and framework region 4 (FW4) represents most of the C-terminal portion, and the CDRs are interspersed between the framework regions, i.e., between FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0153] Whether reference is made to the framework region (FW) or the complementarity determining region (CDR) of the heavy or light chain variable domain is clear from the context, but the FW and CDR are distinguished herein by the labels "H" or "L". For example, the components FW and CDR of the heavy chain variable domain are referred to herein as being schematically represented as follows. (FW-H1)-(CDR-H1)-(FW-H2)-(CDR-H2)-(FW-H3)-(CDR-H3)-(FW-H4)

[0154] Similarly, the components FW and CDR of the light chain variable domain are referred to herein as being schematically represented as follows. (FW-L1)-(CDR-L1)-(FW-L2)-(CDR-L2)-(FW-L3)-(CDR-L3)-(FW-L4)

[0155] In an embodiment, the VH of the α-1,6-core-fucosylated AFP-specific antibody or antigen-binding fragment of the present invention contains a framework region (FW) and has the following structure. FW-H1-CDR-H1-FW-H2-CDR-H2-FW-H3-CDR-H3-FW-H4

[0156] FW-H1 can include or consist of the amino acid sequence of SEQ ID NO: 15, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0157] FW-H2 can include or consist of the amino acid sequence of SEQ ID NO: 16 or 17, or a variant of SEQ ID NO: 16 or 17 having at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0158] FW-H3 can include or consist of the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0159] FW-H4 can include or consist of the amino acid sequence of SEQ ID NO: 19, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0160] In an embodiment, the framework sequence of VH has at most two or at most one amino acid substitution, insertion, and / or deletion event.

[0161] In an embodiment, the VL of the α-1,6-core-fucosylated AFP-specific antibody or antigen-binding fragment of the present invention includes a framework region (FW) and has the following structure. FW-L1-CDR-L1-FW-L2-CDR-L2-FW-L3-CDR-L3-FW-L4

[0162] FW-L1 includes, or can consist of, the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0163] FW-L2 includes, or can consist of, the amino acid sequence of SEQ ID NO: 21 or 22, or a variant of SEQ ID NO: 21 or 22 having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0164] FW-L3 includes, or can consist of, the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% identity thereto.

[0165] FW-L4 can include or consist of a variant having the amino acid sequence of SEQ ID NO: 24, or at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, or even more preferably at least 99% thereof.

[0166] In embodiments, the framework sequence of VH has at most 4, at most 3, at most 2 or at most 1 amino acid substitution, insertion and / or deletion events.

[0167] In embodiments, the framework region of VH and / or VL of the antibody or antigen-binding fragment of the invention may include the rabbit-derived framework sequences FW1, FW2, FW3 and FW4.

[0168] In embodiments, the framework region of VH and / or VL of the antibody or antigen-binding fragment of the invention is derived from the same (rabbit) germline as the framework region of 19B12 or 3C5.

[0169] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention can be rabbit antibodies.

[0170] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention can have a detection label attached thereto.

[0171] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention can have a capture label attached thereto.

[0172] The inventors have found that using a multivalent 1,6fucAFP antibody containing more than twice the F V domain of 19B12 (as in the case of conventional antibodies), for example more than eight times (also referred to herein as p8), can increase the signal-to-noise ratio of immunoassays as described in the appended examples.

[0173] Thus, in embodiments, the 1,6fucAFP antibody of the present invention is at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 Fs formed by any combination of the VH domain and the VL domain as described above herein in relation to the heavy and light chains of the antibody of the present invention V domains, which can be a multivalent antibody. In embodiments, the 1,6fucAFP antibody of the present invention can contain any combination of the VH domain and the VL domain described herein at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, or at least 8 times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 times). In embodiments, the 1,6fucAFP antibody of the present invention is an F formed by any combination of the VH domain and the VL domain described herein V and can contain at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 Fab domains containing the same. In one embodiment, the multivalent antibody of the present invention is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of the F of antibody 19B12 or its variant as defined above herein V domain (formed by VH and VL).

[0174] As used herein, a multivalent antibody relates to an antibody containing at least 3 Fv domains. In a preferred embodiment, the multivalent antibody is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 10 copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of the same F V containing.

[0175] Exemplary but non-limiting embodiments for multivalent antibodies and methods for making such antibodies are disclosed in International Publication No. WO 2019 / 057816, which is hereby incorporated by reference in its entirety. Specifically, all embodiments regarding the structural constitution of such multivalent antibodies and methods for making such multivalent antibodies (also referred to as p3, p4, p5, p6, p7 or p8) are hereby disclosed by reference.

[0176] In embodiments, the multivalent antibodies of the invention can comprise a heavy chain comprising a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies, 8 in certain embodiments) of VH-CH1 domains (e.g., all comprising the (preferably the same) VH of the 1,6fucAFP antibody or antigen-binding fragment of the invention) adjacent to a linker sequence (e.g., one of the linker sequences described in International Publication No. WO 2019 / 057816, which is hereby incorporated by reference). The additional VH-CH1 domains compared to conventional antibodies can be located upstream and / or downstream of the hinge-CH2-CH3 sequence. The light chain in such multimeric antibodies can be a conventional light chain consisting of a VL and a constant domain.

[0177] Alternative methods for making multivalent antibodies include chemical polymerization / cross-linking of antibodies or antigen-binding fragments.

[0178] The antibodies of the invention can be made using immunization and selection methods as described in the appended examples.

[0179] Another suitable method for generating or isolating the antibodies and antibody-antigen binding fragments of the present invention includes, but is not limited to, selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to, bacteriophage, ribosome, oligonucleotide, RNA, cDNA or yeast display libraries) using the binding activity of interest. For example, an antibody or antigen-binding fragment can be selected from such libraries by positive selection for specific binding to the glycopeptide of formula I and negative selection for binding to the glycopeptide of formula III and / or the core-fucosylated glycan of formula IV and / or the peptide of SEQ ID NO: 2 or 25. Display libraries are well known in the art and are available from various commercial vendors including, for example, but not limited to, Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsried / Planegg, Del.), Biovation (Aberdeen, Scotland, UK) and Bioinvent (Lund, Sweden). Again, the selected clones can be processed according to conventional methods for subsequent recombinant processing.

[0180] Variants of the antibodies or antigen-binding fragments disclosed herein can be generated using routine recombinant DNA techniques using the sequences disclosed herein. For example, recombinant DNA techniques can be used to retrieve or modify DNA sequences encoding the antibodies and / or antibody-antigen binding fragments disclosed herein, such as the heavy and / or light chain variable domains described above herein. For example, recombinant DNA techniques can be used to substitute or remove portions of the coding sequence(s) that are not necessary to maintain specific and selective binding to the antigen(s) of interest. Molecules expressed from such modified or truncated DNA molecules are also encompassed by the antibodies of the present invention.

[0181] The antibodies of the present invention can be recombinantly expressed. Thus, in certain embodiments, the monoclonal antibodies of the present invention can be recombinant antibodies. Methods for producing recombinant antibodies are known in the art. Exemplary embodiments for antibody expression are disclosed in the accompanying examples and elsewhere in this specification.

[0182] In a second aspect, the present invention provides a nucleic acid molecule or set of polynucleotides encoding a 1,6fucAFP antibody or 1,6fucAFP antigen-binding fragment disclosed herein. In particular, polynucleotides encoding 1,6fucAFP heavy and / or light chain variable domains as defined above herein are provided. As used herein, the terms "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and similar terms include both genomic DNA and cDNA, as well as RNA capable of driving the expression of the antibodies or antigen-binding fragments of the present invention. As used herein, the term "RNA" is understood to include all forms of RNA including mRNA, tRNA and rRNA, and also genomic RNA in the case of RNA of RNA viruses. Preferably, embodiments described as "RNA" relate to mRNA. The nucleic acid molecules / nucleic acid sequences of the present invention can be of natural as well as synthetic or semi-synthetic origin. Thus, the nucleic acid molecule can be, for example, a nucleic acid molecule synthesized according to recombinant methods, or semi-synthetically, for example by combining chemical synthesis and recombinant methods, according to conventional protocols of organic chemistry. Those skilled in the art are proficient in the preparation and use of such nucleic acid molecules. A "set of polynucleotides" relates to at least two polynucleotides encoding a part of an antibody or antigen-binding fragment. For example, a first polynucleotide encoding a heavy chain variable domain and a second polynucleotide encoding a light chain variable domain may be used.

[0183] In an embodiment, a polynucleotide or a set of polynucleotides encoding one of the combinations of VH and VL disclosed in connection with the first aspect of the present invention is provided. Accordingly, for each of the aspects and embodiments regarding the 1,6fucAFP antibody or antigen-binding fragment thereof described herein, corresponding polynucleotides encoding each antibody or antigen-binding fragment are provided with the necessary modifications.

[0184] In certain embodiments, a polynucleotide or a set of polynucleotides encoding the heavy chain variable domain VH of SEQ ID NO: 11 or 12 or a variant thereof as defined in connection with the first aspect of the present invention, and / or the light chain variable domain of SEQ ID NO: 13 or 14 or a variant thereof as defined in connection with the first aspect of the present invention is provided.

[0185] In another specific embodiment, a polynucleotide or a set of polynucleotides encoding VH of SEQ ID NO: 11 and VL of SEQ ID NO: 13 is provided.

[0186] In another specific embodiment, a polynucleotide or a set of polynucleotides encoding VH of SEQ ID NO: 12 and VL of SEQ ID NO: 14 is provided.

[0187] In some embodiments, the polynucleotide may include additional sequences to ensure the expression of not only the heavy and light chain variable domains but also the remaining heavy and / or light chain constant regions so that a full-length antibody (e.g., IgG) containing the heavy and / or light chain variable domains of the present invention is expressed.

[0188] In a third aspect, vectors are provided herein that include a polynucleotide or set of polynucleotides according to the second aspect of the invention. In particular, vectors are provided that include a nucleic acid molecule encoding an antibody or antigen-binding fragment of an antibody of the invention. As used herein, the term "vector" refers to a circular or linear nucleic acid molecule capable of self-replication in a host cell into which it has been introduced. Non-limiting examples of vectors suitable for use in the present invention include cosmids, plasmids (e.g., naked or encapsulated in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) and bacteriophages. However, the art provides many suitable vectors and the choice depends on the desired function. The development and use of suitable vectors is well documented in the art and reference may be made, for example, to the techniques described in Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Vectors used in connection with the present invention include a nucleic acid sequence encoding the 1,6fucAFP antibody or antigen-binding fragment disclosed herein. Accordingly, for each of the aspects and embodiments relating to monoclonal antibodies or antigen-binding fragments that specifically bind to α-1,6-core-fucosylated AFP described herein, vectors are provided herein that include the corresponding polynucleotide encoding each antibody or antigen-binding fragment.

[0189] As used herein, with respect to the term "vector comprising", it is understood in the art that additional nucleic acid sequences that are necessary and / or sufficient for the desired vector activity in a host cell are present in the vector, for example, driving the replication of the vector (and thus, the nucleic acid sequence encoding) and / or directing the host cell to express the antibody or antigen-binding fragment of the present invention. Such additional nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or the expression of the desired sequence in a particular cell line. For example, a vector may comprise a nucleic acid molecule encoding an antibody or antibody antigen-binding fragment of the present invention that is operably linked and / or under the control of regulatory sequences. The term "regulatory sequence" refers to a DNA sequence necessary to effect the expression of a coding sequence to which it is operably linked. The term "control sequence" is intended to include at least all the components whose presence may also be necessary for expression, and may further include additional advantageous components, for example, for enabling replication. As understood in the art, the nature of such regulatory and control sequences varies depending on the host organism. For example, in prokaryotes, control sequences generally include a promoter, a ribosome-binding site, and a terminator. In eukaryotes, control sequences generally include a promoter, a terminator, and in some cases, an enhancer, a transcriptional activator, or a transcription factor.

[0190] The vector used in the present invention is preferably an expression vector. The expression vector can direct the replication and expression of the nucleic acid molecule of the present invention in a host cell, and thus, for example, provide the expression of the heavy chain variable domain and / or the light chain variable domain of the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention. In some embodiments, the vector includes not only the heavy chain and light chain variable domains, but also additional sequences to ensure the expression of the remaining heavy chain and light chain constant regions such that a full-length antibody (e.g., IgG) containing the heavy chain and / or light chain variable domains of the present invention is expressed. Suitable expression vectors are widely described in the literature, and the determination of a suitable expression vector for a particular cell line can be readily performed by those skilled in the art using conventional methods. Preferably, the vectors disclosed herein include a recombinant polynucleotide (i.e., a nucleic acid sequence encoding the monoclonal antibody of the present invention) and a control sequence operably linked thereto. The vectors provided herein preferably further include a promoter. The vectors described herein may also include a selectable marker gene and an origin of replication to ensure replication in the host. Furthermore, the vectors provided herein may also include a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in a host cell.

[0191] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein. Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nucleofection), non-chemical methods (electroporation, sonoporation, phototransfection, gene electrophoresis, hydrodynamic delivery, or natural transformation when cells are contacted with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetofection, impalfection), phage vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, Semliki Forest viruses, or bovine papillomaviruses may be used for transfection of nucleic acid molecules into target cell populations. Furthermore, baculovirus systems can also be used as vectors in eukaryotic expression systems for the nucleic acid molecules of the present invention.

[0192] The term "prokaryote" is meant to include all bacteria that can be transformed, transduced or transfected with DNA or DNA or RNA molecules for the expression of the proteins of the invention. Prokaryotic hosts include Gram-negative bacteria as well as Gram-positive bacteria, such as Escherichia coli (E. coli), Salmonella typhimurium (S. typhimurium), Serratia marcescens, Corynebacterium (glutamicum), Pseudomonas (fluorescens), Lactobacillus, Streptomyces, Salmonella and Bacillus subtilis. The term "eukaryote" is meant to include yeast, higher plants, insects and mammalian cells. Non-limiting examples of host cells typically used in the art include Hela, HEK293, H9, Per.C6 and Jurkat cells, mouse NIH3T3, NS / 0, SP2 / 0 and C127 cells, COS cells, such as COS1 or COS7, CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bos melanoma cells and Chinese hamster ovary (CHO) cells.

[0193] According to a fourth aspect, the invention relates to a host cell comprising a polynucleotide or set of polynucleotides of the second aspect of the invention, or a vector of the third aspect. The host cell can be configured such that the 1,6fucAFP antibody or antigen-binding fragment provided herein is expressed. The host cell can be a prokaryotic cell or a eukaryotic cell. In a preferred embodiment, the host cell is a eukaryotic cell. Exemplary prokaryotic and eukaryotic cells are disclosed above in the context of the third aspect of the invention. In a particular embodiment, the cell is a HEK cell. In another particular embodiment, the host cell is a CHO cell.

[0194] The host cell according to the present invention is preferably a eukaryotic cell (e.g., HEK or CHO). Although antibodies and antigen-binding fragments as disclosed herein can be expressed in both prokaryotic and eukaryotic host cells, expression of antibodies in eukaryotic cells is preferred, and expression of antibodies in mammalian host cells is most preferred. This is because such eukaryotic cells (particularly mammalian cells are most preferred) are more likely to express properly folded antibodies / antibody fragments that contain appropriate post-translational modifications to be immunologically active.

[0195] When a recombinant expression vector encoding the antibody heavy chain and / or light chain variable domain disclosed herein is introduced into a host cell, the antibody or antibody antigen-binding fragment is produced by culturing the host cell for a period sufficient to allow the host cell to express the antibody or antigen-binding fragment, or preferably, to secrete the antibody or antigen-binding fragment into the medium in which the host cell is growing. The antibody and / or antigen-binding fragment can be recovered from the medium using standard protein purification methods.

[0196] Accordingly, in a fifth aspect, the present invention also provides a method for the production of a 1,6fucAFP antibody or 1,6fucAFP antibody antigen-binding fragment as disclosed herein, comprising culturing a host cell of the present invention that expresses an antibody or antigen-binding fragment of the present invention under appropriate conditions, and isolating the produced 1,6fucAFP antibody or antigen-binding fragment.

[0197] The transformed host cells can be grown in a bioreactor and cultured by techniques known in the art to achieve optimal cell growth. The antibodies and / or antibody antigen-binding fragments of the present invention can then be purified by any conventional means, such as, but not limited to, affinity chromatography (e.g., Strep-tag II or His 6 It can be isolated from cell fractions or growth media by using fusion tags such as tags), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high performance liquid chromatography (HPLC), reverse phase HPLC or immunoprecipitation.

[0198] In a sixth aspect, provided herein is a 1,6-fucAFP antibody or antigen-binding fragment obtainable or obtained by the method according to the fifth aspect of the invention.

[0199] In a seventh aspect, the invention relates to a composition comprising a 1,6fucAFP antibody or antigen-binding fragment of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention. In a preferred embodiment, the composition is a diagnostic composition, i.e., a composition for use in diagnostic applications. In a preferred embodiment, the composition is for use in an in vitro diagnostic test for detecting α-1,6-core-fucosylated AFP (i.e., the clinically important part of AFP-L3). In a preferred embodiment, the diagnostic composition can be a reagent for immunoassay for detecting α-1,6-core-fucosylated AFP (i.e., the clinically important part of AFP-L3). The diagnostic composition is preferably configured to enable the detection of α-1,6-core-fucosylated AFP (i.e., the clinically important part of AFP-L3) in a sample obtained from a subject. The sample may be a body fluid. In certain embodiments, the sample can be a blood sample (e.g., whole blood, serum or plasma).

[0200] In an embodiment, the composition of the present invention is preferably a composition for in vitro detection (preferably quantification) of α-1,6-core-fucosylated AFP (i.e., the clinically important part of AFP-L3) in a sample, using an immunoassay. In an embodiment, the immunoassay is a heterologous immunoassay. In an embodiment, the immunoassay is a sandwich immunoassay. In an embodiment, the immunoassay is a competitive immunoassay. In an embodiment, the immunoassay is the immunoassay of the present invention.

[0201] The 1,6fucAFP antibodies and antibody-antigen binding domains of the present invention, as well as methods for their production and use, are provided not only as diagnostic tools, but are also envisioned to have applicability in the treatment and amelioration of diseases and disease syndromes, as well as in model systems for investigating disease therapies. Accordingly, the present invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and (i) a 1,6fucAFP antibody or an antigen-binding fragment thereof; (ii) a polynucleotide encoding the antibody or antigen-binding fragment of (i); (iii) a vector comprising the polynucleotide of (ii); or (iv) a host cell comprising the vector of (iii) that expresses the polynucleotide of (ii) and / or the antibody or antigen-binding fragment of (i).

[0202] The pharmaceutical compositions disclosed herein are formulated for administration to human or animal subjects. In the manufacture of pharmaceutical formulations, the antibodies or antigen-binding fragments of the present invention are admixed with pharmaceutically acceptable carriers, excipients, and / or diluents. The carriers, excipients, and / or diluents must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and not deleterious to the subject. Examples of pharmaceutical carriers suitable for use with antibody-based compositions are well known in the art and can be formulated by conventional methods.

[0203] In an eighth aspect, the use of a 1,6fucAFP antibody or antigen-binding fragment according to the invention, or a composition according to the invention, for an in vitro immunoassay, particularly an in vitro assay for detecting (and optionally quantifying) α-1,6-core-fucosylated AFP (or a subsequence thereof comprising α-1,6-core-fucosylated AFP) in a sample, is provided herein. Since α-1,6-core-fucosylated AFP is a core component of AFP-L3, the immunoassay may also be referred to as an immunoassay for detecting (and optionally quantifying) AFP-L3 in a sample.

[0204] The sample can be a tissue slide or a body fluid such as, but not limited to, a blood sample, cerebrospinal fluid, semen, saliva or urine. In an embodiment, the sample is a blood sample such as whole blood, serum or plasma. In an embodiment, the sample is serum or plasma.

[0205] Non-limiting examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot assay, or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence or electrochemiluminescence.

[0206] Further exemplary immunoassay formats using a 1,6fucAFP antibody or antigen-binding fragment (different antibodies provided herein) for detection and / or quantification are also described in European Patent No. 3252073, US Patent Application Publication No. 2018 / 0110889 and Egashira Y et al. (Scientific Reports, 2019, 9:12359), all of which are hereby incorporated by reference in their entirety.

[0207] The principle of the immunoassay using the 1,6fucAFP antibody or antigen-binding fragment (or composition containing the same) according to the present invention is to incubate the 1,6fucAFP antibody or antigen-binding fragment (or composition containing the same) with a sample (suspected of containing or containing α-1,6-core-fucosylated AFP). When the analyte of interest (i.e., α-1,6-core-fucosylated AFP) is present, a detection complex containing the 1,6fucAFP antibody or antigen-binding fragment and α-1,6-core-fucosylated AFP is formed by an antibody-antigen binding reaction / binding. To detect and / or quantify α-1,6-core-fucosylated AFP, the formation of the detection complex may be detected. Methods for detecting the formation of the detector complex are well known. For example, the detection may involve a detection label (e.g., one that binds to the antibody or antigen-binding fragment of the present invention).

[0208] Accordingly, the use according to the eighth aspect of the present invention a) incubating a sample containing α-1,6-core-fucosylated AFP with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention such that a detection complex containing α-1,6-core-fucosylated AFP and the 1,6-fucAFP antibody or antigen-binding fragment of the present invention is formed; b) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex, may be included.

[0209] As shown in the attached examples, by using a pretreatment (e.g., reduction pretreatment) of a sample containing α-1,6-core-fucosylated AFP, the signal in the immunoassay could be significantly increased. The pretreatment seems to facilitate the accessibility of the epitope of the antibody or antigen-binding fragment of the present invention.

[0210] Accordingly, the use according to the eighth aspect preferably includes adding a pretreatment agent according to the present invention (as described elsewhere herein) to the sample prior to incubation of the sample with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention.

[0211] The use may also include further dilutions of the sample after pretreatment, for example, by adding a specific volume before or simultaneously with the addition of the sample of the 1,6-fucAFP antibody or antigen-binding fragment of the invention to the sample, preferably. In other words, the concentration of the pretreatment agent can be reduced to a concentration acceptable for the formation of the detection complex. For example, the concentration of the components of the pretreatment agent may be diluted 2 to 3 times.

[0212] In an embodiment, the pretreatment can be a reduction pretreatment, that is, it may include the addition of a reducing agent (for example, a protein reducing agent) to the sample. In other words, the pretreatment reagent may contain or consist of a reducing agent.

[0213] A protein reducing agent is a reducing agent capable of reducing / decomposing disulfide bonds within or between protein / polypeptide chains.

[0214] Non-limiting examples of the reducing agent or protein reducing agent used for pretreatment are dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethanol, and dibutylamine disulfide (DTBA). In an embodiment, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and dibutylamine disulfide (DTBA). In an embodiment, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In an embodiment, the reducing agent or protein reducing agent can be DTT.

[0215] In an embodiment, a protein reducing agent having a redox potential at pH 7 that is 50% to 150% of the redox potential of DTT at pH 7 is used. The redox potential of DTT at pH 7 is -0.33V.

[0216] The pH during pretreatment is selected such that the reducing compound has an efficient reducing power. As is well known in the art, DTT has efficient reducing activity only, for example, at pH 6.5 to 9.0 (preferably pH 7 to 8). TCEP is redox active, for example, in a pH range of about 1.5 to about 8.5.

[0217] (Protein) The concentration of the reducing agent can be varied and depends on the exact reducing agent used. As shown in the attached examples of DTT, the concentration used for efficient pretreatment can be determined by comparing the immunoassay signals of samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of (protein) reducing agent before performing immunoassays that are identical in other respects.

[0218] Exemplarily, when DTT is used as the (protein) reducing agent, the final concentration of DTT in the sample during pretreatment can be 0.1 mM to 100 mM, 0.1 mM to 60 mM, 0.1 mM to 50 mM, or 0.1 mM to 47.8 mM. In embodiments, the concentration of DTT can be at least 0.2 mM, at least 0.3 mM, at least 0.5 mM, or at least 1 mM. In embodiments, the DTT concentration can be at most 100 mM, at most 60 mM, at most 50 mM, or at most 47.8 mM. In embodiments, the concentration of DTT can be 2.2 mM. In embodiments, the concentration of DTT can be 2.3 mM. In embodiments, for DTT, the final concentration of DTT in the mixture of the pretreatment agent and the sample can be 0.6 mM to 107.8 mM. In embodiments, for DTT, the final concentration of DTT in the mixture of the pretreatment agent and the sample can be 0.7 mM to 107.8 mM.

[0219] The concentration of DTT in the pretreatment agent is higher than that in the final mixture of the sample during pretreatment. For example, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold concentrate), i.e., it can be the stock solution.

[0220] The pretreatment agent used in the pretreatment of the sample may further contain a chelating agent such as a chelating agent that binds to ions. It has been found that adding such a chelating agent improves the stability of the pretreatment agent. In certain embodiments, the chelating agent may be a chelating agent that binds to ions. In a more specific embodiment, the chelating agent may be a chelating agent that binds to divalent ions.

[0221] Non-limiting examples of chelating agents for binding to (divalent) ions include aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrin, polyamine, crown ether, ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

[0222] Further non-limiting examples of chelating agents are DTPA, EC, DMSA, EDTA, EGTA, Cy-EDTA, EDTMP, DTPA, DTPA, CyDTPA, Cy2DTPA, BOPTA, DTPA-MA, DTPA-BA, DTPMP, DOTA, TRITA, TETA, DOTMA, DOTA-MA, HP-DO3A, pNB-DOTA, DOTP, DOTMP, DOTEP, DOTPP, DOTBzP, DOTPME, HEDP, DTTP, anN3S triamidothiol, DADS, MAMA, DADT, N2S4 diaminedithiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propyleneamine oxime, tetraamine, cyclam, or combinations thereof.

[0223] In some embodiments, the chelating agent is EDTA.

[0224] The concentration of the chelating agent during the pretreatment of the sample can be varied and may depend on the exact chelating agent and reducing agent used. The useful concentration range can be determined by comparing the immunoassay signals of separate samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of the chelating agent.

[0225] For example, when using EDTA, exemplary concentrations during sample pretreatment are 0.1 mM to 50 mM, 0.1 mM to 20 mM, 0.1 mM to 10 mM, 0.1 mM to 5 mM, and 0.1 mM to 4 mM.

[0226] In an embodiment, the pretreatment agent further comprises a buffering substance (also referred to herein as a buffer). The buffer can be selected according to the pH required for the protein reducing agent to reduce disulfide bonds.

[0227] In an embodiment, the pretreatment agent comprises or consists of a protein reducing agent and a chelating agent for ions (e.g., divalent ions) and optionally a buffer.

[0228] In an embodiment, the pretreatment agent comprises DTT and EDTA and optionally a buffer solution.

[0229] The pretreatment of the sample may be performed at different times. For example, the pretreatment can be performed for 1 minute to 30 minutes, 2 minutes to 20 minutes, or 3 minutes to 10 minutes. In some embodiments, the pretreatment may be performed for 9 minutes.

[0230] In an embodiment, the pretreatment can be incubating the sample with DTT at pH 6.5 to 9.0 (preferably pH 7 to 8) for a predetermined time (e.g., at least 2, 3, 4, 5, 6, 7, 8, or 9 minutes). Optionally, a chelating agent (e.g., EDTA) as described above may also be present. In an embodiment, the pretreatment may also include the addition of a buffer solution (e.g., Tris at a concentration of 10 mM to 200 mM, 30 mM to 150 mM, 50 mM to 130 mM, or 100 mM) that adjusts the pH in the sample to above 7 to the sample. After the pretreatment, the concentration of DTT may be reduced so that the detection complex can still be formed.

[0231] In embodiments, the use may further comprise incubating the sample with an AFP-specific antibody that does not compete with the binding of the sample to the 1,6-fucAFP antibody or antigen-binding fragment of the present invention to AFP. In these embodiments, the detection complex further comprises a sandwich comprising the AFP-specific antibody, i.e., the 1,6-fucAFP antibody of the present invention, α-1,6-core-fucosylated AFP, and an AFP-specific antibody that does not compete with the binding of the 1,6-fucAFP antibody or antigen-binding fragment of the present invention to AFP. In embodiments using a pretreatment, an AFP-specific antibody is selected such that the antibody can bind to AFP even in the presence of the pretreatment agent.

[0232] Non-limiting examples of AFP-specific antibodies that do not compete with the binding of the antibodies of the present invention include the commercially available anti-AFP antibody Tu11 (Roche Diagnostics Deutschland GmbH, material number: 11492080103). Thus, an antibody that does not compete with the binding of the antibodies of the present invention to AFP can be an antibody or antigen-binding fragment thereof having the same epitope as Tu11, or can compete with the binding of the Tu11 antibody to AFP. As demonstrated by the appended examples, the Tu11 antibody is also compatible with the pretreatment of samples according to the present invention.

[0233] In embodiments, the use may comprise digesting α-1,6-core-fucosylated AFP contained in the sample into glycopeptides and peptides (e.g., using a protease) prior to incubation with the antibody or antigen-binding fragment of the present invention, the glycopeptide comprising a partial AFP sequence comprising α-1,6-core-fucosylated Asn-251 of AFP (e.g., as described elsewhere herein). Optionally, the agent for generating the glycopeptide or peptide can be removed or inactivated prior to adding the antibody or antigen-binding fragment of the present invention. Subsequently, the presence or level of α-1,6-core-fucosylated AFP in the sample can be determined based on the detection of a detection complex comprising a partial AFP sequence comprising α-1,6-core-fucosylated Asn-251 of AFP and the antibody or antigen-binding fragment of the present invention.

[0234] The immunoassay using the 1,6fucAFP antibody or antigen-binding fragment of the present invention may be a heterologous immunoassay.

[0235] Additionally or alternatively, the 1,6fucAFP antibody or antigen-binding fragment according to the present invention can be used in a sandwich immunoassay (e.g., a heterologous sandwich immunoassay).

[0236] In an embodiment, the immunoassay can be an immunohistochemistry (IHC) assay.

[0237] In an embodiment, the immunoassay can be for detecting a glycopeptide of Formula I or a glycoprotein comprising the glycopeptide of Formula I.

[0238] As is apparent from the present disclosure, the antibodies and antigen-binding fragments of the present invention can distinguish α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation from (i) AFP or a partial AFP sequence lacking α-1,6-core-fucosylation and / or (ii) an α-1,6-core-fucosylated protein other than AFP. Accordingly, the use of the 1,6fucAFP antibody and antigen-binding fragment for distinguishing α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation from (i) AFP or a partial AFP sequence lacking α-1,6-core-fucosylation and / or (ii) an α-1,6-core-fucosylated protein other than AFP is also provided herein.

[0239] In a ninth aspect, the present invention provides an in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation in a sample using the 1,6-fucAFP antibody or antigen-binding fragment of the present invention.

[0240] The embodiments disclosed in connection with the use according to the eighth aspect are applied with necessary modifications. In an embodiment, the method according to the ninth aspect is (i) binding the antibody or antigen-binding fragment of the present invention to α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation contained in a sample so as to form a detection complex; (ii) detecting the detection complex, thereby detecting the presence and optionally the amount of α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation in the sample by determining the presence and optionally the amount of α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation.

[0241] In an embodiment, the method according to the ninth aspect is a) incubating a sample containing α-1,6-core-fucosylated AFP with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention so that a detection complex containing α-1,6-core-fucosylated AFP and the 1,6-fucAFP antibody or antigen-binding fragment of the present invention is formed; b) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex.

[0242] As shown in the attached examples, by using a pretreatment (e.g., a reduction pretreatment) of a sample containing α-1,6-core-fucosylated AFP, the signal in the immunoassay could be significantly increased. The pretreatment seems to facilitate the accessibility of the epitope of the antibody or antigen-binding fragment of the present invention in relation to full-length α-1,6-core-fucosylated AFP.

[0243] Therefore, the method according to the ninth aspect of the present invention may preferably include adding a pretreatment agent according to the present invention to the sample before incubation of the sample with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention.

[0244] Therefore, the method according to the ninth aspect is a) incubating a sample (e.g., a sample containing α-1,6-core-fucosylated AFP) in the presence of a pretreatment reagent according to the present invention; b) incubating the pretreated sample with a 1,6-fucAFP antibody or antigen-binding fragment of the present invention such that a detection complex comprising α-1,6-core-fucosylated AFP and the 1,6-fucAFP antibody or antigen-binding fragment of the present invention is formed; c) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex.

[0245] The method may include a further dilution of the pretreated sample, e.g., a further dilution of the sample by adding a specific volume, preferably before or simultaneously with the addition of the antibody or antigen-binding fragment of the present invention to the sample. In other words, the concentration of the pretreatment agent may be reduced to a concentration acceptable for the formation of the detection complex.

[0246] In an embodiment, the pretreatment may be a reduction pretreatment, i.e., it may include the addition of a reducing agent (e.g., a protein reducing agent) to the sample. In other words, the pretreatment reagent may contain or consist of a reducing agent (e.g., a protein reducing agent).

[0247] A protein reducing agent is a reducing agent capable of reducing / decomposing disulfide bonds within or between protein / polypeptide chains.

[0248] Non-limiting examples of reducing agents or protein reducing agents used in the pretreatment are dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethanol, and dithiobutylamine (DTBA). In embodiments, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and dithiobutylamine (DTBA). In embodiments, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In embodiments, the reducing agent or protein reducing agent can be dithiothreitol (DTT).

[0249] In embodiments, a protein reducing agent having a redox potential at pH 7 that is 50% to 150% of the redox potential of DTT at pH 7 is used. The redox potential of DTT at pH 7 is -0.33 V.

[0250] The pH during pretreatment is selected such that the reducing compound has an efficient reducing power. As is well known in the art, DTT has efficient reducing activity at a pH value of, for example, 6.5 to 9.0 (preferably pH 7 to 8). TCEP is redox active in a pH range of, for example, about 1.5 to about 8.5.

[0251] (Protein) Reducing agent concentrations can vary and depend on the exact reducing agent used. As shown in the attached example of DTT, the concentration used for efficient pretreatment can be determined by comparing the immunoassay signals of samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of (protein) reducing agent before performing immunoassays that are identical in other respects.

[0252] Exemplarily, when DTT is used as a (protein) reducing agent, the final concentration of DTT in the sample during pretreatment can be 0.1 mM to 100 mM, 0.1 mM to 60 mM, 0.1 mM to 50 mM, or 0.1 mM to 47.8 mM. In embodiments, the concentration of DTT can be at least 0.2 mM, at least 0.3 mM, at least 0.5 mM, or at least 1 mM. In embodiments, the DTT concentration can be at most 100 mM, at most 60 mM, at most 50 mM, or at most 47.8 mM. In embodiments, the concentration of DTT can be 2.2 mM. In embodiments, the concentration of DTT can be 2.3 mM. In embodiments, for DTT, the final concentration of DTT in the mixture of the pretreatment agent and the sample can be 0.6 mM to 107.8 mM. In embodiments, for DTT, the final concentration of DTT in the mixture of the pretreatment agent and the sample can be 0.7 mM to 107.8 mM.

[0253] The concentration of DTT in the pretreatment agent is higher than that in the final mixture of the sample during pretreatment. For example, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold concentrate), i.e., it can be the stock solution.

[0254] The pretreatment agent used for the pretreatment of the sample may further contain a chelating agent such as a chelating agent that binds to ions. Such chelating agents have been found to improve the stability of the pretreatment agent containing a reducing agent such as DTT. In certain embodiments, the chelating agent may be a chelating agent that binds to ions. In more specific embodiments, the chelating agent may be a chelating agent that binds to divalent ions.

[0255] Non-limiting examples of chelating agents for binding to (divalent) ions are aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrin, polyamine, crown ether, ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

[0256] Further non-limiting examples of chelating agents are DTPA, EC, DMSA, EDTA, EGTA, Cy-EDTA, EDTMP, DTPA, CyDTPA, Cy2DTPA, BOPTA, DTPA-MA, DTPA-BA, DTPMP, DOTA, TRITA, TETA, DOTMA, DOTA-MA, HP-DO3A, pNB-DOTA, DOTP, DOTMP, DOTEP, DOTPP, DOTBzP, DOTPME, HEDP, DTTP, anN3S triamidothiol, DADS, MAMA, DADT, N2S4 diaminedithiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propyleneamineoxime, tetraamine, cyclam, or combinations thereof.

[0257] In some embodiments, the chelating agent is EDTA.

[0258] The concentration of the chelating agent during sample pretreatment can be varied and may depend on the exact chelating agent and reducing agent used. The useful concentration range can be determined by comparing the immunoassay signals of separate samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of the chelating agent.

[0259] For example, when using EDTA, exemplary concentrations during sample pretreatment are 0.1 mM to 50 mM, 0.1 mM to 20 mM, 0.1 mM to 10 mM, 0.1 mM to 5 mM.

[0260] In an embodiment, the pretreatment agent further includes a buffering substance (also referred to herein as a buffer). The buffer can be selected according to the pH required for the protein reducing agent to reduce disulfide bonds.

[0261] In an embodiment, the pretreatment agent comprises or consists of a protein reducing agent, a chelating agent for ions (e.g., divalent ions), and optionally a buffer.

[0262] In an embodiment, the pretreatment agent includes DTT and EDTA, and optionally a buffer solution.

[0263] The pretreatment of the sample may be performed at different times. For example, the pretreatment can be performed for 1 minute to 30 minutes, 2 minutes to 20 minutes, or 3 minutes to 10 minutes. In some embodiments, the pretreatment may be performed for 9 minutes.

[0264] In an embodiment, the pretreatment can be incubating the sample with DTT at pH 6.5 to 9.0 (preferably pH 7 to 8) for a predetermined time (e.g., at least 2, 3, 4, 5, 6, 7, 8, or 9 minutes). Optionally, a chelating agent (e.g., EDTA) as described above may also be present. In an embodiment, the pretreatment may also include adding to the sample a buffer solution (e.g., Tris at a concentration of 10 mM to 200 mM, 30 mM to 150 mM, 50 mM to 130 mM, or 100 mM) that adjusts the pH in the sample above 7. Such a pH ensures the reduction potential of DTT. Preferably, the pH can be adjusted to a pH of 7 to 10, 7 to 9, or 7 to 8.

[0265] After the pretreatment, the concentration of DTT may be reduced so that the detection complex can still be formed.

[0266] In embodiments, the method of the ninth aspect may further include incubating the sample with an AFP-specific antibody that does not compete with the binding of the 1,6-fucAFP antibody or antigen-binding fragment of the invention to AFP. In these embodiments, the detection complex further includes a sandwich comprising the AFP-specific antibody, i.e., the 1,6-fucAFP antibody of the invention, α-1,6-core-fucosylated AFP, and an AFP-specific antibody that does not compete with the binding of the 1,6-fucAFP antibody or antigen-binding fragment of the invention to AFP. In embodiments using a pretreatment, an AFP-specific antibody is selected such that the antibody can bind to AFP even in the presence of the pretreatment agent.

[0267] Non-limiting examples of AFP-specific antibodies that do not compete with the binding of the antibodies of the invention include the commercially available anti-AFP antibody Tu11 (Roche Diagnostics Deutschland GmbH, material number: 11492080103). Thus, an antibody that does not compete with the binding of the antibodies of the invention to AFP may be an antibody or antigen-binding fragment thereof having the same epitope as Tu11, or may compete with the binding of the Tu11 antibody to AFP. As demonstrated by the appended examples, the Tu11 antibody is also compatible with the pretreatment of samples according to the invention.

[0268] In embodiments, the method of the ninth aspect may include digesting α-1,6-core-fucosylated AFP contained in the sample into glycopeptides and peptides (e.g., using a protease) prior to incubation with the antibody or antigen-binding fragment of the invention, the glycopeptide comprising a partial AFP sequence comprising α-1,6-core-fucosylated Asn-251 of AFP (e.g., as described elsewhere herein). Optionally, the agent for generating the glycopeptide or peptide may be removed or inactivated prior to adding the antibody or antigen-binding fragment of the invention. Then, the presence or level of α-1,6-core-fucosylated AFP in the sample may be determined based on the detection of a detection complex comprising a partial AFP sequence comprising α-1,6-core-fucosylated Asn-251 of AFP and the antibody or antigen-binding fragment of the invention.

[0269] In an embodiment, the method according to the ninth aspect can be a heterogeneous immunoassay. In an embodiment, the method according to the ninth aspect can be a sandwich immunoassay (e.g., a heterogeneous sandwich immunoassay). In an embodiment, the method according to the ninth aspect can be an immunohistochemistry (IHC) assay, and the sample can be a tissue slide. In an embodiment, the method according to the ninth aspect is a body fluid immunoassay, and the sample is a body fluid.

[0270] The sample used in the method according to the ninth aspect can be, but is not limited to, a tissue slide or a body fluid such as a blood sample, cerebrospinal fluid, semen, saliva, or urine. In an embodiment, the sample is a blood sample such as whole blood, serum, or plasma. In an embodiment, the sample is serum or plasma.

[0271] The method according to the ninth aspect is an in vitro method for a sample previously obtained from a subject.

[0272] As is known in the art, AFP-L3 is a useful marker, and preferably, α-1,6-core-fucosylated AFP, in combination with other markers for the detection of early hepatocellular carcinoma (HCC), is the core component of AFP-L3.

[0273] Accordingly, provided herein is an in vitro method for detecting or assisting in the detection of HCC, a) determining the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using the method according to the ninth aspect; b) comparing the determined level of α-1,6-core-fucosylated AFP in the sample with a reference level of α-1,6-core-fucosylated AFP; c) assisting in the detection of HCC or detecting HCC based on the comparison.

[0274] In particular, AFP-L3, and thus α-1,6-core-fucosylated AFP, are also frequently used in combination with other biomarkers and / or clinical or demographic information in a score for detecting HCC - an exemplary score is the so-called GALAD score that includes gender, age, AFP-L3, AFP and DCP (also known as PIVKA-II).

[0275] As a result, an in vitro method for assisting in the detection of HCC or for detecting HCC, a) determining the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using the method according to the ninth aspect of the invention; b) calculating a score for HCC detection taking into account the determined level of α-1,6-core-fucosylated AFP; c) assisting in the detection of HCC or detecting HCC based on the calculated score (e.g., by comparing with a reference value of the score indicative of HCC), is provided herein.

[0276] The score for HCC detection may take into account the level of at least one additional biomarker (e.g., AFP or DCP) for HCC detection. Thus, the method may further comprise determining or receiving the level of the at least one additional HCC biomarker in the sample or in another sample taken from the subject (e.g., simultaneously).

[0277] The score for HCC detection may additionally or alternatively take into account clinical parameters of the subject (e.g., ultrasound data) or demographic information (e.g., age and / or gender). Thus, the method may further comprise receiving the clinical parameters or the demographic information of the subject.

[0278] In certain embodiments, the score for HCC detection may include the levels of AFP and DCP in the sample, as well as the gender and age of the subject, i.e., it may be the GALAD score. Thus, the method may further include determining or receiving the levels of AFP and DCP (PIVKA-II) in a sample taken from (e.g., simultaneously) or another sample from the subject, as well as receiving the age and gender of the subject.

[0279] A computer-implemented method for detecting HCC, a) receiving the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using the method according to the ninth aspect of the present invention; b) calculating a score for HCC detection taking into account the determined level of α-1,6-core-fucosylated AFP; c) assisting in the detection of HCC or detecting HCC based on the calculated score (e.g., by comparing the score to a reference value indicative of HCC), is provided herein.

[0280] The above-described in vitro method embodiment for detecting HCC using a score taking into account the determined level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using the method according to the ninth aspect of the present invention is applied with the necessary modifications, with the only exception being that the levels of further biomarkers are received and not determined.

[0281] In a tenth aspect, the present invention provides a pretreatment reagent for use in an immunoassay for detecting α-1,6-core-fucosylated AFP (e.g., using the antibody or antigen-binding fragment of the present invention) that includes a reducing agent.

[0282] The term "pretreatment reagent" is used interchangeably in this specification with the terms "pretreatment agent", "pretreating agent", and "pretreatment reagent", and relates to a pretreatment composition applied to a sample (e.g., a blood sample such as serum or plasma) to facilitate the binding of a 1,6fucAFP antibody to α-1,6-core-fucosylated AFP contained in the sample.

[0283] In some embodiments, for the pretreatment reagent of the tenth aspect of the present disclosure, the reducing agent may be a protein reducing agent. In other words, the pretreatment reagent may contain or consist of a protein reducing agent.

[0284] A protein reducing agent is a reducing agent capable of reducing / decomposing disulfide bonds within or between protein / polypeptide chains.

[0285] Non-limiting examples of the reducing agent or protein reducing agent are dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethanol, and dibutylamine disulfide (DTBA). In an embodiment, the reducing agent or protein reducing agent may be selected from dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), and dibutylamine disulfide (DTBA). In an embodiment, the reducing agent or protein reducing agent may be selected from dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP). In an embodiment, the reducing agent or protein reducing agent may be dithiothreitol (DTT).

[0286] In an embodiment, the reducing agent may be a protein reducing agent having a redox potential at pH 7 that is 50% to 150% of the redox potential of DTT at pH 7. The redox potential of DTT at pH 7 is -0.33V.

[0287] The pH of the pretreatment reagent can be selected within a wide range and may vary depending on the components contained in the pretreatment agent. If necessary, the pH may be selected so that the reduction potential is as high as possible, or the pH can be selected so that the stability (shelf life) of the reducing agent is maintained. In an embodiment, the pH can be selected such that the pH of the sample to which the pretreatment reagent is added is adjusted to a pH that enables the reducing activity of the reducing agent after being added to the sample.

[0288] Exemplarily, the reducing agent is DTT, and the pH of the pretreatment agent is selected to be less than pH 6.5 (for example, pH 4 - 6.5, pH 5 - 6.5, pH 5 - 6.5 or pH 5.5 - 6.5). This range of pH values stabilizes DTT, i.e., enables longer storage of the pretreatment agent.

[0289] The concentration of the (protein) reducing agent in the pretreatment agent can be varied and depends on the exact reducing agent used and the factor by which the pretreatment agent is concentrated over the final use concentration. Furthermore, the stability of the reducing agent may depend on the exact concentration. One skilled in the art can select the concentration of the reducing agent in the pretreatment agent so as to facilitate the binding of the 1,6fucAFP antibody or antigen-binding fragment to α-1,6-core-fucosylated AFP in a sample (e.g., a blood sample such as plasma or serum). For example, a control sample containing α-1,6-core-fucosylated AFP can be used, and the signals of immunoassays using different concentrations of the reducing agent in the pretreatment agent can be compared, and the concentration with the highest immunoassay signal can be selected. Exemplary assays are described in the attached examples.

[0290] Exemplarily, when DTT is used as a reducing agent, the concentration in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of 0.1 mM to 100 mM, 0.1 mM to 60 mM, 0.1 mM to 50 mM, or 0.1 mM to 47.8 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the concentration in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of at least 0.2 mM, at least 0.3 mM, at least 0.5 mM, or at least 1 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the DTT concentration in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of at most 100 mM, at most 60 mM, at most 50 mM, or at most 47.8 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the concentration in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of 2.2 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the concentration in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of 2.3 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the concentration of DTT in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of 0.7 mM to 107.8 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent). In an embodiment, the concentration of DTT in the pretreatment agent is selected such that the final concentration of DTT in the sample during pretreatment of 0.6 mM to 107.8 mM can be achieved (e.g., without diluting the sample by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, or more than 10-fold by the addition of the pretreatment reagent).

[0291] Exemplary concentrations of the reducing agent (e.g., DTT) in the pretreatment agent can be 0.1 mM to 150 mM, 0.1 mM to 110 mM, 0.5 mM to 100 mM, 1 mM to 50 mM, 1 mM to 20 mM, or 1 mM to 10 mM. In an embodiment, the concentration of the reducing agent (e.g., DTT) in the pretreatment agent can be 1 mM to 10 mM (e.g., 10 mM). In an embodiment, the concentration of the reducing agent (e.g., DTT) in the pretreatment agent can be 3 mM to 110 mM.

[0292] In an embodiment, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold concentrate), i.e., it may be the stock solution. In some embodiments, the pretreatment agent can be a 4- to 8-fold concentrate.

[0293] The pretreatment agent according to the tenth aspect of the present invention may further contain a chelating agent such as a chelating agent that binds to ions. It has been found that the addition of such a chelating agent improves the stability of the pretreatment reagent containing a reducing agent such as DTT. In a specific embodiment, the chelating agent may be a chelating agent that binds to ions. In a more specific embodiment, the chelating agent may be a chelating agent that binds to divalent ions.

[0294] Non-limiting examples of chelating agents for binding to (divalent) ions are aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrin, polyamine, crown ether, or ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

[0295] Further non-limiting examples of chelating agents are DTPA, EC, DMSA, EDTA, EGTA, Cy-EDTA, EDTMP, DTPA, CyDTPA, Cy2DTPA, BOPTA, DTPA-MA, DTPA-BA, DTPMP, DOTA, TRITA, TETA, DOTMA, DOTA-MA, HP-DO3A, pNB-DOTA, DOTP, DOTMP, DOTEP, DOTPP, DOTBzP, DOTPME, HEDP, DTTP, anN3S triamidothiol, DADS, MAMA, DADT, N2S4 diaminedithiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propyleneamine oxime, tetraamine, cyclam, or combinations thereof.

[0296] In some embodiments, the chelating agent is EDTA.

[0297] The concentration of the chelating agent in the pretreatment agent can be varied and depends on the exact chelating agent used and the factor by which the pretreatment agent is concentrated over the final use concentration. One of ordinary skill in the art can select the concentration of the chelating agent in the pretreatment agent to facilitate the binding of the 1,6fucAFP antibody or antigen-binding fragment to α-1,6-core-fucosylated AFP in a sample (e.g., a blood sample such as plasma or serum). For example, a control sample containing α-1,6-core-fucosylated AFP can be used and the signals of immunoassays using different concentrations of the chelating agent in the pretreatment agent can be compared, and the concentration with the highest immunoassay signal and / or higher storage stability can be selected. Exemplary assays are described in the appended examples.

[0298] For example, when EDTA is used as the chelating agent, exemplary concentrations during sample pretreatment are 0.1 mM to 50 mM, 0.1 mM to 20 mM, 0.1 mM to 10 mM, 0.1 mM to 5 mM (e.g., 4 mM).

[0299] In an embodiment, the pretreatment agent further includes a buffering substance (also referred to herein as a buffer). The buffer can be selected according to the pH required for the protein reducing agent to reduce the disulfide bond. For example, Tris, Hepes, or citrate may be used.

[0300] In an embodiment, the pretreatment agent comprises or consists of a protein reducing agent and a chelating agent (e.g., divalent ions) for ions and optionally a buffer.

[0301] In an embodiment, the pretreatment agent includes DTT and EDTA, and optionally a buffer solution. The pretreatment agent can be a 4- to 5-fold concentrate and can contain 8-12 mM DTT (e.g., 10 mM) and 1-5 mM EDTA (e.g., 4.5 mM). The pH of the pretreatment reagent may be 5.5 or less (e.g., pH 4-5.5, or pH 5.5).

[0302] In a specific embodiment, a set of pretreatment reagents for use in an immunoassay for the detection of α-1,6-core-fucosylated AFP (e.g., the step of using the antibody or antigen-binding fragment of the present invention) is provided. The set of pretreatment reagents can include first and second pretreatment compositions provided in separate containers. The first pretreatment composition can be any of the pretreatment agents defined in connection with the tenth aspect and includes a reducing agent. The pH of the first pretreatment composition is preferably selected so that the stability of the components is high.

[0303] The second pretreatment composition may include a buffer solution and may have a pH and buffer concentration that enable the reducing activity of the reducing agent when both the first and second pretreatment compositions are added to the sample.

[0304] In certain embodiments, the first pretreatment composition may include DTT and EDTA. The pretreatment agent may be a 4- to 5-fold concentrate and may include 8-12 mM DTT (e.g., 10 mM) and 1-5 mM EDTA (e.g., 2 mM). The pH of the pretreatment reagent may be 5.5 or less (e.g., pH 4-5.5, or pH 5.5). The second pretreatment composition may include a buffer and may have a pH of 6.5-9, such as 7.5-8.5. The buffer concentration may be 10 mM-150 mM, 20 mM-100 mM, or 30 mM-80 mM (e.g., 100 mM). The buffer may be a buffer having a pKa of 7-9, such as 7.5-8.5. In an embodiment, the buffer may be Tris (e.g., pH 8.5).

[0305] In an eleventh aspect, the present invention provides a kit comprising the 1,6fucAFP antibody or antigen-binding fragment of the present invention. The kit may be a kit for the detection and / or quantification of α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation in a sample (e.g., a blood sample such as plasma or serum). The kit may be a kit for the detection and / or quantification of α-1,6-core-fucosylated AFP in a sample (e.g., a blood sample such as plasma or serum).

[0306] In an embodiment, the kit may be an immunoassay kit such as a kit for a sandwich immunoassay (e.g., a heterologous sandwich immunoassay).

[0307] In certain embodiments, the kit according to the eleventh aspect of the present invention further comprises the pretreatment agent of the present invention (see above) or a set of pretreatment agents (see above). In an embodiment, the antibody or antigen-binding fragment and the pretreatment reagent are provided in separate containers.

[0308] As demonstrated in the attached examples, in particular, the combination of the 1,6fucAFP antibody or antigen-binding fragment of the present invention and the pretreatment agent of the present invention enables the detection and quantification of native α-1,6-core-fucosylated AFP with high signals using immunoassays, such as those described in connection with the present invention.

[0309] The present invention also particularly relates to the following items.

[0310] 1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing said α-1,6-core-fucosylation.

[0311] 2. (i) A heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions; and CDR-H3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution, and (ii) A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8 or a variant of SEQ ID NO: 7 or 8 modified by up to two amino acid substitutions; CDR-L2 having the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and CDR-L3 having the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution, The monoclonal antibody or antigen-binding fragment according to item 1.

[0312] 3. (i) A CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution, and a heavy chain variable domain (VH), (ii) A CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by up to two amino acid substitutions; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution, and a light chain variable domain (VL), The monoclonal antibody or antigen-binding fragment according to item 1 or 2, comprising

[0313] 4. The monoclonal antibody or antigen-binding fragment according to item 2 or 3, wherein the total number of amino acid substitutions in all CDR sequences is at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, or at most 2.

[0314] 5. (i) A CDR-H1 having the amino acid sequence of SEQ ID NO: 3; a CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one amino acid substitution; and a heavy chain variable domain (VH) comprising a CDR-H3 having the amino acid sequence of SEQ ID NO: 6, (ii) A CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution; a CDR-L2 having the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 having the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain (VL), The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 4, comprising

[0315] 6. (i) A heavy chain variable domain (VH) comprising a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5, or a variant of SEQ ID NO: 4 or 5 modified by one amino acid substitution; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6, and (ii) A light chain variable domain (VL) comprising a CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by one amino acid substitution; a CDR-L2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 10, and The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 5, comprising

[0316] 7. The amino acid substitution(s) in the variant of SEQ ID NO: 4 or 5 include(s) an amino acid substitution at position 5 of SEQ ID NO: 4 or position 4 of SEQ ID NO: 5, and / or the amino acid substitution(s) in the variant of SEQ ID NO: 7 or 8 include(s) an amino acid substitution at position 7 of SEQ ID NO: 7 or 8, respectively. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 6.

[0317] 8. The amino acid substitution(s) in the variant of SEQ ID NO: 4 or 5 include(s) a conservative amino acid substitution at position 5 of SEQ ID NO: 4 or a conservative amino acid substitution at position 4 of SEQ ID NO: 5, and / or the amino acid substitution(s) in the variant of SEQ ID NO: 7 or 8 include(s) a conservative amino acid substitution(s) at position 7 of SEQ ID NO: 7 or 8. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 7.

[0318] 9. All of the above amino acid substitutions are conservative amino acid substitutions or highly conservative amino acid substitutions. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 7.

[0319] 10. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 8, wherein the amino acid in the variant of SEQ ID NO: 4 corresponding to the 5th position of SEQ ID NO: 4 or the 4th position of SEQ ID NO: 5, or the amino acid in the variant of SEQ ID NO: 7 or 8 corresponding to the 7th position of SEQ ID NO: 7 or 8 is serine or asparagine, and / or the amino acid in the variant of SEQ ID NO: 7 or 8 corresponding to the 7th position of SEQ ID NO: 7 or 8 is serine or glycine.

[0320] 11. The monoclonal antibody or antigen-binding fragment according to any one of items 7, 8, and 10, wherein all amino acid substitutions other than the 5th position of SEQ ID NO: 4 or the 4th position of SEQ ID NO: 5 and / or the 7th position of SEQ ID NO: 7 or 8 are conservative or highly conservative amino acid substitutions.

[0321] 12. The conservative substitution(s) is / are substitution by another amino acid selected from the same group of amino acids, and the group of amino acids is a) Nonpolar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, and Met; b) Polar neutral amino acids consisting of Ser, Thr, Asn, and Gln; c) Basic amino acids having a positive charge consisting of Arg, Lys, and His, and d) Acidic amino acids having a negative charge consisting of Asp and Glu, and when Cys is conservatively substituted, it is substituted with Ser or Ala, and when Pro is conservatively substituted, it is substituted with Ala. The monoclonal antibody or antigen-binding fragment according to any one of items 8, 9, or 11.

[0322] 13. The monoclonal antibody or antigen-binding fragment antibody according to item 9, 11, or 12, wherein the highly conservative amino acid substitution is selected from the following: a) Substitution of Ala with Val, Leu, Ile, or Gly; b) Substitution of Arg with Lys; c) Substitution of Asn with Gln; d) Substitution of Asp with Glu; e) Substitution of Cys with Ser; f) Substitution of Gln with Asn; g) Substitution of Glu with Asp; h) Substitution of Gly with Ala; i) Substitution of His with Arg; j) Substitution of Ile with Leu, Val or Ala; k) Substitution of Leu with Ile, Val or Ala; l) Substitution of Lys with Arg; m) Substitution of Met with Leu, Ile or Val; n) Substitution of Phe with Tyr or Trp; o) Substitution of Pro with Ala; p) Substitution of Ser with Thr; q) Substitution of Thr with Ser; r) Substitution of Trp with Phe or Tyr; s) Substitution of Tyr with Phe or Trp; and t) Substitution of Val with Leu, Ile or Ala.

[0323] 14. A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 13, comprising: (i) A heavy chain variable domain (VH) having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.5% sequence identity with SEQ ID NO: 11 or 12, and

[0324] (ii) A light chain variable domain (VL) having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.3% sequence identity with SEQ ID NO: 13 or 14.

[0325] 15. The monoclonal antibody or antigen-binding fragment according to item 14, wherein said CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are as described in any one of items 2 to 13.

[0326] 16. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 15, wherein the heavy-chain variable domain (VH) contains a framework region (FW) and has the following structure: FW-H1-CDR-H1-FW-H2-CDR-H2-FW-H3-CDR-H3-FW-H4 (wherein FW-H1 has the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, FW-H2 has the amino acid sequence of SEQ ID NO: 16 or 17 or a variant of SEQ ID NO: 16 or 17 having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, FW-H3 has the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, (FW-H4 has the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof).

[0327] 17. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 16, wherein the light chain variable domain (VL) contains a framework region (FW) and has the following structure: FW-L1-CDR-L1-FW-L2-CDR-L2-FW-L3-CDR-L3-FW-L4 (wherein FW-L1 has the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, FW-L2 has the amino acid sequence of SEQ ID NO: 21 or 22, or a variant of SEQ ID NO: 21 or 22 having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, FW-L3 has the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof, (FW-L4 has the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 98%, more preferably 99% thereof)).

[0328] 18. (i) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 11 or 12, and (ii) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 13 or 14, and The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 17, comprising

[0329] 19. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 18, wherein the partial sequence of AFP comprises or consists of SEQ ID NO: 2.

[0330] 20. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 19, wherein the α-1,6-core-fucosylated AFP or the partial sequence of AFP containing the α-1,6-core-fucosylation comprises or consists of the glycopeptide of Formula I.

Chemical formula

[0331] 21. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 20, wherein the α-1,6-core-fucosylated AFP or the partial sequence of AFP containing the α-1,6-core-fucosylation comprises or consists of the glycopeptide of Formula II.

Chemical formula

[0332] 22. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 21, wherein the antibody or antigen-binding fragment distinguishes (i) the α-1,6-core-fucosylated AFP or the partial sequence of AFP containing the α-1,6-core-fucosylation from (ii) AFP lacking the α-1,6-core-fucosylation residue or a partial sequence thereof.

[0333] 23. The monoclonal antibody or antigen-binding fragment according to any one of items 20 to 22, wherein the partial sequence of AFP comprises or consists of SEQ ID NO: 2.

[0334] 24. The monoclonal antibody or antigen-binding fragment according to item 22 or 23, wherein the AFP or a partial sequence thereof lacking an α-1,6-core-fucose residue comprises or consists of the glycopeptide of formula III.

Chemical formula

[0335] 25. The monoclonal antibody or antigen-binding fragment according to any one of items 22 to 24, wherein the antibody or antigen-binding fragment binds with an equilibrium dissociation constant (K D ) that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, or at least 245-fold lower than the K D for binding to (i) the α-1,6-core-fucosylated AFP or the partial AFP sequence containing α-1,6-core-fucosylation and (ii) the AFP lacking an α-1,6-core-fucose residue or the partial sequence of AFP lacking an α-1,6-core-fucose residue, and the binding affinities for (i) and (ii) are determined under the same conditions.

[0336] 26. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 25, wherein the antibody or antigen-binding fragment distinguishes between (i) the α-1,6-core-fucosylated AFP or a partial sequence of AFP containing α-1,6-core-fucosylation and (ii) the α-1,6-core-fucosylated glycan of formula (IV).

Chemical formula

[0337] 27. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 25, wherein the antibody or antigen-binding fragment binds to (i) the α-1,6-core-fucosylated AFP or the partial AFP sequence containing α-1,6-core-fucosylation and (ii) the α-1,6-core-fucosylated glycan of formula (IV) with an equilibrium dissociation constant (K D at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, or at least 245-fold lower than said K D which binds and wherein the binding affinity for (i) and (ii) is determined under the same conditions, the monoclonal antibody or antigen-binding fragment according to item 26.

[0338] 28. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 27, wherein the antibody or antigen-binding fragment discriminates between (i) said α-1,6-core-fucosylated AFP, or said partial sequence of AFP containing α-1,6-core-fucosylation, and (ii) the AFP peptide of SEQ ID NO: 2 or 25.

[0339] 29. The antibody or antigen-binding fragment, for the binding of (i) said α-1,6-core-fucosylated AFP, or a partial AFP sequence containing α-1,6-core-fucosylation, to (ii) the AFP peptide of SEQ ID NO: 2 or 25, has an equilibrium dissociation constant (K D at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or at least 5000-fold lower than said K D which binds and wherein the binding affinity for (i) and (ii) is determined under the same conditions, the monoclonal antibody or antigen-binding fragment according to item 28.

[0340] 30. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 29, wherein the antibody or antigen-binding fragment discriminates between the glycopeptide according to formula I and both the glycopeptide of formula III and the core-fucosylated glycan of formula IV.

[0341] 31. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 30, wherein the antibody or antigen-binding fragment discriminates between the glycopeptide according to formula I and all three of the following: the glycopeptide of formula III, the core-fucosylated glycan of formula IV, and the AFP peptide of SEQ ID NO: 2 or 25.

[0342] 32. The sugar peptide of formula I binds with a K of 100 nM or less, 20 nM or less, preferably 10 nM or less, more preferably 3.1 nM or less or 2.5 nM or less D (Optionally, when the K D is measured by the affinity in surface plasmon resonance spectroscopy, 0.9 nM or less or 0.4 nM or less), and optionally the K D is measured at 37°C, the monoclonal antibody or antigen-binding fragment according to any one of items 1 to 31.

[0343] 33. The association rate constant k for the sugar peptide of formula I a is at least 2.0×10 4 M -1 s -1 , in an embodiment at least 10 5 M -1 s -1 , in an embodiment at least 2.5×10 5 M -1 s -1 , in an embodiment at least 6.0×10 5 M -1 s -1 , in an embodiment at least 1.0×10 6 M -1 s -1 and optionally the k a is measured at 37°C, the monoclonal antibody or antigen-binding fragment antibody according to any one of items 1 to 32.

[0344] 34. The dissociation rate constant k for the sugar peptide of formula I d is at most 1.2×10 -2 s -1 , at most 8.0×10 -3 s -1 , at most 7.3×10 -3 s -1 , at most 3.2×10 -3 s -1 or at most 1.5×10 -3 s -1 and optionally the k d A monoclonal antibody or antigen-binding fragment antibody according to any one of items 1 to 33, which is measured at 37°C.

[0345] 35. (When referring to one or more K D values and / or k a values and / or k d values) said K D values and / or k a values and / or k d values (plural possible) are a monoclonal antibody or antigen-binding fragment according to any one of items 1 to 34, determined by surface plasmon resonance spectroscopy.

[0346] 36. The surface plasmon resonance spectroscopy includes capturing the monoclonal antibody or antigen-binding fragment on a C1 sensor chip and injecting each of the sugar peptides or peptides (e.g., the sugar peptide of formula I) as an analyte, and optionally the determination is performed at a temperature of 37°C, optionally using HBS-ET pH 7.4 (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% w / v Tween 20 (registered trademark)) as the system buffer, and a system buffer supplemented with 1 mg / ml carboxymethyl dextran as the sample buffer, for the monoclonal antibody or antigen-binding fragment according to item 35.

[0347] 37. The K D values and / or k a values and / or k d values (plural possible) are determined at 37°C for the monoclonal antibody or antigen-binding fragment according to item 35 or 36

[0348] 38. The surface plasmon resonance spectroscopy is performed using a Biacore T200 or 8K instrument for the monoclonal antibody or antigen-binding fragment according to any one of items 35 to 37.

[0349] Said K D and / or said k a The determination involves optionally fitting surface plasmon resonance data using a Langmuir fitting model to R max A monoclonal antibody or antigen-binding fragment according to any one of items 35 to 38, which comprises globally fitting.

[0350] 40. The monoclonal antibody or antigen-binding fragment according to any one of item 35, wherein the K D is determined by the affinity in the affinity in solution method.

[0351] 41. The monoclonal antibody or antigen-binding fragment according to item 40, wherein the affinity in solution method comprises competition regarding the binding of the antibody or antigen-binding fragment to an analyte bound to a chip and the same analyte in solution.

[0352] 42. The K of a rabbit IgG antibody comprising the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 13 for the glycopeptide of formula I D is less than 10-fold, less than 8-fold, less than 6-fold, less than 4-fold, or less than 2-fold of the K D that binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 41.

[0353] 43. The K of a rabbit IgG antibody comprising the heavy chain variable domain of SEQ ID NO: 12 and the light chain variable domain of SEQ ID NO: 14 for the glycopeptide of formula I D is less than 10-fold, less than 8-fold, less than 6-fold, less than 4-fold, or less than 2-fold of the K D that binds to the glycopeptide of formula I, and the K D value is measured using the same method under the same conditions. A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 42.

[0354] A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 43, which binds to α-1,6-core-fucosylated alpha-fetoprotein (AFP) pretreated with the pretreating agent according to any one of 44.74 to 78.

[0355] A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 43, which binds better to α-1,6-core-fucosylated alpha-fetoprotein (AFP) pretreated with the pretreating agent according to any one of 45.74 to 78 than without the pretreatment.

[0356] 46. A polynucleotide or set of polynucleotides, (i) the heavy chain or heavy chain variable domain of the monoclonal antibody or antigen-binding fragment according to any one of items 1 to 45, and / or (ii) the light chain or light chain variable domain of the monoclonal antibody or antigen-binding fragment according to any one of items 1 to 45 A polynucleotide or set of polynucleotides encoding the same.

[0357] 47. A vector comprising the polynucleotide or set of polynucleotides according to item 46.

[0358] 48. A host cell comprising the polynucleotide or set of polynucleotides according to item 46, or the vector according to item 47.

[0359] 49. The host cell according to item 48, which is a prokaryotic cell or a eukaryotic cell.

[0360] 50. The host cell according to item 48, which is a eukaryotic cell and the cell is a CHO cell.

[0361] A method for producing a monoclonal antibody or antigen-binding fragment according to any one of Items 1 to 45, comprising culturing a host cell according to any one of Items 48 to 50, and isolating said antibody or antigen-binding fragment.

[0362] 52. An antibody according to any one of Items 1 to 46, obtainable by the method according to Item 51.

[0363] 53. A composition comprising an antibody or antigen-binding fragment according to any one of Items 1 to 45 and 52, a polynucleotide or set of polynucleotides according to Item 46, a vector according to Item 47, or a host cell according to any one of Items 48 to 50.

[0364] 54. A composition comprising an antibody or antigen-binding fragment according to any one of Items 1 to 45 and 52, which is a diagnostic composition.

[0365] 55. Use of an antibody or antigen-binding fragment according to any one of Items 1 to 45 and 52, or a composition according to Item 53 or 54, for an in vitro immunoassay, particularly for an in vitro immunoassay for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP) or AFP-L3.

[0366] 56. Use according to Item 55, wherein said immunoassay is a heterologous immunoassay.

[0367] 57. Use according to Item 55, wherein the immunoassay is an immunohistochemistry (IHC) assay.

[0368] 58. Use according to any one of Items 55 to 57, wherein the sample for said immunoassay consists of or is prepared from blood, plasma or serum.

[0369] Use according to any one of items 55 to 58 for detecting α-1,6-core-fucosylated AFP or a partial sequence of AFP containing said α-1,6-core-fucosylation.

[0370] 60. Use according to any one of items 55 to 59, wherein the immunoassay is an immunoassay for detecting a glycopeptide of formula I or a glycoprotein containing a glycopeptide of formula I.

[0371] 61. Use of an antibody according to any one of items 55 to 60 for distinguishing α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation from (i) AFP or a partial AFP sequence lacking α-1,6-core-fucosylation, and / or (ii) an α-1,6-core-fucosylated protein other than AFP.

[0372] 62. An in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation in a sample using an antibody or antigen-binding fragment according to any one of items 1 to 45 and 52.

[0373] 63. A method according to item 62, comprising: (i) binding an antibody or antigen-binding fragment according to any one of items 1 to 45 and 52 to α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation contained in the sample so as to form a detection complex; and (ii) detecting the detection complex, thereby determining the presence and optionally the amount of α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation in the sample.

[0374] 64. The method according to item 63, wherein the method is an IHC assay and the sample is a tissue slide.

[0375] 65. The method according to item 63, wherein the method is a serum immunoassay and the sample is a body fluid.

[0376] 66. The method according to item 65, wherein the body fluid is a blood sample, semen or urine.

[0377] 67. The method according to item 65 or 66, wherein the body fluid is a blood sample that is whole blood, capillary blood, serum or plasma (preferably serum or plasma).

[0378] 68. The method according to any one of items 65 to 67, comprising: (i) pretreating the sample with a pretreatment agent; and (ii) incubating the pretreated sample with an antibody or antigen-binding fragment described in any one of items 1 to 45 and 52.

[0379] 69. The method according to item 68, wherein the pretreatment agent is a pretreatment reagent described in any one of items 74 to 78.

[0380] 70. The method according to any one of items 63 to 69, wherein the method distinguishes α-1,6-core-fucosylated AFP or a partial AFP sequence containing the α-1,6-core-fucosylation from AFP lacking α-1,6-core-fucosylation or a partial sequence thereof lacking α-1,6-core-fucosylation.

[0381] 71. The method according to any one of items 65 to 70, wherein the method is a sandwich immunoassay and comprises incubating the sample with an AFP-specific antibody or antigen-binding fragment that does not compete for binding with the antibodies described in items 1 to 45 and 52 and binds to AFP independently of the α-1,6-core-fucosylation.

[0382] 72. The method according to item 71, wherein the AFP-specific antibody is anti-AFP (Tu-11).

[0383] 73. The method according to any one of items 63 to 72, wherein the method is for detecting or assisting in the detection of hepatocellular carcinoma, in an embodiment, early hepatocellular carcinoma.

[0384] 74. A pretreatment reagent for treating a sample containing α-1,6-core-fucosylated AFP, wherein the pretreatment reagent contains a reducing agent, particularly a protein reducing agent.

[0385] 75. The pretreatment reagent according to item 74, wherein the reducing agent is selected from the group consisting of dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), β-mercaptoethanol, and dibutylamine dithiol (DTBA).

[0386] 76. The pretreatment reagent according to item 74 or 75, wherein the reducing agent is DTT.

[0387] 77. The pretreatment reagent according to any one of items 74 to 76, wherein the pretreatment agent further contains a chelating agent, in an embodiment, a chelating agent for ions, particularly a chelating agent for divalent ions, and in an embodiment, a chelating agent selected from ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), porphyrin, polyamine, crown ether, or ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA).

[0388] 78. The pretreatment reagent according to any one of items 74 to 77, wherein the pretreatment agent further contains a buffer solution such as Tris.

[0389] 79. A kit containing the antibody or antigen-binding fragment according to any one of items 1 to 45 and 52.

[0390] 80. The kit according to item 79, which is an immunoassay kit.

[0391] 81. The kit according to item 79 or 80, which is for detecting or quantifying α-1,6-core-fucosylated AFP or a partial AFP sequence containing said α-1,6-core-fucosylation.

[0392] 82. The kit according to any one of items 79 to 81, wherein the kit contains a pretreatment agent, for example, the pretreatment agent according to any one of items 74 to 78.

[0393] 83. The kit according to item 82, wherein the antibody or antigen-binding fragment and the pretreatment reagent are provided in separate containers.

[0394] The following definitions and embodiments apply to all aspects and embodiments of the present invention provided above and in the claims.

[0395] As is known in the art, human AFP is a glycoprotein having the amino acid sequence of SEQ ID NO: 1 (or its natural variant described in Uniprot ID P02771 (version 209)), and contains a single N-glycosylation site corresponding to Asn-251 of Uniprot ID P02771. The N-glycan of Asn-251 can contain a core fucose residue (see Figure 1). As further understood in the art, the term "core fucosylation" within a glycan indicates that a fucose residue is α-1,6-linked to a core GlcNac residue bound to Asn-251 of the AFP protein or to a partial sequence thereof containing Asn corresponding to Asn-251 (see Figure 1). The terms "core-fucosylation" and "α-1,6-core-fucosylation" are recognized as interchangeable. Thus, the term "specific (or specifically binding) to core-fucosylated AFP and / or a partial AFP sequence containing core-fucosylation" is interchangeable with the term "specific (or specifically binding) to α-1,6-core-fucosylated AFP and its partial sequence containing α-1,6-core-fucosylation". The antibodies and antibody antigen-binding fragments of the present invention are also interchangeably referred to herein as 1,6fucAFP antibodies and their antigen-binding fragments.

[0396] Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the sugar chain structures of glycoproteins, it is possible to separate and concentrate marker glycoprotein fraction(s) having specific sugar chain structures. In the case of AFP, lectins derived from Lens culinaris agglutinin-A (LCA) are widely used. Using LCA, AFP can be fractionated into three variants L1, L2, and L3, and AFP-L3 has the highest affinity for LCA. The AFP-L3 fraction is composed of AFP, and AFP is N-glycosylated at Asn-251 using an N-glycan containing α-1,6-core-fucosylated (i.e., AFP in which a fucose sugar is bound to N-acetylglucosamine (GlcNAc) located via an α-1,6 bond at the reducing end of the N-type sugar chain). Therefore, since AFP-L3 is composed of α-1,6-core-fucosylated AFP, the terms AFP-L3 and α-1,6-core-fucosylated AFP are used interchangeably herein. The Lens culinaris agglutinin (LCA) reactive fraction of α-fetoprotein (AFP-L3) specifically increases in HCC patients (Khien VV et al., The International Journal of Biological Markers. 2001;16(2):105-111).

[0397] As used herein, the terms "antibody," "antibodies," and similar terms refer to complete immunoglobulin molecules, antibodies in their naturally occurring forms (including, but not limited to, IgG, IgA, IgM, IgE), as well as recombinant antibody constructs including, but not limited to, single-chain antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, multispecific antibodies, and multivalent antibodies, and all antigen-binding fragments and derivatives thereof as described above. As used herein, the terms "antibody," "antibodies," and similar terms also refer to their antigen-binding fragments when not explicitly mentioned otherwise. Antigen-binding fragments of antibodies may be referred to herein as antibody antigen-binding fragments and / or simply antigen-binding fragments. These terms refer to one or more fragments of an antibody that retain the ability to specifically bind to a target antigen, i.e., α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., the glycopeptides of Formula I or Formula II), and as is known in the art, include antigen-binding fragments such as the Fv domain (i.e., paired heavy-chain variable domain and light-chain variable domain), e.g., Fab, Fab’, F(ab’) 2 and antigen-binding fragments including Fv fragments, as well as recombinant constructs such as single-chain Fv domains known in the art as scFv. This term also includes antibody antigen-binding fragments containing a single unpaired heavy-chain or light-chain variable domain known in the art that retain the ability to specifically and selectively bind to an antigen as defined herein, including, but not limited to, single-domain antibodies based on camelid heavy chains (also referred to in the art as sdAb, dAb, and / or nanobody) and V H H domains are included.

[0398] In certain embodiments, the monoclonal antibodies of the invention can be complete immunoglobulins, Fab, Fab’, F(ab’) 2 , Fv or scFv. In a specific embodiment, the monoclonal antibodies of the invention can be Fab fragments.

[0399] In certain embodiments, the monoclonal antibodies of the invention can be multivalent antibodies.

[0400] As used herein, "multivalent antibody" refers to an antibody that includes at least three Fv or Fab domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies). In preferred embodiments, the multivalent antibody has at least three, at least four, at least five, at least six, at least seven, at least eight, or at least ten copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies) of the same F V and contains.

[0401] Exemplary but non-limiting embodiments for multivalent antibodies and methods for making such antibodies are disclosed in International Publication No. WO 2019 / 057816, which is hereby incorporated by reference in its entirety. Specifically, all embodiments regarding the structural composition of such multivalent antibodies and methods for making such multivalent antibodies (also referred to as p3, p4, p5, p6, p7, or p8) are hereby incorporated by reference.

[0402] In embodiments, the multivalent antibodies of the invention can include a heavy chain that includes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies, 8 copies in certain embodiments) of VH-CH1 domains (e.g., each containing the (preferably the same) VH of the 1,6fucAFP antibody or antigen-binding fragment of the invention) adjacent to, for example, a linker sequence (e.g., one of the linker sequences described in International Publication No. WO 2019 / 057816, which is hereby incorporated by reference). The additional VH-CH1 domains compared to conventional antibodies can be located upstream and / or downstream of the hinge-CH2-CH3 sequence. The light chain in such multimeric antibodies can be a conventional light chain consisting of a VL and a constant domain.

[0403] Alternative methods for making multivalent antibodies include chemical polymerization / cross-linking of antibodies or antigen-binding fragments.

[0404] The antibody can be polyclonal or monoclonal. The antibodies of the present invention are monoclonal. As used herein with respect to an antibody or antigen-binding fragment thereof, the term "monoclonal" refers to a population of antibody polypeptides or fragments thereof produced from a single B cell clone, the population of which contains only one specificity of antigen-binding site capable of immunoreacting with a particular epitope of an antigen. This is in contrast to "polyclonal" antibodies and compositions, which is a term (s) referring to a population of antibody polypeptides or antigen-binding fragments that contain antigen-binding sites of multiple specificities. Also included are modified forms of the monoclonal antibodies of the present invention, such as humanized or chimeric versions thereof, and recombinant antibody constructs, such as antibody (or antigen-binding fragment) fusion proteins, where the antibody or antigen-binding fragment contains additional domain (s) for, e.g., isolation and / or preparation of the recombinantly produced antibody / fragment / construct.

[0405] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or antibody light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (VH and VL, respectively) generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three hypervariable regions (HVRs), e.g., complementarity determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single VH domain or VL domain may be sufficient to confer antigen-binding specificity. Further, an antibody that binds a particular antigen can be isolated using the VH domain or VL domain of the antibody that binds the antigen, and libraries of complementary VL or VH domains, respectively, can be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0406] As used herein, the terms "hypervariable region" or "HVR" refer to each region of an antibody variable domain where the sequence is hypervariable and which determines antigen-binding specificity, e.g., "complementary determining regions" (CDRs).

[0407] Generally, an antibody contains six CDRs, three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2) and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)).

[0408] Unless otherwise specified, CDRs are determined according to Kabat et al. above. Those skilled in the art will understand that the CDR notations can be determined according to Chothia above, MacCallum above, or any other scientifically approved nomenclature system.

[0409] "Framework" or "FR" refers to variable domain residues other than the complementarity determining regions (CDRs). The FRs of the variable domain generally consist of four FR domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR sequences generally occur in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0410] As used herein, the phrase "specifically binds" in connection with an antibody or an antibody antigen-binding fragment indicates that each antigen binds to the antibody or antibody antigen-binding fragment via an antigen-antibody reaction. The term "specifically binds" also represents that the antibody or antigen-binding fragment binds to a structure that is preferentially shown over other structures that may exhibit cross-reactivity. As also explained herein, the term to distinguish from / to indicates that the antibodies and antigen-binding fragments of the present invention specifically bind to a target antigen (i.e., core-fucosylated AFP and / or its core-fucosylated partial sequence, most preferably the glycopeptide of formula I or II), but do not specifically bind to an AFP / AFP partial sequence lacking a core-fucose residue and / or a core-fucosylated glycan in another situation such as a single core-fucosylated asparagine shown in formula IV.

[0411] As used herein, the term "distinguish from / over" and similar terms regarding two antigens, e.g., an antibody distinguishes antigen X from / over antigen Y, indicates that the antibody or antigen-binding fragment specifically binds to the target antigen X but does not specifically bind to the non-target antigen Y. Thus, as used herein, the term "distinguish" and similar terms mean that an antibody or antigen-binding fragment "does not specifically bind" / "does not significantly bind" (these are used interchangeably) to non-target antigens. The terms "specifically bind" and "does not significantly bind" are well known in the art to represent the degree to which an antibody distinguishes between two antigens. This is because it is known that antibodies do not have absolute specificity in that they react with only one epitope under any conditions. That is, in the presence of other (non-target) antigens, an antibody or antigen-binding domain can react to some extent with similar epitopes on these other (non-target) antigens. However, the affinity of a monoclonal antibody or monoclonal antigen-binding fragment for its target epitope / antigen is significantly greater than its affinity for related epitopes. This difference in affinity is used to establish assay conditions under which an antibody or antigen-binding fragment binds almost exclusively to a particular epitope. In this regard, the binding (or non-binding) of an antibody or antigen-binding fragment to an antigen is not understood as absolute. That is, the 1,6fucAFP antibody and / or antigen-binding fragment may exhibit some (residual) binding activity to other (non)-targets, but at a level significantly lower compared to the binding activity to core-fucosylated AFP or the core-fucosylated partial sequence of AFP, preferably the glycopeptide of formula I or the glycoprotein containing the glycopeptide of formula I.

[0412] For example, the characteristic of distinguishing a target antigen from / over a non-target antigen may be characterized by a 1,6fucAFP antibody or antibody antigen-binding fragment having an affinity for the target antigen that is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold, or at least 245-fold superior to its affinity for the non-target antigen (i.e., the K of binding to the target antigen D is at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 150-fold, at least 180-fold or at least 245-fold lower than the KD of the binding to the non-target antigen). The formulation is at least "XX", the K for the non-target antigen D is very high and embodiments where it cannot be detected by the method used are also included. Thus, whether an antibody or antigen-binding fragment can distinguish a target structure from a non-target structure is determined by the same method (e.g., K as described below herein D for determining) for each binding to determine the K D value.

[0413] In embodiments, the ability of an antibody to distinguish a target antigen from a non-target antigen / target antigen can be evaluated using an immunoassay in which the binding of the antibody or antigen-binding fragment being tested to the target structure is detected. Using such an immunoassay, the immunoassay signal obtained in a first sample containing a defined concentration of the target antigen can be compared with the immunoassay signal obtained in a second sample containing the same concentration of the non-target antigen. That the immunoassay signal of the first sample is higher than the immunoassay signal from the second sample indicates discrimination between the target antigen and the non-target antigen. In embodiments, the antibody or antigen-binding fragment being tested can distinguish the target structure from the non-target structure if the immunoassay signal of the first sample is at least 5-fold, at least 10-fold, at least 20-fold, at least 40-fold, at least 50-fold or at least 100-fold higher than that of the second sample. Exemplary but non-limiting immunoassays that can be used for such analysis are provided in Example 7. Exemplary concentrations of the target antigen and the non-target antigen can be 12 nm.

[0414] When used herein in connection with amino acids, the terms "substitute", "exchange" or "mutate" refer to replacing an amino acid with another amino acid. Deletion of an amino acid at a particular position and insertion of one (or more) amino acids at different positions are not explicitly encompassed by the term "substitute". As stated, the present invention encompasses conservative or highly conservative amino acid substitutions as defined above herein.

[0415] Amino acids are herein spelled or abbreviated using the one-letter code or the three-letter code.

[0416] In the context of the present invention, it refers to variants of sequences (especially CDRs). These variants typically contain one or more amino acid substitutions. It is clear that the variant CDRs are functional variants, i.e., they may differ from the reference amino acid sequence, but the different sequences have an amino acid sequence that exhibits or maintains the same functional activity as the reference sequence in the context of the described heavy and / or light chain variable domains. Specifically, as used herein, the term same functional activity means that an antibody or antibody binding fragment of the present invention comprising one or more variant CDRs has specific binding to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing said α-1,6-core-fucosylation (e.g., a glycopeptide of formula I or formula II), and maintains the property of distinguishing these structures from non-α-1,6-core-fucosylated AFP or partial sequences (e.g., a glycopeptide of formula III or a peptide of SEQ ID NO: 2 or 25) and / or a glycan of formula IV. In embodiments, functional activity may also mean that the kinetic parameters referred to herein for binding to the glycopeptide of formula I are conserved.

[0417] When used in the context of the present invention, "conservative amino acid substitution" means substitution of an amino acid with another amino acid selected from the same physicochemical group, and the physicochemical groups of amino acids are as follows. a) Non-polar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met; b) Polar neutral amino acids consisting of Ser, Thr, Asn and Gln; c) Basic amino acids with a positive charge, consisting of Arg, Lys and His, and d) Acidic amino acids with a negative charge, consisting of Asp and Glu, and When Cys is conservatively substituted, it is substituted with Ser or Ala, and when Pro is conservatively substituted, it is substituted with Ala.

[0418] When used in the context of the present invention, "highly conserved amino acid substitution" means the following amino acid substitutions: a) Substitution of Ala with Val, Leu, Ile or Gly; b) Substitution of Arg with Lys; c) Substitution of Asn with Gln; d) Substitution of Asp with Glu; e) Substitution of Cys with Ser; f) Substitution of Gln with Asn; g) Substitution of Glu with Asp; h) Substitution of Gly with Ala; i) Substitution of His with Arg; j) Substitution of Ile with Leu, Val or Ala; k) Substitution of Leu with Ile, Val or Ala; l) Substitution of Lys with Arg; m) Substitution of Met with Leu, Ile or Val; n) Substitution of Phe with Tyr or Trp; o) Substitution of Pro with Ala; p) Substitution of Ser with Thr; q) Substitution of Thr with Ser; r) Substitution of Trp with Phe or Tyr; s) Substitution of Tyr with Phe or Trp; and t) Substitution of Val with Leu, Ile or Ala.

[0419] As used herein, the term "percent sequence identity" in relation to an amino acid sequence and / or nucleic acid sequence or nucleic acid molecule of a polypeptide / peptide refers to the number of matching identical amino acids or nucleic acid residues compared to the number of residues that make up the full length of the sequences being compared (or the portion thereof being compared in its entirety) of two or more aligned sequences. The percentage of residues that are identical is determined using an alignment of two or more sequences or subsequences, by comparing the (sub)sequences over a comparison window or over a specified region measured using sequence comparison algorithms known in the art, and aligning for maximum match, or by aligning manually and visual inspection. Non-limiting examples of algorithms for use in determining sequence identity include, for example, those based on the NCBI BLAST algorithm (Altschul et al., Nucleic Acids Res 25(1997), 3389-3402), the CLUSTALW computer program (Thompson, Nucl.Acids Res.2(1994), 4673-4680) or FASTA (Pearson and Lipman, Proc.Natl.Acad.Sci., 85(1988), 2444). The FASTA algorithm typically does not consider internal non-matching deletions or additions, i.e., gaps, in the sequence in its calculations, although this can be corrected manually to avoid overestimation of % sequence identity. However, CLUSTALW considers sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul et al., Nucl Acids Res., 25(1977), 3389) are also available.

[0420] In connection with the present invention, glycopeptides of AFP (e.g., Formula I, Formula II, Formula III) and N-glycan structures (e.g., Formula IV) are disclosed. In each of the formulas described hereinabove, the type of bond is not indicated. Preferred bonds are those shown in the exemplary glycan structures shown in FIG. 1.

[0421] Thus, in certain embodiments, the glycopeptide of formula I may have the following structure Ia.

Chemical formula

[0422] Thus, in certain embodiments, the glycopeptide of formula II may have the following structure IIa.

Chemical formula

[0423] Thus, in certain embodiments, the glycopeptide of formula III may have the following structure IIIa.

Chemical formula

[0424] Thus, in certain embodiments, the N-glycan of formula IV may have the following structure IVa.

Chemical formula

[0425] As used herein, the terms "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and like terms include both genomic DNA and cDNA, as well as RNA that can drive the expression of an antibody or antigen-binding fragment of the invention. As used herein, the term "RNA" includes mRNA, tRNA and rRNA, and is understood to include all forms of RNA, including genomic RNA, such as in the case of the RNA of an RNA virus. Preferably, embodiments described as "RNA" relate to mRNA. The nucleic acid molecules / nucleic acid sequences of the invention can be of natural as well as synthetic or semi-synthetic origin. In embodiments, the nucleic acids / nucleic acid sequences of the invention can be isolated. Thus, a nucleic acid molecule can be, for example, a nucleic acid molecule synthesized according to conventional protocols of organic chemistry, synthesized according to recombinant methods, or produced semi-synthetically, for example by combining chemical synthesis and recombinant methods. Those skilled in the art are proficient in the preparation and use of such nucleic acid molecules.

[0426] As used herein, "immunoassay" is a well-established biological assay method in which the detection or quantification of an analyte relies on the reaction of the analyte with at least one analyte-specific binding agent, thus forming an analyte:binding agent complex (also referred to as a detection complex). In the context of the present invention, at least one of the at least one analyte-specific binding agents is an antibody of the present invention. Specific embodiments of "sandwich" immunoassays can be used for analytes having multiple recognition epitopes. Thus, a sandwich assay requires at least two binding agents that attach to non-overlapping epitopes on the analyte. In a "heterogeneous sandwich immunoassay", one of the binding agents has the functional role of an analyte-specific capture binding agent, which is immobilized on a solid phase or (during the course of the assay) becomes immobilized on the solid phase. The second analyte-specific binding agent is supplied in dissolved form in the liquid phase. When each analyte is bound by the first and second binding agents, a sandwich-like complex (binding agent-1:analyte:binding agent-2) is formed. The sandwich-like complex is also referred to as a "detection complex". Within the detection complex, the analyte is sandwiched between the binding agents, i.e., in such a complex, the analyte represents the connecting element between the first binding agent and the second binding agent.

[0427] (As opposed to "homogeneous") The term "heterogeneous" indicates two essential and separate steps in the assay procedure. In the first step, a detection complex containing a label is formed and immobilized, but unbound label still surrounds the complex. The unbound label is removed from the immobilized detection complex before determining the label-dependent signal, corresponding to the second step. In contrast, a homogeneous assay generates an analyte-dependent detectable signal by single-step incubation and does not require a washing step.

[0428] In a heterogeneous immunoassay, the solid phase is functionalized so that a functional capture binder (first binder) can bind to its surface before contacting the analyte, or the surface of the solid phase is functionalized so that it can tether the first binder after reacting with the analyte. In the latter case, the tethering process must not interfere with the ability of the binder to specifically capture and bind the analyte. A second binder present in the liquid phase is used to detect the bound analyte. Thus, in a heterogeneous immunoassay, the analyte is bound to a first binder (capture) and a second binder (detector). This forms a "detection complex" in which the analyte is sandwiched between the capture binder and the detector binder. In a typical embodiment, the detector binder is labeled before contacting the analyte, or a label is specifically attached to the detector binder after analyte binding. When the detection complex is immobilized on the solid phase, the amount of the label detectable on the solid phase corresponds to the amount of the sandwiched analyte. After removing the unbound label, the immobilized label indicating the presence and amount of the analyte can be detected.

[0429] As used herein, a "competitive immunoassay" preferably uses a single binder that directly interacts with the analyte. A "competitive heterogeneous immunoassay" typically detects a signal of a detection label that is inversely proportional to the amount of analyte in the sample.

[0430] As used herein, a "detectable label" relates to a label that enables detection. According to one embodiment of the present invention, the detectable label is an enzyme, or in one embodiment, a label that emits fluorescence, luminescence, chemiluminescence, electrochemiluminescence or radioactivity. In a preferred embodiment, the label is an electrochemiluminescence label, which is tris(2,2'-bipyridyl)ruthenium(II) complex (Ru(bpy)) in one embodiment. Since interference is caused by the three-dimensional structure of the label molecule that attracts autoantibodies and similar interfering molecules, and not by the signal emission mechanism of the label such as light or radioactivity, all of the above labels can be used in the present invention.

[0431] As used herein, "capture label" relates to a label that can immobilize a capture agent (e.g., an antibody bearing the capture label) on a surface (e.g., on magnetic particles such as microbeads). Non-limiting examples are members of a binding pair. Non-limiting examples of capture labels are biotin or its derivatives, which can interact with streptavidin or its derivatives. Various capture labels are well known in the art.

[0432] The term "and / or" is to be understood as meaning either one or both of the alternatives.

[0433] As used herein, unless otherwise specified, the term "about" is to be understood as synonymous with the term "approximately". By way of example, unless otherwise specified, the use of the term "about" when used in combination with a recited numerical value or range indicates being somewhat greater or somewhat less than the recited value or range, and is within ±15% of the recited value or range, ±10% of the recited value, ±5% of the recited value, or ±2% of the recited value for convenience. Thus, such values are encompassed in the claims that recite the term "about" or "approximately".

[0434] As used herein, the terms "biomarker" or "marker" generally refer to a gene, protein, carbohydrate structure, or glycolipid, metabolite, mRNA, miRNA, protein, DNA (cDNA or genomic DNA), molecule including DNA copy number, or epigenetic change, such as an increase, decrease or change in DNA methylation (e.g., cytosine methylation, or CpG methylation, non-CpG methylation); histone modification (e.g., (de)acetylation, (de)methylation, (de)phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation); change in nucleosome arrangement, and their expression or presence in or on mammalian tissues or cells can be detected by standard methods (or methods disclosed herein), which can be predictive, diagnostic and / or prognostic of an individual's health or disease. Thus, hereinafter in this specification, the more general term "marker" may also be used when explaining more general terms and definitions. The term marker also includes glycan structures or glycans, or glycopeptides analyzed in the present disclosure.

[0435] The term "in vitro method" is used to indicate that this method is performed outside of a living organism, preferably on body fluids, isolated tissues, organs or cells. In vitro methods are sometimes also referred to as ex vivo methods.

[0436] Hepatocellular carcinoma (HCC) is the main histological type among primary liver cancers occurring worldwide, accounting for 70% - 85% of the total burden. It is known that underlying liver diseases such as hepatic fibrosis and cirrhosis are the main risk factors for the development of HCC. HCC can be treated by resection, liver transplantation, or local ablation using high frequency for patients diagnosed early. If this malignant tumor is diagnosed early, the 5-year survival rate of HCC patients can be as high as 70%. However, the 5-year survival rate of HCC patients significantly decreases as the diagnosis of the disease is delayed, and it drops to only 15% when HCC is diagnosed at a late stage of the disease (Tsuchiya N, Sawada Y, Endo I, et al. Biomarkers for the early diagnosis of hepatocellular carcinoma. World J Gastroenterol. 2015;21(37):10573 - 83; Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA: A Cancer Journal for Clinicians. 2013;63(1):11 - 30).

[0437] The term "assisting in the detection of hepatocellular carcinoma (HCC)" is used to indicate that the method according to the invention serves / assists, for example, a medical professional such as a doctor in evaluating whether an individual has HCC or is at risk of developing HCC. As is understood, to detect or rule out the presence of HCC, a doctor can use several alternative methods (e.g., ultrasound, radiography, MRT, or CT), and the alternative methods can be combined with in vitro biomarker data such as glycan structure data. The final diagnosis of HCC is usually made by tissue biopsy or tissue samples after surgery. The term "assisting in the detection of HCC" includes the use of this method as a single diagnostic utility or as one of multiple diagnostic utilities. The amount of the marker does not exceed the reference level in all HCC patients (100%), and the level of the marker does not fall below the reference level or cut-off level in all healthy individuals. As those skilled in the art understand, in many diseases, biochemical markers do not have 100% specificity and simultaneously 100% sensitivity. Rather, the fact that the analyzed marker or a combination of markers including this marker is at, for example, a given specificity level or a given sensitivity level indicates a certain likelihood that the individual from whom the sample was analyzed has a particular clinical condition, e.g., has HCC. Those skilled in the art are fully proficient in the mathematical / statistical methods used to calculate specificity, sensitivity, positive predictive value, negative predictive value, reference value, or total error. Any of these parameters can be calculated and used to obtain an indicator of the presence or absence of HCC.

[0438] One convenient goal for quantifying the diagnostic accuracy of laboratory tests is to represent their performance by a single number. The most common global measure is the area under the curve (AUC) of the ROC plot. The area under the ROC curve is a measure of the probability that a measured value enables correct discrimination of a state (or distinction between one state and another). The value typically ranges from 1.0 (perfect separation of the test values of two groups) to 0.5 (no clear distribution difference between the two groups of test values). The area depends not on specific parts of the plot, such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative descriptive representation of how close the ROC plot is to being perfect (area = 1.0). In the context of the present invention, the two different states can be whether or not a patient has HCC.

[0439] As used herein, the terms "subject" or "individual" refer to a single human being. The subject can be healthy or a patient, e.g., a patient who has cirrhosis, is at risk of developing HCC, and has experienced or has experienced one or more signs, symptoms, or other indicators of HCC. Intended to be included as a subject is any subject participating in a clinical research study that shows no clinical signs of any disease, or a subject participating in an epidemiological study, or a subject whose sample serves as a control. In some embodiments, the subject may be at risk of developing HCC, for example, due to chronic alcohol consumption, hepatitis B and / or C infection, non-alcoholic fatty liver disease, Wilson's disease, hereditary hemochromatosis, alpha1-antitrypsin deficiency, primary biliary cirrhosis, autoimmune hepatitis, and other risk factors. Other risk factors may include obesity and / or liver transplantation. In embodiments, the subject may be known to be at risk of developing HCC, for example, via hepatic fibrosis, non-cirrhotic liver disease, NASH, chronic HBV, and chronic HCV.

[0440] In one embodiment according to the present disclosure, the subject from whom the sample to be investigated is obtained is a healthy subject and is screened for the presence of HCC as part of routine oncological surveillance.

[0441] In one embodiment according to the present disclosure, the subject from whom the sample to be investigated is obtained is a subject at risk of developing HCC and is screened for the presence of HCC as part of routine oncological surveillance.

[0442] If it is known that the subject suffers from chronic liver disease, viral or non-viral hepatitis and / or cirrhosis, the subject may be at risk of developing HCC.

[0443] In one embodiment according to the present disclosure, the subject from whom the sample to be investigated is obtained has chronic liver disease, viral or non-viral hepatitis, cirrhosis, and is used for differential diagnosis of the presence or absence of HCC.

[0444] As used in connection with the present disclosure, a "sample" can be a liquid sample that contains, or is expected to contain, α-1,6-core-fucosylated AFP, or a subsequence thereof that includes α-1,6-core-fucosylation. The sample can be, without particular limitation, a body fluid such as a blood sample, cerebrospinal fluid, semen, saliva, or urine. In a preferred embodiment, the sample is a blood sample such as whole blood, serum, or plasma. In an even more preferred embodiment, the sample is serum or plasma.

[0445] As used herein, the terms "amount" or "level" of an analyte in a sample refer to any absolute scale that corresponds to or is proportional to the amount or concentration of the analyte in the sample, or any relative scale, i.e., a scale representing the amount or concentration relative to the reference amount or concentration of the analyte, respectively.

[0446] As used herein, the term "reference amount" (or "reference level") with respect to an analyte (e.g., a glycan structure or a glycopeptide) refers to a predetermined amount of the analyte that has been independently established. One of ordinary skill in the art will understand that the reference amount is predetermined and set to meet established requirements with respect to specificity and / or sensitivity, for example, for the purpose of detecting HCC (e.g., early HCC). Thus, the reference amount can be selected such that it indicates HCC (e.g., early HCC). The requirements for detecting HCC can vary, for example, for each regulatory agency. For example, the sensitivity or specificity of an assay may each need to be set at a particular limit, such as 80%, 90%, 95%, or 98%. These requirements may also be defined with respect to the positive predictive value or the negative predictive value. For example, for any requirement selected with respect to the level of sensitivity or specificity, the reference range (when the evaluated, decreased value indicates an abnormal condition) or the reference level or cutoff level (when the evaluated or decreased value indicates an abnormal condition) can be determined by one of ordinary skill in the art. Similarly, a reference ratio can be determined according to the same principle.

[0447] As used herein, the term "reference value" in relation to, for example, a reference value of a score, relates to a predetermined value independently established for each parameter (e.g., a score). One of ordinary skill in the art will understand that the reference value is predetermined and set to meet established requirements with respect to specificity and / or sensitivity, for example, for the purpose of detecting HCC (e.g., early HCC). Thus, the reference value can be selected such that it indicates HCC (e.g., early HCC). What has been described above with respect to the reference amount applies mutatis mutandis.

[0448] The terms "glycan structure" or "glycan" are used interchangeably herein. In the present disclosure, the glycan under investigation is an N-glycan. Thus, the terms glycan and N-glycan herein are used interchangeably. A glycan or glycan structure consists of various types of sugar moieties. The linkages are preferably as shown in Figure 1. The N-glycan structure is attached to the side chain of the amino acid asparagine.

[0449] The term "glycopeptide" is used to refer to a peptide or peptide fragment of a larger polypeptide that includes an amino acid to which a glycan is covalently attached.

[0450] It is understood that the words "comprise", and variations such as "comprises" and "comprising", mean the inclusion of the stated integer or step or group of integers or steps, but do not mean the exclusion of any other integer or step or group of integers or steps.

[0451] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0452] Concentrations, amounts, and other numerical data may be expressed or presented herein in the form of a "range". It should be understood that such a range format is merely used for convenience and brevity, and thus, not only includes the numerical values explicitly listed as the boundaries of the range, but should be interpreted flexibly to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly listed. By way of illustration, the numerical range of "150 mg to 600 mg" should be interpreted to include not only the explicitly listed values of 150 mg to 600 mg, but also the individual values and sub-ranges within the indicated range. Thus, this numerical range includes individual values such as 150, 160, 170, 180, 190, ··· 580, 590, 600 mg, etc., and sub-ranges such as 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to ranges that list only one numerical value. Further, such an interpretation should apply regardless of the width of the range or the characteristics described.

[0453] The term "about", when used in relation to a numerical value, means a value within a range having a lower limit that is 5% less than the indicated numerical value and an upper limit that is 5% greater than the indicated numerical value.

[0454] In the foregoing detailed description of the present invention, several individual elements, features, techniques, and / or steps are disclosed. It is readily recognized that each of these, when considered or used alone, as well as when considered and used in combination with one another, has benefits. Thus, to avoid excessive repetition and redundancy, this description has avoided repeating all possible combinations and permutations. Nevertheless, it is understood that such combinations, whether or not explicitly listed, are fully within the scope of the subject matter of this disclosure.

[0455] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques herein are intended to refer to techniques commonly understood in the art, including variations of those techniques or substitutions of equivalent techniques that would be apparent to one of ordinary skill in the art.

[0456] All amino acid sequences provided herein begin with the most N-terminal residue and end with the most C-terminal residue, as is customary in the art, and the one-letter or three-letter code abbreviations used to identify amino acids throughout the present invention correspond to those commonly used for amino acids.

[0457] Numerous documents are cited herein, including patent applications and manufacturer manuals. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0458] Now, the elements of the present invention will be described. These elements are listed as aspects having specific embodiments, but it should be understood that they can be combined in any way and in any number to create additional aspects and embodiments. In particular, embodiments disclosed in the context of one aspect can be applied to other aspects with the necessary modifications. The various described examples and preferred embodiments should not be construed as limiting the present invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all the elements described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0459] Description of Sequences The following amino acid sequences are referred to in connection with the present disclosure.

[0460] SEQ ID NO:1: AFP sequence (see UniProt: P02771; version 209); Asn-251, which is a site for N-glycosylation, is underlined MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYKEVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEGRHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLWAARYDKIIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITVTKLSQKFTKV N FTEIQKLVLDVAHVHEHCCRGDVLDCLQDGEKIMSYICSQQDTLSNKITECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFNQFSSGEKNIFLASFVHEYSRRHPQLAVSVILRVAKGYQELLEKCFQTENPLECQDKGEEELQKYIQESQALAKRSCGLFQKLGEYYLQNAFLVAYTKKAPQLTSSELMAITRKMAATAATCCQLSEDKLLACGEGAADIIIGHLCIRHEMTPVNPGVGQCCTSSYANRRPCFSSLVVDETYVPPAFSDDKFIFHKDLCQAQGVALQTMKQEFLINLVKQKPQITEEQLEAVIADFSGLLEKCCQGQEQEVCFAEEGQKLISKTRAALGV

[0461] SEQ ID NO:2: Peptide sequence of AFP (from AS243 to 261 of SEQ ID NO:1); the Asn corresponding to Asn-251 is underlined LSQKFTKV N FTEIQKLVLD

[0462] SEQ ID NO:3: CDR-H1 of 19B12 and 3C5 TYGMG

[0463] CDR-H2 of SEQ ID NO: 4 19B12 IIGDNGSTYYANWA

[0464] CDR-H2 of SEQ ID NO: 5 3C5 IIDSGSTYYANWA

[0465] CDR-H3 of SEQ ID NO: 6 19B12 and 3C5 DRDPSSSGYYFKM

[0466] CDR-L1 of SEQ ID NO: 7 19B12 QASQSISSYLA

[0467] CDR-L1 of SEQ ID NO: 8 3C5 QASQSIGSYLA

[0468] CDR-L2 of SEQ ID NO: 9 19B12 and 3C5 GASNLES

[0469] CDR-L3 of SEQ ID NO: 10 19B12 and 3C5 QTAFYIFSSDNA

[0470] VH sequence of SEQ ID NO: 11 19B12 (CDRs underlined) QSVEESGGRLVAPGTPLTLTCTVSGIDLS TYGMG WVRQAPGKGLEWIG IIGDNGSTYYANWA KGRFTISKTSTTVDLKMTSLTTEDTATYFCAR DRDPSSSGYYFKM WGPGTLVTVSL

[0471] VH sequence of SEQ ID NO: 12 3C5 (CDRs underlined) QSVEESGGRLVAPGTPLTLTCTVSGIDLS TYGMG WVRQAPGKGLEYIG IIDSGSTYYAN WAKGRFTISKTSTTVDLKMTSLTTEDTATYFCAR DRDPSSSGYYFKM WGPGTLVTVSL

[0472] Sequence number 13 VL sequence 19B12 (CDR underlined) ALVMTQTPSSVSAAVGGTVTINC QASQSISSYLA WYQQKPGQPPKLLIF GASNLES GVPSRFKGSGSGTEFTLTISDLECDDAATYYC QTAFYIFSSDNA FGGGTEVVVK

[0473] Sequence number 14 VL sequence 3C5 (CDR underlined) ALVMTQTPSSVSAAVGGTVTINC QASQSIGSYLA WYQQKPGQPPRLLIY GASNLES GVPSRFKGSGSGTEFTLTISDLECDDAATYYC QTAFYIFSSDNA FGGGTEVVVK

[0474] Sequence number 15 FW1 of VH of 19B12 and 3C5 QSVEESGGRLVAPGTPLTLTCTVSGIDLS

[0475] Sequence number 16 FW2 of VH of 19B12 WVRQAPGKGLEWIG

[0476] Sequence number 17 FW2 of VH of 3C5 WVRQAPGKGLEYIG

[0477] Sequence number 18 FW3 of VH of 19B12 and 3C5 KGRFTISKTSTTVDLKMTSLTTEDTATYFCAR

[0478] Sequence number 19 FW4 of VH of 19B12 and 3C5 WGPGTLVTVSL

[0479] Sequence number 20 FW1 of VL of 19B12 and 3C5 ALVMTQTPSSVSAAVGGTVTINC

[0480] VL FW2 of SEQ ID NO: 21 19B12 WYQQKPGQPPKLLIF

[0481] VL FW2 of SEQ ID NO: 22 3C5 WYQQKPGQPPRLLIY

[0482] VL FW3 of SEQ ID NO: 23 19B12 and 3C5 GVPSRFKGSGSGTEFTLTISDLECDDAATYYC

[0483] VL FW4 of SEQ ID NO: 24 19B12 and 3C5 FGGGTEVVVK

[0484] Peptide sequence of SEQ ID NO: 25 AFP (AS 248 - 256 of AFP shown in SEQ ID NO: 1); Asn corresponding to Asn - 251 is underlined TKV N FTEIQ

[0485] Peptide sequence of SEQ ID NO: 26 AFP (AS 243 - 256 of AFP shown in SEQ ID NO: 1); Asn corresponding to Asn - 251 is underlined LSQKFTKV N FTEIQ

[0486] Exemplary linker in a multivalent antibody GGGSGGGSGGGSGGGS

Example

[0487] The following examples are provided to assist in the understanding of the present invention.

[0488] Example 1: Synthesis of Peptides for Immunization and Screening Synthesis of Glycopeptides The peptide was synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis on a peptide synthesizer (e.g., manufactured by Protein Technologies, Inc.). For amino acid coupling, 5 equivalents of each amino acid derivative were used. The amino acid derivative was dissolved in dimethylformamide containing 1 equivalent of 1-hydroxy-7-azabenzotriazole (HOAt). The peptide was synthesized on Sieber Amide resin. The coupling reaction was carried out in dimethylformamide (DMF) for 5 minutes using 5 equivalents of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and 10 equivalents of N,N-diisopropylethylamine (DIPEA) with respect to the resin loading. The Fmoc group was cleaved for 8 minutes after each synthesis step using 20% piperidine in DMF. For the synthesis of the disaccharide-containing peptide, Fmoc-protected sugar amino acid components of Asn were used (Fmoc-Asn(α-L-Fuc(Ac)3(1-6)-β-D-GlcNAc(Ac)2)-OH and Fmoc-Asn(β-GlcNAc(Ac)3β(1-4)GlcNAc(Ac)2)-OH, respectively) and coupled according to the above standard conditions. In the case of the peptide containing the complex branched glycan (G0F and GF), the phenylalanine-threonine dipeptide was used as an Fmoc-protected pseudoproline derivative and Asn was introduced as Fmoc-Asn(ODmap). The assembly of the peptide upon cleavage of the resin of Asp(ODmab) was achieved by washing the resin with 2% hydrazine in DMF (5×5 minutes), followed by treatment with 5 mM sodium hydroxide in water / methanol (1:1) for 1 hour. The release of the peptide from the synthetic resin was achieved by incubation with 1% trifluoroacetic acid in dichloromethane (10×3 minutes). Subsequently, the reaction solution was extracted with water and evaporated to dryness. The crude material was purified by flash chromatography. The identity of the purified material was analyzed by ion spray mass spectrometry.

[0489] By adding 1,3-propanedithiol (40 equivalents) and DIPEA (30 equivalents) in methanol, the bi-antennary glycosyl azides of G0F and G2 *) were reduced to the corresponding amines. After stirring for 4 hours, the sugar was precipitated by adding cold diisopropyl ether. Sugar coupling to the peptide was achieved using DMF / DMSO (1:1) containing 2 equivalents of sugar amine (G0F or G2), 2 equivalents of HATU, 2 equivalents of HOAt, and 8 equivalents of DIPEA overnight. Subsequently, cleavage of the acid-labile protecting group was achieved using 9.5 ml of trifluoroacetic acid, 0.25 ml of triisopropylsilane, and 0.25 ml of water at room temperature for 2 hours. The reaction solution was then mixed with cold diisopropyl ether to precipitate the peptide. The precipitate was filtered, washed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid solution, and lyophilized. The crude material was purified by preparative reverse-phase HPLC using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid. The identity of the purified material was analyzed by ion spray mass spectrometry. An overview of all peptides synthesized and used for immunization and screening is shown in Table 1. *) Literature for chemical synthesis of bi-antennary fucosylated N-glycosyl azides: J.Seifert, C.Unverzagt, Tetrahedron Lett. 1997, 38, 7857-7860.

[0490] Synthesis of biotinylated Asn(G0F) Asp-OBzl, biotin-PEG12-NHS ester (1 equivalent), and trimethylamine (8 equivalents) were dissolved in DMF and stirred for 2.5 hours. The crude product was purified by preparative reverse-phase HPLC using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid.

[0491] G0F-azide was reduced to the corresponding amine by adding 1,3-propanedithiol (40 equivalents) and DIPEA (30 equivalents) in methanol. After stirring for 4 hours, the sugar was precipitated by adding cold diisopropyl ether. Sugar coupling to biotin-PEG12-Asp-OBzl was achieved overnight using 0.5 equivalent of the sugar amine, 1 equivalent of HATU, 1 equivalent of HOAt, and 4 equivalents of DIPEA in DMF / DMSO (1:1). The reaction solution was then mixed with cold diisopropyl ether. The precipitate was filtered, washed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid solution, and lyophilized. The crude material was purified by preparative reverse-phase HPLC using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid. The identity of the purified material was analyzed by ion spray mass spectrometry. A schematic diagram showing the N-glycan structure of AFP at Asn-251 (N251), which highlights α-1,6-core-fucosylation, is shown in Figure 1.

[0492] Synthesis of immunogen To a solution of keyhole limpet hemocyanin (KLH) in phosphate buffer (20 mM, pH 7.2), N-hydroxysuccinimide ester of 3-(maleimido)propionic acid was added. The reaction was incubated at room temperature for 5 hours and then dialyzed against phosphate buffer (0.1 M, pH 7.0). The cysteine-containing sugar peptide was dissolved in DMSO and added to a solution of maleimide-activated KLH containing 0.1 M ethylenediaminetetraacetic acid (EDTA). The solution was incubated at room temperature for 5 hours and then dialyzed against phosphate buffer (0.1 M, pH 7.0) to obtain the KLH-peptide conjugate.

[0493] Sugar peptide and peptide [Table 1-1] [Table 1-2]

[0494] Abbreviations Ac: N-terminal acetylation Ahx: 6-aminohexanoic acid βAla: beta-alanine Bi: Biotin Fuc: L-fucose G0F: Fucosylated branched N-glycan G2: Non-fucosylated branched N-glycan GlcNAc: N-acetylglucosamine IMG: Immunogen MP: 3-(Maleimido)propionic acid -NH2: C-terminal carboxamide PEG: Polyethylene glycol

[0495] Structure of glycopeptide and peptide BMO 35.000146:

Chem.

[0496] BMO 35.000148:

Chem.

[0497] BMO 35.000151:

Chem.

[0498] BMO 35.000315:

Chem.

[0499] BMO 35.000150:

Chem.

[0500] BMO 35.000373:

Chem.

[0501] BMO 35.100001:

Chem.

[0502] BMO 35.100002:

Chem.

[0503] Example 2: Antibody Generation Immunization Twelve- to sixteen-week-old 2x2 New Zealand White (NZW) rabbits were immunized with AFP-L3 glycopeptide (AFP(243-256)-G0F-IMG). To enhance the immunogenicity of the peptide, it was conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. During the first month, the animals were immunized weekly. From the second month onwards, the immunization schedule was reduced to once a month. For the first immunization, 500 μg of KLH-conjugated AFP(243-256)-G0F-IMG was dissolved in 0.9% NaCl and emulsified in 2 ml of complete Freund's adjuvant (CFA). For all subsequent immunizations, CFA was replaced with 1 mL of incomplete Freund's adjuvant (IFA) emulsion.

[0504] Titer Analysis Titer analysis was performed using an ELISA protocol. Serum titration was performed using biotinylated AFP(243-261)-G0F-Bi screening peptide as a positive control.

[0505] Since the biotinylated peptide was used for screening, a 31, 25 ng / ml solution at 100 μl / well was immobilized on the surface of a 96-well streptavidin-coated microtiter plate by incubating at room temperature for 60 minutes. Subsequent washing was performed using an automated instrument (Biotek) according to the manufacturer's instructions. A small amount of serum (2 - 3 ml per animal) from each rabbit was collected on the 45th and 105th days after the start of the immunization campaign. Serum from each rabbit was diluted 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700, and 1:656100 in PBS containing 1% BSA. 100 μl of each dilution was added to plates pre-prepared with screening peptides and incubated at room temperature for 60 minutes. Bound antibodies were detected with HRP-labeled F(ab’)2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche). The titer of the analyzed animals was set at 50% signal reduction of the dilution curve.

Table 2

[0506] As demonstrated by the results in Table 2, the polyclonal sera from immunized animals bound to the AFP(243 - 261)-G0F-Bi screening peptide. However, the titers were extremely low, which was expected since the glycopeptide is a poorly immunogenic immunogen and it is extremely difficult to develop antibodies that bind to such molecules. Furthermore, it is expected that only a very small number of antibodies specifically bind to the core-fucosylated peptide and show low cross-reactivity to the non-fucosylated AFP-L1 variant.

[0507] Enrichment and single cell sorting of antigen-reactive B cells To enrich antigen-reactive B cells, 31.25 ng / ml of biotinylated AFP(243-261)-G0F-Bi peptide was pre-incubated with a pool of peripheral blood mononuclear cells (PBMCs) from immunized animals for 15 minutes at room temperature. After a washing step, antigen-reactive B cells loaded with the peptide were incubated with streptavidin-coated beads (Miltenyi) for 15 minutes at room temperature. Selection of positive B cells using a MACS column (Miltenyi) and subsequent incubation were performed as described in Seeber et al., PLoS One 9(2014), issue 2, e86184, except that the MACS column (Miltenyi), rather than plate binding, was involved in the selection of positive B cells. After culturing B cells in 96-well plates for 7 days, supernatants were collected for subsequent ELISA analysis and cells were lysed for cloning. In total, extraction and enrichment of B cells from immunized animals were performed approximately 21 times and 84 times per immunized animal. A major advantage of the method for antibody development described herein is that animals can be bled several times and antibody maturation can be followed over the course of an immunization campaign. The large number of clones per animal also shows how difficult it was to identify clones with the desired specificity for AFP-L3, which has low cross-reactivity to AFP-L1.

[0508] Antibody screening Subsequently, B cells expressing antibodies with the desired binding properties, i.e., antibodies that bind the AFP(243-261)-G0F-Bi peptide with core-fucosylation but do not bind the AFP(243-261)-G2-Bi glycopeptide, which is a core-fucose-free glycopeptide, were identified using Hit-ELISA (i.e., ELISA testing for binding to the screening reagent). Peptides were immobilized on the surface of streptavidin-coated 384-well plates (Nunc) by incubating 100 μl / well of 31, 25 ng / ml solutions for 60 minutes at room temperature. The plates were washed, and 30 μl of rabbit B cell culture supernatant was transferred to each well and incubated for 1 hour at room temperature. For detection of antibodies bound to the screening agent, HRP-labeled F(ab’)2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche) were used according to the manufacturer's instructions. Several clones that bound to the glycopeptide with core-fucosylation but not to the peptide without core-fucosylation were identified (out of 84 B cell sorting experiments with a total of 4 immunized rabbits) (Table 2). The V regions of several clones were cloned into mammalian expression vectors and subsequently expressed in 2 ml of HEK293 cells (as described in Seeber et al., PLoS One 9(2014), issue 2, e86184.). One week after expression, the supernatants of transfected HEK293 cells containing rabbit IgG were used for the initial SPR Biacore-based selection of a subset of antibodies meeting the performance criteria for detailed kinetic analysis (see Table 3).

Table 3

[0509] Example 3: Kinetic Screening and Further Evaluation of Kinetic Properties Surface plasmon resonance (SPR)-spectroscopy-assisted kinetic screening and detailed evaluation of kinetic properties were performed to select antibodies with excellent kinetic profiles and target specificities.

[0510] Dynamic screening was performed at 25 °C using a GE Healthcare Biacore™ B4000 instrument. A Biacore CM5 series S sensor was attached to the instrument, hydrodynamically addressed, and preconditioned according to the manufacturer's instructions. The system buffer was HBS ET pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (w / v) Tween 20. The system buffer was supplemented with 1 mg / mL CMD (carboxymethyl dextran, Sigma, catalog number 86524) and used as the sample buffer for preparing the dilution series.

[0511] A rabbit antibody capture system was immobilized on the sensor surface. The polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (catalogue number 111-005-046; Jackson Immuno Research, lot number 105332) was amine-coupled using EDC / NHS chemistry according to the manufacturer's instructions.

[0512] 30 μg / mL of goat anti-rabbit Fc gamma (GARbFcγ) in 10 mM sodium acetate buffer pH 4.5 was preconcentrated onto spots 1, 2, 4, and 5 of flow cells 1, 2, 3, and 4 and covalently bound to the sensor surface at a density of 12,000–13,000 RU. Free activated carboxyl groups were saturated with 1 M ethanolamine pH 8.5.

[0513] Spots 1 and 5 were used for interaction measurement, and Spots 2 and 4 were used as references. Each rabbit antibody from the primary cell supernatant was diluted 1:2 with sample buffer and injected at a flow rate of 10 μL / min for 2 minutes. The rabbit antibody capture level (CL) in resonance units (RU) was monitored. To achieve high enough sensitivity, additional molecular mass loading of biotinylated peptides AFP(243 - 261)-G0F-Bi, Roche BMO 35.000148, 4.3 kDa and AFP(243 - 261)-G2-Bi, Roche BMO 35.000151, 4.5 kDa was generated by streptavidin (SA) grafting. Biotinylated peptides, streptavidin and amino-PEO-biotin (Thermo Fisher, catalog number 21346) were mixed at a ratio of 1:10:5 and incubated at room temperature (RT) for 2 hours. The molecular weights of SA-grafted-AFP(243 - 261)-G0F-Bi and SA-grafted AFP(243 - 261)-G2-Bi were calculated at 64 kDa. When the SA-grafted peptides were injected onto their respective surfaces as 150 nM analyte, anti-AFP-L3 antibody was shown at 30 μL / min. The association and dissociation phases were monitored for 5 minutes.

[0514] The rabbit clone was regenerated from the sensor surface by injecting 10 mM glycine pH 1.5 at 20 μL / min for 20 seconds, followed by injecting 10 mM glycine pH 1.7 twice for 60 seconds each. The kinetics were monitored by BIAcore™ B4000 control SW V1.1 and evaluated with Evaluation SW V1.1. The reporting points of Binding Late (BL) just before the end of analyte injection and Stability Late (SL) just before the end of the dissociation phase were monitored. The antibody / antigen binding stability was characterized using the BL and SL data. Also, the dissociation rate constant k d [s -1 was calculated using the Langmuir 1:1 model. The antigen / antibody complex stability half-life (minutes) was calculated according to the formula ln(2) / 60*k d . The molar ratio representing the binding stoichiometry was calculated using the following formula. MW(antibody) / MW(antigen)*BL(antigen) / CL(antibody)

[0515] From a plurality of antibodies, specific antibody kinetics were identified, and the antibodies distinguished between fucosylated AFP-L3 peptide and non-fucosylated AFP-L3 peptide (see Figure 2).

[0516] Evaluation of kinetic characteristics Detailed kinetic studies were performed at 37 °C using a BIAcore™ T200 instrument manufactured by GE Healthcare. Five rabbit mAbs identified by kinetic screening <afp-l3>19B12, 3C5, 3D2, 3A3 and 6B6 were kinetically characterized for their binding to the AFP-L3 peptide. ·AFP(243-261)-G0F-Bi ·Asn(G0F)-Bi ·AFP(243-261)-G2-Bi ·AFP(243-261)-(Fuc-GlcNAc)-Bi

[0517] Measurements were made at 37 °C using a Series S C1 sensor.

[0518] The rabbit antibody capture system was immobilized on flow cells 1-4 as described at 700 RU - 800 RU. Using flow cell 1 as a reference, flow cells 2, 3 and 4 were used for interaction measurements. 150 nM antibody 1-5 was injected at 10 μL / min for 30 seconds. The capture level (CL) in resonance units RU was monitored. A peptide analyte concentration series from 3.3 - 270 nM was injected at 60 μl / min. The association phase was monitored for 3 minutes and the dissociation phase was monitored for 10 minutes. Regeneration was performed by injecting 10 mM glycine pH 2.0 at 20 μL / min for 30 seconds, followed by two injections of 10 mM glycine pH 2.25 for 60 seconds each. The kinetic rate constants and dissociation equilibrium constant K D were determined using the Langmuir 1:1 fitting model according to BIAcore™ T200 Evaluation SW 3.2. Next, the Langmuir 1:1 fitting Scrubber-SW V2.0c was applied.

[0519] The kinetic profiles of antibodies 19B12, 3C5, 3D2, 3A3 and 6B6 that bind to the peptide are shown in Figure 3. The antibody 19B12 binding kinetics vs AFP(243-261)-G0F-Bi was determined with k a 6.0E+05±0.07%M -1 s -1 ,k d 1.5E-03±0.05%s -1 ,t / 2diss =8±0.05 minutes, MR = 1.4. The affinity was K D It was 2.5 ± 0.08% nM (see Table 4 and Figure 4). By visual inspection, the kinetics signature of antibody 3C5 is close to antibody saturation at the highest concentration shown, 90 nM. This interaction shows complex binding behavior. Therefore, the constants described represent apparent values (see Table 4 and Figure 3).

[0520] By visual inspection, antibodies 3D2 and 3A3 show slower complex formation when binding to AFP(243 - 261)-G0F-Bi and do not approach antibody saturation at the highest concentration. The interaction does not follow the Langmuir law (see Figure 3). By visual inspection, antibody 6B6 shows the weakest interaction with fucosylated AFP(243 - 261)-G0F-Bi (see Figure 3).

[0521] These analyses revealed that clones 19B12 and 3C5 show the best performance with respect to their specificity and affinity. Both clones are specific for core-fucosylation and furthermore bind to the AFP peptide backbone. The latter is important as a clone, binding only to core-fucose and not to any other core-fucosylated protein in the sample, such as IgG-type molecules. [Table 4]

[0522] The non-fucosylated AFP(243 - 261)-G2-Bi binding kinetics for antibody 19B12 could not be determined. Based on the rate constants determined for the measured concentration range, simulations for concentrations in the range of 7.3 μM to 10 nM estimate a weak affinity greater than 620 nM for non-fucosylated AFP(243 - 261)-G2-Bi. The theoretical response maximum R of the simulated data max 9.3 RU is based on the calculation of the experimental capture level (CL) of 155 RU and the molecular weight of the analyte of 4.3 kDa. Figure 4 shows an overlay for both the simulated and experimental concentration ranges. The black line resembles the same concentration of 270 nM for both data sets.

[0523] Example 4: Affinity in Solution Analysis Dissociation equilibrium constant K D was determined by the Affinity in Solution method (AiS). According to the instructions of the vendor of the CAP-Kit (Cytiva), the peptide AFP(243-261)-G0F-Bi was captured on the surface of the CAP chip sensor. A mixture of 10 nM of antibodies 19B12 and 3C5, each having various concentrations of non-biotinylated AFP(243-261)-G0F from 120 nM to 0.01 nM, and various concentrations of non-biotinylated AFP(248-256) from 200 μM to -0.1 nM were incubated until equilibrium was achieved. The binding events of the mixture to the AFP(243-261)-G0F-Bi displayed on the surface were monitored.

[0524] As the peptide concentration as a competitor increases, the "free" antibody in the solution decreases. The free antibody concentration determined for the competition experiment was plotted against the peptide competitor concentration. Regeneration was performed using the guanidinium / NaOH solution supplied by Cytiva. Two independent series were analyzed for each interaction. The data were evaluated using the affinity model in solution from the Biacore Evaluation software. [Table 5]

[0525] AiS-based K for antibody 19B12 binding peptide AFP(243-261)-G0F D 0.9 ± 0.1 nM, R 2 0.99702 RU, B tot 9.8 ± 0.1 nM is in the same range as the concentration-dependent kinetics described previously in this document, and thus, confirms the experimental approach (see Table 5). Furthermore, the Affinity in Solution approach shows excellent specificity of antibodies 19B12 and 3C5 for fucosylated AFP(243-261)-G0F by complete competition.

[0526] For antibody 3C5, K D 0.4 ± 0.1 nM, R 2 0.98917 RU, B tot AiS was determined using 9.7 ± 0.1 nM (see Table 5). The AiS-based K for each D 5.7 ± 0.5 μM, R 2 0.98766 RU, B tot 10.1 ± 0.1 nM, 12.6 ± 2.9 μM, R 2 0.94759 RU, B tot 11.5 ± 0.4 nM demonstrates weak affinity for antibodies 19B12 and 3C5 that bind to peptide AFP(248 - 256).

[0527] Example 5: Cloning of Antibodies and Antibody Formats, Expression of Antibodies, Purification of Antibodies, and Labeling Cloning and Expression of IgG-Type Antibodies as IgG(P8) Multivalent Formats As described in International Publication No. WO 2019 / 057816 A1, multivalent anti-AFP-L3 antibodies were cloned and expressed. Specifically, to generate multivalent anti-AFP-L3 antibodies, multiple VH-CH1 sequences adjacent to a linker sequence (e.g., (G3S)4; SEQ ID NO: 27) were added upstream and downstream of the hinge-CH2-CH3 coding sequence, thereby creating a heavy chain encoding several VH-CH1 domains, which was cloned into an expression vector. This heavy chain vector was co-expressed with a light chain expression vector encoding a standard light chain consisting of VL and constant domains. Recombinant expression was performed transiently in human embryonic kidney (HEK) cells or transiently or non-transiently in CHO cells. The transformed cells secreted multivalent monospecific anti-AFP-L3 antibodies into the serum-free culture supernatant from which they were isolated. The final multivalent P8 antibody format contains 8 different Fab moieties, all of which have the same sequence (i.e., 19B12 or 3C5).

[0528] Purification of Antibodies and Conjugation with Ruthenium Monoclonal polyclonal anti-AFP-L3 antibodies of clone 19B12 and 3C5 were produced in sufficient yield and without significant loss during the purification period using protein. Affinity chromatography of the culture supernatant using MabSelect SuRe (Cytiva) was performed according to the supplier's instructions. The purified polyclonal recombinant anti-AFP-L3 antibody was reacted with the sulfonated form of the labeling reagent tris-bipyridyl-ruthenium (also called "sBPRu") using standard NHS ester coupling chemistry. Under these conditions, the ruthenium label is covalently bound to the functional groups of lysine amino acid residues in the antibody backbone.

[0529] mAbTU11(mAB <afp>M-TU11-F(ab’) 2 -Purification, fragmentation, and conjugation with biotin of -Bi) The monoclonal anti-AFP antibody clone TU11 was expressed in hybridoma cells and purified by standard ion exchange chromatography using SP and Q resins. The resulting purified IgG was further treated with pepsin to generate F(ab’) 2 fragments. The F(ab’) 2 -fragments of -TU11 were finally conjugated with biotin-PEG24-NHS label via surface-exposed lysine using standard NHS ester coupling chemistry. And the F(ab’) 2 -Bi-PEG24 conjugate was further purified using affinity chromatography with mSA (streptavidin mtein) to remove unconjugated F(ab’) 2 -fragments.

[0530] Expression and purification of anti-AFP-L3 antibody The monoclonal anti-AFP-L3 antibody was transiently expressed in HEK293F and purified by standard protein A affinity chromatography using MabSelect SuRe (Cytiva) according to the supplier's instructions.

[0531] Purification and conjugation of anti-rabbit Fc antibody A polyclonal antibody against rabbit Fc was prepared by immunizing sheep with purified rabbit Fc. The polyclonal anti-rabbit Fc antibody was further purified by positive immunoadsorption with a rabbit Fc immunosorbent and labeled via surface-exposed lysine with its sulfonated form of trisbipyridyl-ruthenium (also called “sBPRu”) using standard NHS ester coupling chemistry.

[0532] Example 6: Purification of AFP-L3 and L1 and protein labeling Purification of AFP-L1 and AFP-L3 Purified AFP from a human hepatocellular carcinoma cell line was purchased from either BioRad (catalog number 13752600) or Scripps (catalog number 90492-0050). The AFP was subjected to affinity chromatography using lentil (lens culinaris) agglutinin (LCA) (Vector Laboratories, catalog number AL-103). The AFP run on an LCA column containing running buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 ) could be separated into the LCA-bound (AFP-L3) and unbound (AFP-L1) forms of AFP. The bound AFP was eluted from the LCA column by adding a mixture of 200 mM α-methyl mannoside and 200 mM α-methyl glucoside to the running buffer.

[0533] Biotinylation of AFP Purified AFP was biotinylated with a biotin-DDS-NHS label via surface-exposed lysine using standard NHS ester coupling chemistry. Subsequently, unreacted label was removed by dialysis.

[0534] Example 7: Comparison of selected antibody clones for binding to AFP-L3 analyte in an immunological assay (Elecsys® assay format) Five different antibody clones selected based on kinetic screening were evaluated for their AFP-L3 specificity and their utility for generating signal kinetics in an Elecsys®-based immunoassay on an Elecsys® instrument, cobas® e601, for mAb <afp-l3>Tested with rRb-xx-IgG (xx: 6B6, 3A3, 3C5, 3D2, 19B12).

[0535] Briefly, the biotinylated peptide or protein (sample) was incubated for 9 minutes with the tested 1,6-fucAFP antibodies (6B6, 3A3, 3C5, 3D2, 19B12) and ruthenated anti-rabbit Fc antibody (see Example 6). Subsequently, 40 μl of Elecsys® beads (coated with streptavidin; "SA beads") were added and the mixture was incubated for 9 minutes so that the detection complex formed in the first step binds to the solid phase via the interaction of biotin and streptavidin. After incubation, the reaction mixture was aspirated into the measurement cell where the Elecsys® beads were magnetically captured on the surface of the electrode. Then, unbound substances were removed. Next, application of a voltage to the electrode induces electrochemiluminescence that is measured by a photomultiplier tube.

[0536] Test protocol and sandwich principle First incubation: 40 μl sample + 60 μl reagent 1 + 60 μl reagent 2, 9-minute incubation

[0537] Second incubation: + 40 μl SA beads. After addition of streptavidin-coated microparticles, the complex binds to the solid phase via the interaction of biotin and streptavidin and is incubated for 9 minutes.

[0538] After incubation, the reaction mixture was aspirated into the measurement cell where the microparticles were magnetically captured on the surface of the electrode. Then, unbound substances were removed. Next, application of a voltage to the electrode induces electrochemiluminescence that is measured by a photomultiplier tube.

[0539] Reagent 1: mAb <afp-l3>rRb-xx-IgG (1 μg / mL) (xx: 6B6, 3A3, 3C5, 3D2, 19B12) Reagent 2: pAb <rrb-fcg>-S-IgG(IS)-sulfoRu(Sux)(0.5 μg / mL)

[0540] Test buffer: 40 mM NaPi pH 7.5 / 150 mM NaCl / 0.1% MIT / 0.1% oxy-pyrithione / 0.1% polidocanol / 0.1% PAK-R-IgG / 2% RPLA 4

[0541] Samples: The following biotinylated peptides diluted in PBS / 0.05% Tween-20 (two concentration levels) were tested. AFP-(243-261)-G0F-Bi: with core-fucosylation 2. AFP-(243-261)-G2-Bi: without core-fucose (AFP-L1 variant) 3. AFP-(243-261)-(Fuc-GlcNAc)-bisaccharide L3 4. AFP(243-256)-(GlcNAc-GlcNAc)-Bi: disaccharide L1

[0542] Furthermore, the following biotinylated proteins were also tested. 5. Total α-fetoprotein (AFP) containing the AFP-L3 fraction derived from a human hepatoma cell line

[0543] Lens culinaris agglutinin A (LCA) affinity chromatography was performed on AFP total.

[0544] Two fractions were isolated: a) α-Fetoprotein (AFP-L3) is the fucosylated variant of AFP that reacts with Lens culinaris agglutinin A b) α-Fetoprotein (AFP-L1) is the non-fucosylated variant of AFP that should not react with Lens culinaris agglutinin A

[0545] As shown in Table 6, all the tested antibodies bind to the peptides AFP-(243-261)-G0F-Bi and AFP-(243-261)-(Fuc-GlcNAc)-Bi. The highest signal kinetics were observed with clones 3C5 and 19B12, confirming the findings in the kinetic analysis and suggesting that these two clones are the best binders.

Table 6

[0546] At the peptide level, the selected antibodies can distinguish between the core-fucosylated and non-fucosylated variants. At the level of the native antigen, very low signals were observed, indicating that the epitope may not be readily accessible. Nevertheless, the AFP-L3 fraction yielded higher signals for antibodies 19B12 and 3C5 than the AFP-L1 fraction, confirming their specificity at the level of the native antigen.

[0547] Example 8: Pretreatment for improving the detection of AFP-L3 in Elecsys Since the native AFP-L3 antigen showed a very low signal compared to the peptide structure, the inventors hypothesized that the epitope region may not be readily accessible in native AFP-L3 for the selected anti-peptide antibodies. To improve accessibility, the inventors tested various pretreatment conditions applied to AFP-L3-containing samples over a predetermined pretreatment time, with the aim of making the epitope more accessible to the antibody.

[0548] Several pretreatment conditions were first tested, and in particular, pretreatment with a protein reducing agent such as DTT or TCEP was thought to be able to significantly improve the signal.

[0549] To confirm whether protein reducing agents such as DTT are sufficient to promote the binding of antibodies 19B12 and 3C5, and how this compares to other antibodies tested in Example 7, the following experiments were conducted.

[0550] Test protocol: First step: Pretreatment Manual sample pretreatment was performed: 10 μl of protein sample (protein concentration in stock solution 250 μg / mL or 25 μg / ml) + 490 μl of pretreatment solution A or B (see below); incubation of the mixture for 30 minutes at room temperature.

[0551] The following biotinylated proteins were used as samples. 1. Total α-fetoprotein (AFP) containing the AFP-L3 fraction derived from a human hepatoma cell line 2. α-fetoprotein (AFP-L3) as a fucosylated variant of AFP that reacts with Lens culinaris agglutinin A 3. α-fetoprotein (AFP-L1) is a non-fucosylated variant of AFP that should not react with Lens culinaris agglutinin A

[0552] Two different pretreatment solutions were tested with each sample. · 50 mM citrate, 20 mM EDTA, 110 mM DTT pH 4.9 (pretreatment solution A) · And as a negative control: PBS + 0.05% Tween 20 (pretreatment solution B)

[0553] The final concentration of the protein used during sample pretreatment incubation was 5000 ng / mL or 500 ng / mL (which corresponds to protein concentrations of approximately 70 nM and 7 nM during sample pretreatment incubation). The concentration of DTT in the sample pretreatment incubation was 107 mM.

[0554] After manual pretreatment, an assay was performed on the cobas e601 analyzer using the following reagents under the following incubation conditions. Second step: addition of ruthenated AFP-L3 antibody 40 μl of pretreated sample (from the first step) + 60 μl of reagent 1 + 60 μl of reagent 2, incubation for 9 minutes

[0555] The following reagents were used. Reagent 1: mAb <afp-l3>rRb-xx-IgG-SulfoBPRu (1 μg / mL) (tested with 5 different clones: xx: 6B6, 3A3, 3C5, 3D2, 19B12) Reagent 2: 40 mM NaPi pH 7.5 / 150 mM NaCl / preservative / 0.1% polidocanol

[0556] The third step: the mixture of the second step + 40 μl of SA beads (streptavidin-coated microparticles). After the addition of SA beads, the complex binds to the solid phase via the interaction between biotin and streptavidin, and incubated for 9 minutes.

[0557] After incubation, the reaction mixture was aspirated into the measurement cell, where the microparticles were magnetically captured on the surface of the electrode. Then, the unbound substances were removed. Next, the application of voltage to the electrode induced electrochemiluminescence measured by a photomultiplier tube.

[0558] The results are shown in Figure 6.

[0559] Summary of results: · Samples pretreated with the pretreatment solution containing DTT gave a much higher signal than samples pretreated with the control solution (without DTT). This indicates an improvement in the recognition of AFP-L3 by using the pretreatment solution (containing a reducing agent) for better signal detection. · Clones 3C5 and 19B12 delivered the highest signals, confirming better binding of these antibodies to pretreated AFP-L3. · AFP and AFP-L3 proteins (both containing core-fucosylated AFP) delivered a much higher signal than AFP-L1 (non-core-fucosylated) protein. This confirms the specificity of the clones used, as previously shown at the peptide level in Biacore and Elecsys®.

[0560] Example 9: Optimization of Pretreatment In Example 8, 50 mM citrate, 20 mM EDTA, and 110 mM DTT at pH 4.9 were used for pretreatment. As is known in the art, DTT has a much higher redox potential at basic or neutral pH but is much more stable at acidic pH (DTT in a buffer with pH < 5.5 is more stable). Therefore, the pretreatment solution of Example 8 showed good stability but used a very high concentration of DTT.

[0561] Since DTT shows high reducing activity at pH > 7, the inventors speculated that a lower DTT concentration could be used if the pH during sample pretreatment incubation was in this pH range.

[0562] To enable both reducing activity at a much lower DTT concentration and sufficient reagent stability, one pretreatment solution was divided into two different bottles: PT1 and PT2 PT1: 7.4 mM DTT, 2 mM EDTA at pH 5.5 (due to stable DTT storage conditions) PT2: Two different formulations of PT2 were tested: 100 mM Tris at pH 8.5 or 200 mM NaOH at pH > 13

[0563] Using this setup, a pH > 7.5 was achieved in the sample pretreatment incubation (sample + PT1 + PT2).

[0564] These pretreatment conditions were tested using the different assay formats used in Examples 7 and 8. Briefly, in Step 1, the sample (here, human serum without AFP-L3 or spiked with 809 ng / ml of AFP-L3) was mixed with the PT1 and PT2 solutions and incubated for 9 minutes. In Step 2, R1 containing biotinylated AFP-specific antibody TU11 as F(ab’) 2 and R2 containing antibody 19B12 as ruthenated polyvalent antibody (P8 format) were added, and the mixture was incubated for 9 minutes. Finally, in Step 3, magnetic streptavidin microbeads were added, and the resulting mixture was incubated for 9 minutes again.

[0565] Reagents used: R1: mAb <afp>M-TU11-F(ab’)2-Bi(linker)2 2.3 μg / ml R2: mAb <afp-l3>rRb-19B12-IgG(P8)-SulfoBPRu at 15 μg / ml

[0566] Test protocol: 30 μl sample + 13 μl PT1 + 23 μl of PT2, incubation for 9 minutes + 51 μl R1 + 53 μl R2, incubation for 9 minutes + 29 μl SA beads, incubation for 9 minutes

[0567] After incubation, the reaction mixture was aspirated into the measurement cell where the microparticles were magnetically captured on the surface of the electrode. Unbound substances were then removed. Application of a voltage to the electrode then induced electrochemiluminescence that was measured by a photomultiplier tube.

[0568] Results

Table 7

[0569] Both pretreatments yielded measurable signals, but using Tris pH 8.5 showed much better performance (resulting in a higher pH) than using NaOH as PT2.

[0570] The conclusion from the experiment was that for PT2, Tris buffer was more suitable than a more aggressive alkaline solution such as NaOH.

[0571] Furthermore, the inventors noted that in this experiment, the P8 format increased the signal as compared to a similar experiment using IgG instead of the multivalent P8 format of the AFP-L3 antibody.

[0572] Example 10: Evaluation of the importance of the reducing agent DTT in the pretreatment solution using clone 19B12 Examples 8 and 9 both used the chelating agent EDTA separately from DTT in the pretreatment. To gain more insight into the importance of DTT for pretreatment activity, different conditions were tested using human natural serum and spiked samples (the master calibrator was set from MK1 to MK7, and the concentration of human serum spiked with AFP-L3 antigen was increased).

[0573] To confirm the role of DTT in the pretreatment, the following two compositions of PT1 were tested. PT1 Version 1: 10 mM DTT, 2 mM EDTA, pH approximately 4.7: With DTT PT1 Version 2: 2 mM EDTA, pH approximately 4.7: Without DTT PT2: 150 mM Tris pH 8.5

[0574] Test protocol: The assay principle used was the same as in Example 9, i.e., a sandwich immunoassay. Step 1: 30 μl sample + 13 μl PT1 (Version 1 or 2) + 23 μl PT2a, incubation for 9 minutes Step 2: + 51 μl R1 + 53 μl R2, incubation for 9 minutes Step 3: + 29 μl SA-Beads, incubation for 9 minutes R1: Biotinylated monoclonal anti-AFP antibody mAb <afp>M-TU11-F(ab’)2-PEG24-Bi 3.4 mg / L; Tris buffer 200 mM, pH 7.5; preservative R2: Ruthenium complex mAb <afp-l3>Monoclonal anti-AFP-L3 antibody labeled with rRb-19B12-IgG(P8)-SulfoBPRu at 22.0 mg / L; Tris buffer 200 mM, pH 7.5; preservative

[0575] After incubation, the reaction mixture was aspirated into the measurement cell, where the microparticles were magnetically captured on the surface of the electrode. Then, the unbound substances were removed. Next, the application of a voltage to the electrode induced electrochemiluminescence that was measured by a photomultiplier tube.

[0576] The results shown in Figures 9 and 10 demonstrate the important role of reducing agents such as DTT for better AFP-L3 detection for both natural and spiked samples.

[0577] Example 11: Variation of DTT concentration (mM) and Tris buffer concentration (mM) in PT2 The purpose of this experiment was to evaluate different concentrations of DTT in PT1 and Tris in PT2.

[0578] Here too, a sandwich immunoassay format was used.

[0579] Analytical pretreatment conditions: Different concentrations in PT1 were tested. PT1: pH 5.5, DTT variation from 0 to 10 mM + EDTA 2 mM

[0580] In PT2, two different concentrations of Tris were tested. PT2: pH 9.0, Tris buffer (50 or 200 mM)

[0581] For Reagents 1 and 2, constant conditions were used. Reagent 1: mAb <afp>M-TU11-F(ab`)2-Bi(PEG24)(2.5 μg / mL) Reagent 2: mAb <afp-l3>rRb-19 B12-IgG(P8)-SulfoBPRu (15 μg / mL)

[0582] Measurement sample: Human serum spiked with AFP-L3 protein (= "HS"), set at pH 8

[0583] Test protocol: First incubation: 39 μl sample + 16 μl PT1 + 16 μl PT2, incubation for 9 minutes Second incubation: First incubation of the mixture + 50 μl of R1 + 52 μl of R2, incubation for 9 minutes Third incubation: Second incubation of the mixture + 30 μl of SA beads, incubation for 9 minutes

[0584] After incubation, the reaction mixture was aspirated into the measurement cell, where the microparticles were magnetically captured on the surface of the electrode. Then, the unbound substances were removed. Next, the application of voltage to the electrode induced electrochemiluminescence, which was measured by a photomultiplier tube.

[0585] The DTT concentrations tested are summarized in Table 8 below.

Table 8

[0586] Results: The signals measured for different test conditions are summarized in Table 9.

Table 9

[0587] Conclusion: As shown in Table 9, different DTT concentrations can be used for pretreatment. A low concentration of 0.676 mM in the pretreatment sample mixture was sufficient to increase the signal compared to without DTT. This indicates that even lower concentrations of DTT can be used.

[0588] In this example, DTT at a concentration of 1 - 10 mM in PT1 was tested. In the previous example, especially Example 8, a higher DTT concentration of 110 mM in the stock was used. This shows that a wide range of DTT concentrations can be used to improve the signal, even to varying degrees depending on the concentration and pH of the pretreated sample incubation.

[0589] EDTA was found to stabilize DTT in PT1 (i.e., 2 mM was used).

[0590] Example 12: Method Comparison of the Prototype Elecsys® AFP - L3 Assay for the Wako Instrument A clinical panel (1000 clinical samples including HCC cases and controls) was measured with two different prototype Elecsys® AFP - L3 assays and compared with μTAS Wako AFP - L3 (Fujifilm Wako Pure Chemical Corporation). The prototype assays were identical except that either 19B12 or 3C5 P8 format polyclonal antibodies were used.

[0591] The assay settings were as follows.

Table 10

[0592] Reagents used: PT1: 6 mM DTT, 2 mM EDTA, pH 5.5 PT2: 100 mM Tris pH 8.5

[0593] As shown in Table 11, two assay prototypes were evaluated.

Table 11

[0594] The sample was further measured by μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions to determine the content (%) of AFP-L3 and the total concentration (ng / mL) of AFP. The Wako AFP-L3 concentration (ng / mL) was calculated from the μTASWako values of the obtained % of AFP-L3 and total AFP concentration (ng / mL). The Elecsys® AFP-L3 value was directly obtained in ng / mL using both prototypes a and b as described in Example 11.

[0595] Both assay prototypes showed very similar results and showed very good correlation with μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation) (see Figures 7 and 8).

[0596] The results of this method comparison confirm that the prototype shows good correlation with the marketed and clinically validated μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation). This also confirms that AFP-L3 is essentially detected by the assay of the present invention even when the LCA lectin is not used.

[0597] Example 13: Performance of the assay in a clinical panel To further confirm the clinical value of the Elecsys® AFP-L3 prototype assay using antibodies 19B12 and 3C5, a clinical panel was analyzed to evaluate the diagnostic performance for detecting early HCC. This panel included 96 patients with liver diseases, of which 38 had HCC and 58 had liver diseases but no diagnosis of HCC (controls).

[0598] An exemplary 19B12-based prototype was used.

[0599] Test protocol: Sandwich immunoassay Procedure 1: 30 μl sample + 13 μl PT1 + 23 μl PT2a, incubation for 9 minutes Procedure 2: + 51 μl R1 + 53 μl R2, incubation for 9 minutes Procedure 3: + 29 μl SA - Beads, incubation for 9 minutes

[0600] Reagents PT1: 10 mM DTT, 2 mM EDTA, pH < 5.5 PT2: 150 mM Tris pH 8.5 R1: Biotinylated monoclonal anti - AFP antibody TU11; 200 mM Tris buffer, pH 7.5; preservative R2: Monoclonal anti - AFP - L3 antibody 19B12 labeled with ruthenium complex as polyvalent P8 antibody; 200 mM Tris buffer, pH 7.5; preservative

[0601] The ROC curve is shown in Figure 11. The AUC (area under the curve) of the AFP - L3 assay based on 19B12 is 91.4% (CI: 85.8 - 97), which demonstrates very good performance for detecting HCC in this panel of assays.

Table 12

Claims

1. A monoclonal antibody or antigen-binding fragment that specifically binds to an α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP containing the α-1,6-core-fucosylated portion, wherein the monoclonal antibody or antigen-binding fragment is (i) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution; CDR-H2 having the amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by up to two amino acid substitutions and / or up to one insertion or deletion; and CDR-H3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution; (ii) A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8, or a variant of SEQ ID NO: 7 or 8 modified by up to two amino acid substitutions; CDR-L2 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by one amino acid substitution; and CDR-L3 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by one amino acid substitution, A monoclonal antibody or its antigen-binding fragment, including the above.

2. The partial arrangement of the AFP containing the α-1,6-core-fucosylation is, 【Chemistry 1】 (Sequence No. 2) (Equation I) A monoclonal antibody or antigen-binding fragment according to claim 1, comprising or consisting of a glycopeptide.

3. The monoclonal antibody or antigen-binding fragment is a glycopeptide of formula I and formula II as shown in claim 2. 【Chemistry 2】 (Sequence No. 2) (Formula II) A monoclonal antibody or antigen-binding fragment according to claim 1, which specifically binds to a glycopeptide.

4. K regarding the binding of the glycopeptide of formula II to the glycopeptide of formula I Ｄ The monoclonal antibody or antigen-binding fragment according to claim 3, wherein the ratio is at least 2.

5. (i) The heavy chain variable domain (VH) has an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.5%, sequence identity with SEQ ID NO: 11 or 12. (ii) The light chain variable domain (VL) has an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or preferably at least 97.3%, sequence identity with SEQ ID NO: 13 or 14. The monoclonal antibody or antigen-binding fragment according to claim 1.

6. (i) The heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 11 or 12, (ii) The light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 13 or 14 The monoclonal antibody or antigen-binding fragment according to claim 1.

7. The glycopeptide of formula I described in claim 2 contains K at a concentration of 100 nM or less, 20 nM or less, 10 nM or less, or 3.1 nM or less. Ｄ The K Ｄ The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the temperature may be measured at 37°C.

8. A rabbit IgG antibody comprising the heavy chain variable domain of SEQ ID NO: 11 or 12 and the light chain variable domain of SEQ ID NO: 13 or 14 for the glycopeptide of formula I according to claim 2. Ｄ K is less than 10 times, less than 8 times, less than 6 times, less than 4 times, or less than 2 times. Ｄ The K is bound to the glycopeptide of formula I described in claim 2. Ｄ A monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 7, wherein the value is measured under the same conditions and using the same method.

9. A polynucleotide or a set of polynucleotides, (i) the heavy chain or heavy chain variable domain of the monoclonal antibody or antigen-binding fragment according to claim 1, and / or (ii) The light chain or light chain variable domain of the monoclonal antibody or antigen-binding fragment according to claim 1 A polynucleotide or set of polynucleotides that encodes a molecule.

10. A vector comprising a polynucleotide or a set of polynucleotides as described in claim 9.

11. A host cell comprising the polynucleotide or set of polynucleotides described in claim 9, expressing the antibody or antigen-binding fragment described in claim 1.

12. A method for producing a monoclonal antibody or antigen-binding fragment according to claim 1, comprising culturing a host cell according to claim 11 and isolating the antibody or antigen-binding fragment.

13. A host cell comprising the vector according to claim 10 and expressing the antibody or antigen-binding fragment according to claim 1.

14. A method for producing a monoclonal antibody or antigen-binding fragment according to claim 1, comprising culturing the host cell according to claim 13 and isolating the antibody or antigen-binding fragment.

15. A composition comprising the antibody or antigen-binding fragment described in claim 1, the polynucleotide described in claim 9, or the vector described in claim 10.

16. Use of the antibody or antigen-binding fragment according to claim 1 for an in vitro immunoassay, or for an in vitro immunoassay to detect α-1,6-core-fucosylated alpha-fetoprotein (AFP).

17. Use of the composition according to claim 15 for an in vitro immunoassay, or for an in vitro immunoassay for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP).

18. A composition comprising the host cell described in Claim 13.

19. Use of the composition according to claim 18 for an in vitro immunoassay, or for an in vitro immunoassay for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP).

20. A pretreatment reagent for processing a sample containing α-1,6-core-fucosylated AFP, wherein the pretreatment reagent contains a reducing agent or a protein reducing agent.

21. An in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence containing the α-1,6-core-fucosylated AFP in a sample using the antibody or antigen-binding fragment described in claim 1.

22. The in vitro immunoassay method according to claim 21, further comprising pretreatment of the sample with the pretreatment reagent according to claim 20.

23. A kit comprising the antibody or antigen-binding fragment described in claim 1.

24. The kit according to claim 23, further comprising the pretreatment reagent according to claim 20.

Images