High affinity antibodies specifically binding to alpha-1, 6-core-fucosylated alpha fetoprotein

Abstract

The present invention relates to monoclonal antibodies and antigen binding fragments that specifically bind to an alpha-1, 6-core-fucosylated alpha fetoprotein (AFP), which is a 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 invention, host cells expressing the antibodies and antigen-binding fragments of the invention, methods for producing the antibodies and antigen-binding fragments of the invention, and uses of the antibodies and antigen-binding fragments of the invention. Also provided herein are pretreatment agents that promote the binding of the antibodies and antigen binding fragments of the invention to alpha-1, 6-core-fucosylated AFP. The invention further relates to kits comprising the antibodies and antigen binding fragments of the invention and optionally a pre-treatment agent of the invention.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies and antigen-binding fragments that specifically bind to alpha-1,6-core-fucosylated alpha-fetoprotein (AFP), which is the core component of AFP-L3. Therefore, 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 promote the binding of the antibodies and antigen-binding fragments of the present invention to alpha-1,6-core-fucosylated AFP. The present invention further relates to a kit comprising the antibodies and antigen-binding fragments of the present invention and optionally the pretreatment agents of the present invention. Background Art

[0002] Worldwide, liver cancer is the seventh most common cancer and the second leading cause of cancer death.

[0003] Hepatocellular carcinoma (HCC) is the main histological type of primary liver cancer occurring worldwide, accounting for 70% to 85% of the total burden. Underlying liver diseases such as liver fibrosis and cirrhosis are known to be major risk factors for the development of HCC. For patients diagnosed at an early stage, HCC can be treated by resection, liver transplantation, or radiofrequency local ablation. Therefore, early detection and minimally invasive screening methods are essential for HCC.

[0004] The most common methods for diagnosing HCC are ultrasound detection, imaging techniques such as computerized axial tomography (CAT scan) or magnetic resonance imaging (MRI), and serological biomarkers. However, the prognostic value of ultrasound detection is poor because it requires a tumor mass of at least 2 cm, and imaging techniques have poor sensitivity based on each lesion and are costly. On the other hand, in recent years, great attention has been paid to the discovery of new blood biomarkers that can be used for monitoring projects for 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 surveillance of high-risk patients every 6 months using ultrasound or other imaging modalities. However, limitations of imaging methods include poor sensitivity for early-stage tumors, operator dependence, and lower quality in patients with obesity or nonalcoholic steatohepatitis.

[0006] Alpha-fetoprotein (AFP) is currently the most mature biomarker for hepatocellular carcinoma (HCC). However, the sensitivity and specificity of AFP for the diagnosis of HCC, especially for the diagnosis of early HCC, still need to be further improved.

[0007] AFP is a glycoprotein, and various glycosylation 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, marker glycoprotein fractions with specific sugar chain structures can be separated and concentrated. In the case of AFP, lectins derived from Lentil agglutinin-A (LCA) have been widely used. By using LCA, AFP can be fractionated into three variants L1, L2 and L3, of which AFP-L3 has the highest affinity for LCA. The AFP-L3 fraction is composed of AFP N-glycosylated with N-glycans containing α-1,6-core-fucosylation at Asn-251 (i.e., AFP with fucose bound to N-acetylglucosamine (GlcNAc), which is 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 lentil agglutinin (LCA) reactive fraction of alpha-fetoprotein (AFP-L3) is specifically increased in patients with HCC (Khien VV et al., The International Journal of Biological Markers. 2001; 16(2): 105-111).

[0008] Recent publications clearly indicate that scores containing the input variables sex, age, AFP and descarboxyprothrombin (DCP=PIVKA-II) (=GAAD-score) or sex, age, AFP, AFP-L3 and descarboxyprothrombin (DCP=PIVKA-II) (=GALAD-score) can and will improve the results of HCC screening efforts and, in particular, the detection of early HCC (Zhou, JM., Wang, T., Zhang, KH. AFP-L3 for the diagnosis of early hepatocellular carcinoma, Medicine 2021; 100(43): p e27673).

[0009] Therefore, there is a great 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. The assay is based on affinity capillary electrophoresis using LCA to separate AFP-L3. However, this method has certain disadvantages. First, N-glycan branching may lead to the detection of erroneous AFP-L3 levels. Second, the method is cumbersome and requires special instrumentation. Therefore, it is highly desirable to design antibody-based methods (Egashira Y et al., Scientific Reports, 2019, 9: 12359). Third, lectins are less specific than antibodies and generally exhibit only low binding affinity.

[0011] JP S63-307900 discloses an antibody that binds to AFP bound to LCA (AFP-LCA-R) but does not bind to AFP not bound to LCA (AFP-LCA-NR). However, the epitope of the antibody is not clear, and it is not clear whether the antibody also recognizes proteins other than AFP having the same sugar moiety.

[0012] EP 3 252 073, US2018 / 0110889 and Egashira Y et al. (Scientific Reports, 2019, 9: 12359) disclose monoclonal antibodies against core-fucosylated AFP (i.e. AFP-L3) that bind in a core-fucose-dependent manner and also bind to portions of the AFP peptide backbone. However, as is well known in the art and as described by Egashira Y et al. (Scientific Reports, 2019, 9: 12359), most anti-carbohydrate antibodies have low affinity for their antigens. Although the affinity of the antibody FasMab is described as being stronger than that of typical anti-carbohydrate antibodies, K D The value is only 6.5x 10 -7 M. Typically, for fully automated immunoassays (such as The K of the antibody was determined (Roche) D The values ​​are significantly lower (eg, in the low nanomolar range), ie, have a higher affinity for AFP-L3.

[0013] Therefore, there is a need to provide novel antibodies that specifically bind to α-1,6-core-fucosylated alpha-fetoprotein (i.e., AFP-L3) with improved kinetic properties, in particular with higher affinity (i.e., lower K D ). Furthermore, there is a need to provide improved immunoassays for detecting AFP-L3. Summary of the invention

[0014] The monoclonal antibody and antigen-binding fragment thereof and uses thereof provided by the present invention solve the above-mentioned needs.

[0015] In a first aspect of the present invention, 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 comprising the α-1,6-core-fucosylation is provided.

[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 α-1,6-core-fucose residues (i.e., non-glycosylated AFP or AFP with N-glycans lacking α-1,6-core-fucose residues). Secondly, the monoclonal antibodies or antigen-binding fragments provided herein can distinguish α-1,6-core-fucosylated AFP from free N-glycans with α-1,6-core-fucose residues. Thirdly, the monoclonal antibodies or antigen-binding fragments provided herein distinguish α-1,6-core-fucosylated AFP from non-glycosylated AFP peptides containing N-glycosylation site N-251. Based on this binding behavior, it can be concluded that the binding of the antibody and its antigen-binding fragment to α-1,6-core-fucosylated AFP depends on the presence of both α-1,6-core-fucosylation and the AFP peptide sequence (e.g., SEQ ID NO: 2). Therefore, 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 with similar α-1,6-core-fucosylated N-glycan structures and (ii) AFP or its partial sequence lacking α-1,6-core-fucose residues.

[0017] With this specificity, the antibodies and antigen binding fragments provided herein are valuable tools for detecting the level of α-1,6-core-fucosylated AFP (clinically equivalent to AFP-L3) in a sample in an immunoassay. Therefore, the antibodies and antigen binding fragments and immunoassays using them can help detect HCC, especially 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 analysis of binding to α-1,6-core-fucosylated AFP peptides. The K used for this interaction measurement DIn the single-digit nanomolar range, i.e., lower than expected based on the teachings of the prior art for this AFP glycopeptide antibody. D Several orders of magnitude lower (Egashira Y et al., Scientific Reports, 2019, 9: 12359). Egashira Y et al. have reported that the affinity of the antibody against α-1,6-core-fucosylated AFP is 650 nM, which has a surprisingly low K D (i.e. high binding affinity), which highlights the unexpected finding that antibodies with the affinities reported herein can be identified.

[0019] Interestingly, the two best monoclonal antibodies identified here, clones 19B12 and 3C5, show very high sequence homology, also especially in the CDR residues. The CDR residues have a total of 3 amino acid differences: one insertion and two amino acid substitutions.

[0020] Therefore, in an embodiment, the monoclonal antibody or antigen-binding fragment according to the first aspect of the present disclosure comprises

[0021] (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 thereof 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; and

[0022] (ii) a light chain variable domain (VL), which VL comprises: 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.

[0023] In a second aspect, the present invention provides a polynucleotide encoding

[0024] (i) a heavy chain or heavy chain variable domain of a monoclonal antibody or antigen-binding fragment according to the first aspect, and / or

[0025] (ii) a light chain or a light chain variable domain of the monoclonal antibody or antigen-binding fragment according to the first aspect.

[0026] In a third aspect, a vector comprising a polynucleotide according to the second aspect of the invention is disclosed.

[0027] According to the fourth aspect of the present invention, there is provided a host cell comprising the polynucleotide of the second aspect or the vector of the third aspect.

[0028] In a fifth aspect, the present invention relates to a method for producing a monoclonal antibody or antigen-binding fragment according to the first aspect, the method comprising culturing a host cell according to the fourth aspect and isolating the antibody or antigen-binding fragment. Antibodies obtained or obtainable by such a method are also disclosed.

[0029] According to a sixth aspect, a composition is provided, comprising the monoclonal antibody or antigen-binding fragment according to the first aspect, the polynucleotide according to the second aspect, the vector according to the third aspect or the host cell according to the fourth aspect.

[0030] In an embodiment of the sixth aspect, there is provided a diagnostic composition comprising the antibody according to the first aspect of the invention.

[0031] The inventors have generated an immunoassay for determining the level of α-1,6-core-fucosylated AFP using the antibodies or antigen-binding fragments of the invention. The levels of α-1,6-core-fucosylated AFP determined using this assay correlate well with the levels determined using the μTASWako AFP-L3 assay, which is currently the most widely used method for detecting and quantifying AFP-L3. Therefore, the immunoassay using the antibodies of the invention is a highly reliable tool, for example, to aid in HCC detection, overcoming the shortcomings of μTASWako AFP-L3.

[0032] Therefore, 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, in particular for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP) or AFP-L3.

[0033] Similarly, the 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 comprising the α-1,6-core-fucosylation in a sample using the monoclonal antibody or antigen-binding fragment thereof according to the first aspect.

[0034] The inventors have also discovered that pretreatment (eg, 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 are more accessible after such pretreatment.

[0035] Therefore, in a ninth aspect, a pretreatment agent or pretreatment composition comprising a reducing agent (eg, DTT) is also provided.

[0036] In a tenth aspect, the present invention provides a kit of parts comprising the monoclonal antibody or antigen binding fragment of the first aspect of the present invention. In an embodiment, the kit is used for an immunoassay for detecting and / or quantifying α-1,6-core-fucosylated AFP. In an embodiment, the kit further comprises a pretreatment agent or composition of the ninth aspect of the present invention. DETAILED DESCRIPTION

[0037] Elements of the present invention will be described below. These elements are listed with specific aspects and embodiments, however, it should be understood that they can be combined in any manner and in any number to create additional aspects and embodiments.

[0038] 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 comprising said α-1,6-core-fucosylation.

[0039] As is known in the art, human AFP is a glycoprotein having the amino acid sequence SEQ ID NO: 1 (or a naturally occurring variant thereof, as described in Uniprot ID P02771), which comprises a single N-glycosylation site corresponding to Asn-251 of Uniprot ID P02771 (version 209). The N-glycan at Asn-251 may comprise a core fucose residue (see Figure 1 ). As further understood in the art, the term "core fucosylation" within a glycan indicates that the fucosyl residue is an α-1,6-linked core GlcNac residue attached to Asn-251 of the AFP protein or a partial sequence thereof comprising Asn corresponding to Asn-251. The terms "core-fucosylation" and "α-1,6-core-fucosylation" are considered to be interchangeable. Thus, the term "having specificity for (or specifically binding to) core-fucosylated AFP and / or a partial sequence of AFP comprising core-fucosylation" is interchangeable with the term "having specificity for (or specifically binding to) α-1,6-core-fucosylated AFP and a partial sequence thereof comprising α-1,6-core-fucosylation". The antibodies and antibody antigen-binding fragments of the present invention may also be referred to interchangeably herein as 1,6fucAFP antibodies and antigen-binding fragments thereof.

[0040] As mentioned above, the α-1,6-core-fucose referred to in the context of the 1,6fucAFP antibody or antigen-binding fragment is part of the N-glycan attached to residue N251 of human AFP (see SEQ ID NO: 1).

[0041] The position of α-1,6-core-fucose in N-glycans is as follows Figure 1 As displayed.

[0042] The partial sequence of AFP may comprise SEQ ID NO: 2. In a specific embodiment, the peptide portion of the partial sequence of AFP consists of SEQ ID NO:2.

[0043] As demonstrated in the accompanying examples, the inventors have identified two monoclonal antibodies (19B12 and 3C5) 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 that lack α-1,6-core-fucosylation at Asn-251 and other proteins modified with α-1,6-core-fucosylated N-glycans.

[0044] The monoclonal antibodies and antigen fragments of the present invention exhibit particularly high affinity (i.e., low K D ). This is 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). In addition, the monoclonal antibodies of the present invention show favorable kinetic parameters for fully automated high-throughput immunoassays, such as a high association rate constant k a and low dissociation rate constant k d .

[0045] As described above, the monoclonal 1,6fucAFP antibody or antigen-binding fragment provided herein specifically binds to α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising the α-1,6-core-fucosylation. The α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising the α-1,6-core-fucosylation may comprise or consist of a glycopeptide of Formula I:

[0046]

[0047] The glycopeptide of Formula I represents an AFP peptide having the amino acid sequence of SEQ ID NO: 2, comprising an Asn amino acid residue at position 3 corresponding to Asn-251 of AFP, said AFP peptide being modified with an N-glycan comprising α-1,6-core-fucosylation.

[0048] Therefore, the 1,6-fucAFP antibody and the antigen-binding fragment thereof of the present invention can specifically bind to the glycopeptide of Formula I or the glycoprotein comprising the glycopeptide of Formula I.

[0049] In an embodiment, the α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising 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:

[0050]

[0051] The glycopeptide of Formula II represents an AFP peptide having the amino acid sequence of SEQ ID NO: 2 comprising an Asn amino acid residue at position 3 corresponding to Asn-251 of AFP, the AFP peptide being modified with an N-glycan comprising α-1,6-core-fucosylation.

[0052] In embodiments where the α-1,6-core-fucosylated AFP or a partial sequence of the AFP comprising said α-1,6-core-fucosylation comprises a glycopeptide of formula II, for example, further amino acids may be present in the peptide sequence or further sugar moieties may be present in the glycan (preferably attached to GlcNAc).

[0053] Therefore, the 1,6-fucAFP antibody and antigen-binding fragments thereof of the present invention can specifically bind to the glycopeptide of Formula II or a glycoprotein comprising the glycopeptide of Formula II.

[0054] In an embodiment, the 1,6-fucAFP antibody and antigen-binding fragments thereof of the present invention can specifically bind to the glycopeptide of Formula I or II or a glycoprotein comprising the glycopeptide of Formula I or II.

[0055] As demonstrated in the accompanying examples, the 1,6-fucAFP antibodies and antigen-binding fragments of the present disclosure bind with high affinity (i.e., low K D ) binds to both the glycopeptide of Formula I and the glycopeptide of Formula II. It has been surprisingly observed that the binding affinity to the glycopeptide of Formula I is slightly higher than that to the glycopeptide of Formula II (i.e., K DThis suggests that the added sugar moiety in the N-glycans of the glycopeptide of Formula I promotes binding to some extent. Since N-glycan structures are generally more complex in nature, such as those shown in the glycopeptide of Formula I, this binding behavior may be advantageous because it increases specificity for more complex N-glycosylated AFP species.

[0056] Thus, in embodiments, the 1,6-fucAFP antibodies and antigen-binding fragments thereof of the present invention can bind to the glycopeptide of Formula II with higher affinity (i.e., lower K D ) is bound to a glycopeptide of Formula I. In an embodiment, the K for binding to a glycopeptide of Formula II and to a glycopeptide of Formula I D The ratio between 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 a specific embodiment, the K for binding to the glycopeptide of Formula II and to the glycopeptide of Formula I is D The ratio between can be at least 7. In an embodiment, the K for binding to the glycopeptide of Formula II and to the glycopeptide of Formula I is D The ratio between can be at most 11, at most 12, or at most 15. In an embodiment, the K for binding to the glycopeptide of Formula II and to the glycopeptide of Formula I is D The ratio between can be at most 12.

[0057] As already discussed above, the 1,6fucAFP antibodies and antigen-binding fragments thereof of the present invention distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising said α-1,6-core-fucosylation (e.g., having formula I) and (ii) AFP or a partial sequence thereof lacking α-1,6-core-fucose residues.

[0058] The AFP or partial sequence thereof lacking α-1,6-core-fucose residues may include (i) AFP or partial sequence thereof being N-glycosylated at Asn-251 or a position corresponding thereto but lacking core fucose in the N-glycan and (ii) non-glycosylated AFP or partial sequence thereof.

[0059] Furthermore, the 1,6fucAFP antibodies and antigen-binding fragments thereof of the present invention distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising said α-1,6-core-fucosylation (e.g., having formula I) and (ii) α-1,6-core-fucosylated N-glycans isolated or in the presence of proteins other than AFP.

[0060] As also defined below, the term "discriminating" means that the 1,6fucAFP antibodies and antigen-binding fragments bind to specific antigen targets (i.e., core-fucosylated AFP and core-fucosylated partial sequences thereof) with greater affinity and / or selectivity than they bind to other antigens ("non-target antigens") (e.g., AFP / AFP partial sequences lacking core fucosyl residues and / or core-fucosylated glycans (e.g., in isolated form or in the context of other glycosylated proteins or peptides)). For example, the feature of discriminating between target antigens and / or non-target antigens can be that the affinity of the 1,6fucAFP antibody or antibody antigen-binding fragment for the target antigen is at least 10 times, at least 20 times, at least 50 times, or at least 100 times, at least 150 times, at least 180 times, or at least 245 times greater than the affinity for the non-target antigen (i.e., the K for binding to the target antigen is greater than the K for binding to the non-target antigen). D The formulation of at least "XX" also encompasses K values ​​for non-target antigens. D Therefore, whether the antibody or antigen-binding fragment can distinguish target structures from non-target structures can be determined using the same methods (e.g., as described below for determining K D The preferred method) is to determine the K for the corresponding binding D value.

[0061] In an embodiment, an immunoassay can be used to assess the ability of an antibody to distinguish between a target antigen and / or a non-target antigen, in which the binding of the antibody or antigen-binding fragment to be tested to the target structure is detected. Using such an immunoassay, an immunoassay signal obtained with a first sample containing a defined concentration of a target antigen can be compared with an immunoassay signal obtained with a second sample containing the same concentration of a non-target antigen. The immunoassay signal of the first sample is higher than the immunoassay signal of the second sample indicating the distinction between the target antigen and the non-target antigen. In an embodiment, if the immunoassay signal of the first sample is at least 5 times, at least 10 times, at least 20 times, at least 40 times, at least 50 times, or at least 100 times the immunoassay signal of the second sample, the test antibody or antigen-binding fragment can distinguish between the target structure and the non-target structure. An exemplary but non-limiting immunoassay that can be used for such analysis is provided in Example 7. Exemplary concentrations of target antigens and non-target antigens can be 12 nM.

[0062] In an embodiment, the AFP lacking α-1,6-core-fucose residues or a partial sequence thereof may comprise or consist of a glycopeptide of Formula III:

[0063]

[0064] Since Formula III lacks core-fucose, the 1,6fucAFP antibody or antigen-binding fragment thereof can distinguish this glycopeptide of Formula I or a glycoprotein comprising the same from the glycopeptide of Formula III.

[0065] Thus, provided herein are 1,6fucAFP antibodies and antigen-binding fragments thereof that distinguish between the glycopeptide of Formula I (or an AFP sequence comprising the same) and the glycopeptide of Formula III (or an AFP sequence comprising the same).

[0066] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments of the invention distinguish between the glycopeptide of Formula II (or an AFP sequence comprising the same) and the glycopeptide of Formula III (or an AFP sequence comprising the same).

[0067] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments of the invention distinguish between the glycopeptide of Formula I (or an AFP sequence comprising the same) and the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence comprising the same).

[0068] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments of the invention distinguish the glycopeptide of Formula II (or an AFP sequence comprising the same) from the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence comprising the same).

[0069] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments provided herein distinguish between the glycopeptide of Formula I (or an AFP sequence comprising the same) and both the glycopeptide of Formula III (or an AFP sequence comprising the same) and the peptide of SEQ ID NO: 2 or SEQ ID NO: 25 (or an AFP sequence comprising the same).

[0070] In embodiments, the 1,6fucAFP antibodies and antigen-binding fragments of the invention distinguish between the glycopeptide of Formula II (or an AFP sequence comprising the same) and both the glycopeptide of Formula III (or an AFP sequence comprising the same) and the peptide of SEQ ID NO: 25 (or an AFP sequence comprising the same).

[0071] In the examples, the equilibrium dissociation constant (K) of the 1,6fucAFP antibodies and antigen-binding fragments of the invention binding to (ii) AFP lacking α-1,6-core-fucose residues or a partial sequence of AFP lacking α-1,6-core-fucose residues (e.g., the glycopeptide of Formula III or SEQ ID NO: 2 or 25) is D ) is a K that binds to (i) α-1,6-core-fucosylated AFP or a partial AFP sequence comprising α-1,6-core-fucosylation (eg, a glycopeptide of Formula I)D In a specific embodiment, the K D The difference in the dissociation constant 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 the dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0072] In an embodiment, the AFP lacking α-1,6-core-fucose residues or the partial sequence of AFP lacking α-1,6-core-fucose residues is an N-glycosylated AFP lacking core-fucose residues or a partial sequence thereof (e.g., a glycopeptide of Formula III); and the equilibrium dissociation constant (K) of the 1,6fucAFP antibody and antigen-binding fragment of the present invention binding to (ii) AFP lacking α-1,6-core-fucose residues or the partial sequence of AFP lacking α-1,6-core-fucose residues is D ) is a K that binds to (i) α-1,6-core-fucosylated AFP or a partial AFP sequence comprising α-1,6-core-fucosylation (eg, SEQ ID NO: 2); D In a specific embodiment, the K D The difference in the dissociation constant 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 the dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0073] In the examples, the equilibrium dissociation constant (K) of the 1,6-fucAFP antibodies and antigen-binding fragments of the invention binding to the glycopeptide of (ii) Formula III is D ) is the K of the antibody or antigen-binding fragment binding to the glycopeptide of formula (i) D In a specific embodiment, the K D The difference in the dissociation constant 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 the dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0074] In embodiments, the AFP lacking α-1,6-core-fucose residues or the partial sequence of AFP lacking α-1,6-core-fucose residues is a non-glycosylated AFP or a non-glycosylated partial AFP sequence (e.g., SEQ ID NO: 2 or 25), and the equilibrium dissociation constant (K) of the 1,6fucAFP antibody and antigen-binding fragment of the present invention binding to the non-glycosylated AFP or the non-glycosylated partial AFP sequence (e.g., SEQ ID NO: 2 or 25) is D ) is a K that binds to (i) α-1,6-core-fucosylated AFP or a partial AFP sequence comprising α-1,6-core-fucosylation (eg, a glycopeptide of Formula I) D In an embodiment, the K D The difference is at least 1000-fold. In an embodiment, the K D The difference is at least 5000-fold. The binding affinities for (i) and (ii) were determined under the same conditions.

[0075] In the examples, the equilibrium dissociation constant (K) of the 1,6-fucAFP antibodies and antigen-binding fragments of the invention binding to (ii) the AFP peptide of SEQ ID NO: 25 is D ) is the K of the antibody or antigen-binding fragment binding to the glycopeptide of formula (i) D In an embodiment, the K D The difference is at least 1000-fold. In an embodiment, the K D The difference is at least 4000 times. In an embodiment, the K D The difference is at least 5000-fold. The binding affinities for (i) and (ii) were determined under the same conditions.

[0076] As demonstrated by the corresponding glycopeptides and glycans in the accompanying examples and figures, the monoclonal antibodies or antigen-binding fragments of the present invention distinguish between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising said α-1,6-core-fucosylation (e.g., a glycopeptide of formula I) and (ii) isolated α-1,6-core-fucosylated glycans (e.g., having formula (IV)) and other glycoproteins having α-1,6-core-fucosylated glycans (i.e., proteins comprising a glycopeptide of formula IV but not a glycopeptide of formula I).

[0077] The polysaccharide of formula IV has the following structure.

[0078]

[0079] Therefore, in an embodiment, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention distinguishes between (i) α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising the α-1,6-core-fucosylation (e.g., a glycopeptide of Formula I) and (ii) isolated α-1,6-core-fucosylated glycans (e.g., having Formula (IV)) and other glycoproteins having α-1,6-core-fucosylated glycans (i.e., proteins comprising a glycopeptide of Formula IV but not a glycopeptide of Formula I).

[0080] In specific embodiments, the equilibrium dissociation constant (K) of the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention for binding to (ii) an isolated α-1,6-core-fucosylated glycan (e.g., having formula (IV)) and other glycoproteins having α-1,6-core-fucosylated glycans (i.e., a protein comprising a glycopeptide of formula IV but not a glycopeptide of formula I) is D ) is the K of the antibody or antigen-binding fragment binding to (i) α-1,6-core-fucosylated AFP or a partial AFP sequence comprising α-1,6-core-fucosylation (e.g., a glycopeptide of Formula I) D The binding affinities for (i) and (ii) are determined under the same conditions. In a specific embodiment, the K D The difference in the dissociation constant 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 the dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0081] In even more specific embodiments, the equilibrium dissociation constant (K) of the 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention binding to (ii) the isolated α-1,6-core-fucosylated glycan of formula (IV) is D ) is the K of the antibody or antigen-binding fragment binding to the glycopeptide of formula (i) D The binding affinities for (i) and (ii) are determined under the same conditions. In a specific embodiment, the K DThe difference in the dissociation constant 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 the dissociation is at least 183-fold. The binding affinities for (i) and (ii) are determined under the same conditions.

[0082] In embodiments, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention distinguishes between glycopeptides according to Formula I and both glycopeptides of Formula III and core-fucosylated glycans of Formula IV. D The description of the difference multiple of the values ​​applies mutatis mutandis.

[0083] In embodiments, the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention distinguishes the glycopeptide according to Formula I from all three of: the glycopeptide of Formula III, the core-fucosylated glycan of Formula IV, and the AFP peptide of SEQ ID NO: 25 or 2 (eg, SEQ ID NO: 25).

[0084] It has been found that the monoclonal 1,6-fucAFP monoclonal antibodies or antigen-binding fragments of the present invention have surprisingly high affinity (i.e., low K) for the glycopeptide of Formula I (see above), i.e., for the AFP sequence comprising α-1,6-core-fucosylation. D ). Low K D Often useful for immunoassays.

[0085] In embodiments, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention has 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 Binds to the glycopeptide of Formula I. If K is determined by affinity in solution D , then the K D It may also be 0.9 nM or less or 0.4 nM or less. In a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K of 20 nM or less. D In a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K of 10 nM or less. D In a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a K of 5 nM or less. D Conjugated to the glycopeptide of formula I.

[0086] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention is a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13, and the K of the glycopeptide of Formula I is D 1 / 10, 1 / 8, 1 / 6, 1 / 4 or 1 / 2 of K D Binding to the glycopeptide of Formula I, wherein K is measured under the same conditions and using the same method D In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention has a K value equal to or less than that of a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of Formula I. D K D Binding to the glycopeptide of Formula I, wherein K is measured under the same conditions and using the same method D value.

[0087] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention is a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14, and the K of the glycopeptide of Formula I is D 1 / 10, 1 / 8, 1 / 6, 1 / 4 or 1 / 2 of K D Binding to the glycopeptide of Formula I, wherein K is measured under the same conditions and using the same method D In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the invention has a K value equal to or less than that of a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 for the glycopeptide of Formula I. D K D Binding to the glycopeptide of Formula I, wherein K is measured under the same conditions and using the same method D value.

[0088] It has further been found that the monoclonal 1,6-fucAFP monoclonal antibodies or antigen-binding fragments of the present invention have a surprisingly high association rate (k) for binding to the glycopeptide of formula I (see above), i.e. to an AFP sequence comprising α-1,6-core-fucosylation. a ) constant. High k a It is important for rapid binding of antibody to antigen at equilibrium. a Short incubation times are important in immunoassays.

[0089] The association rate constant (k) of the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention binding to the glycopeptide of Formula I a ) can be at least 2.0*10 4 M -1 s -1 , in the embodiment is at least 2.1*10 4 M -1 s -1 , in an embodiment is at least 10 5 M -1 s -1 , in the embodiment is at least 2.5*10 5 M -1 s -1 , in the embodiment is at least 6.0*10 5 M -1 s -1 and in an embodiment is at least 1.0*10 6 M -1 s -1 .

[0090] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention has a higher k value than a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of Formula I. a 3.4%, 5%, 10%, 20%, 50%, 80% or 90% of k a Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method a In a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k value equal to or higher than that of a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of Formula I. a K a Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method a value.

[0091] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention has a higher k value than a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 for the glycopeptide of Formula I. a2.0%, 3.4%, 5%, 10%, 20%, 50%, 80% or 90% of k a Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method a In a specific embodiment, the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k value equal to or higher than that of a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 for the glycopeptide of Formula I. a K a Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method a value.

[0092] It has further been found that the monoclonal 1,6-fucAFP monoclonal antibodies or antigen-binding fragments of the present invention have a surprisingly low off-rate (k ) for binding to the glycopeptide of formula I (see above), i.e. to an AFP sequence comprising α-1,6-core-fucosylation. d )constant.

[0093] The dissociation rate constant (k) of the monoclonal 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention for the glycopeptide of formula I d ) can be at most 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 at most 1.5*10 -3 s -1 .

[0094] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention has a lower k value than a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of Formula I. d 1.5, 2, 2.5, 3, 3.5 or 3.75 times k d Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method dIn a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k value equal to or lower than that of a rabbit IgG antibody (e.g., antibody 19B12) comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of Formula I. d K d Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method d value.

[0095] In an embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the invention has a lower k value than a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 for the glycopeptide of Formula I. d 1.5, 2, 2.5, 3, 3.5, 3.75, 5, 6, 7 or 8 times k d Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method d In a specific embodiment, the monoclonal 1,6-fucAFP monoclonal antibody or antigen-binding fragment of the present invention has a k value equal to or lower than that of a rabbit IgG antibody (e.g., antibody 3C5) comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 for the glycopeptide of Formula I. d K d Binding to the glycopeptide of Formula I, wherein k is measured under the same conditions and using the same method d value.

[0096] In the embodiments, all K disclosed herein D , k a and k d The value can be K at 37°C D , k a and k d value.

[0097] As is understood in the art, 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.

[0098] In the context of the present invention, the K mentioned above is particularly D , k a and k d The value is preferably determined by surface plasmon resonance spectroscopy (e.g., ) OK. Two K DValue between two k a Values ​​between and two k d The fold difference between the values ​​(ie the ratio) can be calculated based on the two corresponding values ​​measured by plasmon resonance spectroscopy, respectively.Preferred peptides and methods of performing the analysis are disclosed in the accompanying examples and figures.

[0099] It is particularly preferred herein to use surface plasmon resonance spectroscopy as described in Example 3 to determine K D , k a and k d value.

[0100] Therefore, for determining K D , k a and / or k d The surface plasmon resonance spectroscopy method of the present invention may include capturing the 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, wherein 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 ) can be used as a system buffer, and the system buffer supplemented with 1 mg / ml carboxymethyl dextran can be used as a sample buffer.

[0101] In an embodiment, combined with the parameter K D and / or k a and / or k d The Langmuir fit can be used to fit the model 1:1 (e.g., according to BIAcore TM T200 Evaluation SW 3.2). Preferably, the fit is performed using R max Alternatively or additionally, a Langmuir 1:1 fit Scrubber-SW V2.0c may be applied.

[0102] Surface plasmon resonance spectroscopy can be performed using various instruments known in the art. In a particularly preferred embodiment, T200 or 8K instrument.

[0103] The concentration of the antibody or antigen binding fragment used for surface plasmon resonance measurements can be 150 nM and can be injected, for example, at 10 μl / min for 30 seconds. For analytes (e.g., peptides or glycopeptides), a concentration series from 3.3 nm to 270 nm can be used. The injection of the analyte can be 60 μl / min.

[0104] The association phase in a surface plasmon resonance spectroscopy measurement can be monitored, for example, for 3 minutes. The dissociation phase can be monitored, for example, for 10 minutes. Regeneration can be performed by injecting 10 mM glycine pH 2.0 at 20 μL / min for 30 seconds, followed by 2 injections of 10 mM glycine pH 2.25 for 60 seconds.

[0105] In a preferred embodiment, K D and / or k a and / or k d It can be determined as follows:

[0106] The Series S C1-sensor can be used at 37°C from GE Healthcare T200 instrument. A rabbit antibody capture system (e.g., as described in the examples) can be fixed on a flow cell, for example, at 700 to 800 RU. One flow cell can be used as a control and three flow cells can be used for measurement. Then 150 nM of the antibody or antigen binding fragment to be tested can be injected at 10 μL / min for 30 seconds. The capture level (CL) can be monitored in resonance units RU. An analyte concentration series from 3.3 nM 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 can be performed by injecting 10 mM glycine pH 2.0 at 20 μL / min for 30 seconds, followed by 2 injections of 10 mM glycine pH 2.25 for 60 seconds. The BIAcore TM T200 Evaluation SW 3.2 uses the Langmuir 1:1 fitting model to determine the kinetic rate constant and dissociation equilibrium constant K D Secondly, a Langmuir 1:1 fit to Scrubber-SW V2.0c can be applied.

[0107] Unless otherwise expressly stated herein, the K D The K value (and rate constant) is determined by surface plasmon resonance spectroscopy using a monoclonal antibody or antigen binding fragment on a capture chip (e.g., a C1 sensor chip) and injecting the analyte to be analyzed. As is well known in the art, there are also methods known in the art for determining K D Alternative methods for determining the values ​​and / or rate constants of antibodies 3C5 and 19B12 may also be used as long as the results obtained with these methods for antibodies 3C5 and 19B12 are consistent with the results reported herein for the antibodies in Example 3.

[0108] The accompanying examples also describe methods for determining K D Alternative method for determining K values: Affinity in solution analysis. As shown in the accompanying example, the K values ​​determined using affinity in solution analysis areD The values ​​are very similar to those for the capture-based approach described above, but may differ to some extent. If this technique is explicitly mentioned, the K values ​​quoted in the paper are D The values ​​refer only to the K measured by affinity in solution. D If a discrepancy exists, then a surface plasmon resonance spectroscopy dataset based on antibody capture as described above should be used.

[0109] Affinity measurements in solution can be performed as described following the supplier's instructions for the CAP-Kit (Cytiva). For example, a biotinylated analyte (e.g., a glycopeptide or peptide) can be captured on the CAP chip sensor surface. A mixture of 10 nM antibody or antigen-binding fragment to be tested and different concentrations of non-biotinylated AFP (243-261)-G0F between 120 nM and 0.01 nM or different concentrations of non-biotinylated AFP (248-256) between 200 μM and 0.1 nM can be incubated until equilibrium is reached. The binding events of the mixture to the analyte displayed on the surface can be monitored. As the concentration of the peptide as a competitor increases, the 'free' antibody in the solution will decrease. The free antibody concentration determined by the competition experiment can then be plotted against the peptide competitor concentration. From The in-solution affinity model of the evaluation software can be used to evaluate the data and determine the K D .

[0110] In an embodiment, the method for determining K is selected D method, so that

[0111] (1) comprising a heavy chain variable domain having a sequence of SEQ ID NO: 11 and a heavy chain variable domain having a sequence of SEQ ID NO:

[0112] A rabbit IgG antibody having a light chain variable domain of the sequence of NO:13; and

[0113] (2) Glycopeptide of Formula I

[0114] The combined K D Determined to be 2.5 (± method error, e.g. 0.08%) nM.

[0115] In an embodiment, the method for determining K is selected D method, so that

[0116] (1) comprising a heavy chain variable domain having a sequence of SEQ ID NO: 12 and a heavy chain variable domain having a sequence of SEQ ID NO:

[0117] A rabbit IgG antibody having a light chain variable domain of the sequence of NO:14; and

[0118] (2) Glycopeptide of Formula I

[0119] The combined K D Determined to be 3.1 (± method error, e.g. 0.13 percent) nM.

[0120] As demonstrated in the accompanying examples and as further discussed herein below, the 1,6fucAFP antibody or antigen-binding fragment can detect α-1,6-core-fucosylated alpha-fetoprotein (AFP) (e.g., native α-1,6-core-fucosylation occurring in a blood sample (e.g., plasma or serum) obtained from a human subject) that has been pretreated with a pretreatment agent according to the present invention (see elsewhere herein) much better than without such pretreatment. Thus, in embodiments, the monoclonal antibody or antigen-binding fragment of the present disclosure binds better to α-1,6-core-fucosylated alpha-fetoprotein (AFP) than to α-1,6-core-fucosylated alpha-fetoprotein (AFP) without such pretreatment. Measures to detect this "better" binding can be signaled using one of the immunoassay setups disclosed in the accompanying examples. The pretreatment agent is preferably a reducing agent, such as DTT.

[0121] Surprisingly, the two best monoclonal 1,6fucAFP antibodies identified herein (i.e., 19B12 and 3C5) show very high sequence homology, but there are certain differences in the framework regions and CDR residues. This indicates that the CDR sequences and VH and VL sequences that form the basis of the identified antibodies define very valuable building blocks for generating 1,6fucAFP antibodies and antigen-binding fragments thereof. However, the sequence variability found in VH and VL also indicates that certain amino acid exchanges can be made in the CDR and framework regions of VH and VL without significantly negatively affecting the antibody properties. It should be understood that a technician can easily generate variants of the antibody or antigen-binding fragment sequences of the present invention, and can determine whether such antibodies retain the favorable properties disclosed herein based on the disclosure herein. For example, affinity maturation can be applied to obtain a variant monoclonal antibody or antigen-binding fragment comprising VH and VL of 19B12 or 3C5 and amino acid substitutions obtained by such affinity maturation, which has the same or improved properties as 19B12 or 3C5.

[0122] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0123] (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 a substitution, insertion or deletion of one amino acid; 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 thereof modified by a substitution, insertion or deletion of up to two amino acids; 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 a substitution, insertion or deletion of one amino acid; and

[0124] (ii) a light chain variable domain (VL), which VL comprises: 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, insertions or deletions; 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, insertion or deletion; 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, insertion or deletion.

[0125] In specific embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0126] (i) a heavy chain variable domain (VH), the 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); and

[0127] (ii) a light chain variable domain (VL), which VL comprises: 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).

[0128] In specific embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0129] (i) a heavy chain variable domain (VH), the 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 substitution of one amino acid and / or insertion or deletion of one amino acid; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6 (CDR-H3 of 19B12 and 3C5); and

[0130] (ii) a light chain variable domain (VL), which VL comprises: 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 one amino acid; 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).

[0131] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0132] (i) a heavy chain variable domain (VH), the 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 thereof 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; and

[0133] (ii) a light chain variable domain (VL), which VL comprises: 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 thereof modified by up to two amino acid substitutions, insertions or deletions; 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 one amino acid substitution, insertion or deletion; 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 one amino acid substitution, insertion or deletion.

[0134] The variation in the CDR sequences of the monoclonal antibody or antigenic fragment may be such that the sum of substitution, insertion and deletion events 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.

[0135] In a specific embodiment, the sum of amino acid substitutions, insertions and deletions in all CDR sequences may be at most 3. In an embodiment, the at most 3 amino acid substitutions, insertions and deletion events may be at most two amino acid substitutions and one insertion or deletion.

[0136] In another specific embodiment, the sum of amino acid substitutions, insertions and deletions in all CDR sequences may be at most 2.

[0137] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0138] (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 thereof 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; and

[0139] (ii) a light chain variable domain (VL), which VL comprises: 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.

[0140] As used herein, the expression "a variant thereof modified by one amino acid substitution" means that the variant sequence comprises exactly one (not more than one) amino acid substitution relative to the specified parent sequence.

[0141] In specific embodiments, the monoclonal antibody or antigen-binding fragment comprises

[0142] (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 thereof 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

[0143] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution.

[0144] The variation in the CDR sequences of the monoclonal antibody or antigenic fragment may be such that the sum 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.

[0145] In a specific embodiment, the sum of amino acid substitutions in all CDR sequences may be at most 3.

[0146] In another specific embodiment, the sum of amino acid substitutions in all CDR sequences may be at most 2.

[0147] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0148] (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 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 thereof modified by up to 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

[0149] (ii) a light chain variable domain (VL), which VL comprises: 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 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.

[0150] In specific embodiments, the monoclonal antibody or antigen-binding fragment comprises

[0151] (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 conservative amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant thereof modified by up to 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

[0152] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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 conservative amino acid substitutions; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one conservative amino acid substitution; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one conservative amino acid substitution.

[0153] In embodiments, the monoclonal antibodies or antigen-binding fragments provided herein comprise

[0154] (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 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 thereof modified by up to two highly 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 highly conservative amino acid substitution; and

[0155] (ii) a light chain variable domain (VL), which VL comprises: 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 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 highly 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 highly conservative amino acid substitution.

[0156] In specific embodiments, the monoclonal antibody or antigen-binding fragment comprises

[0157] (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one highly conservative amino acid substitution; a CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 4 or 5 or a variant thereof modified by up to two highly 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 highly conservative amino acid substitution; and

[0158] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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 highly conservative amino acid substitutions; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one highly conservative amino acid substitution; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one highly conservative amino acid substitution.

[0159] 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) relative to the CDR-H2 of 3C5 (i.e., SEQ ID NO: 5). In addition, 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.

[0160] Therefore, the 1,6fucAFP antibody or antigen-binding fragment provided herein may comprise

[0161] (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 having an amino acid sequence of SEQ ID NO: 3; a CDR-H2 having an 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 an amino acid sequence of SEQ ID NO: 6; and

[0162] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which has 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, which has the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which has the amino acid sequence of SEQ ID NO: 10.

[0163] In specific embodiments, the monoclonal antibody or antigen-binding fragment may comprise

[0164] (i) a heavy chain variable domain (VH), the 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

[0165] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0166] In embodiments, the antibody or antigen-binding fragment comprises

[0167] (i) a heavy chain variable domain (VH), the 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 amino acid substitution; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and

[0168] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which has 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, which has the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which has the amino acid sequence of SEQ ID NO: 10.

[0169] In specific embodiments, the monoclonal antibody or antigen-binding fragment may comprise

[0170] (i) a heavy chain variable domain (VH), the 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 conservative amino acid substitution; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0171] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0172] In embodiments, the antibody or antigen-binding fragment comprises

[0173] (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 having an amino acid sequence of SEQ ID NO: 3; a CDR-H2 having an amino acid sequence of SEQ ID NO: 4 or 5 or a variant of SEQ ID NO: 4 or 5 modified by a conservative or highly conservative amino acid substitution; and a CDR-H3 having an amino acid sequence of SEQ ID NO: 6; and

[0174] (ii) a light chain variable domain (VL), which VL comprises: 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 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.

[0175] In specific embodiments, the monoclonal antibody or antigen-binding fragment may comprise

[0176] (i) a heavy chain variable domain (VH), the 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 conservative or highly conservative amino acid substitution; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0177] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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 or highly conservative amino acid substitution; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0178] The difference in the amino acid sequences in the CDRs of the 1,6fucAFP antibodies 19B12 and 3C5 is three amino acids. Specifically, relative to the CDR-H2 of 3C5 (i.e., SEQ ID NO: 5), there is an amino acid insertion in position 3 of the CDR-H2 of 19B12 (i.e., SEQ ID NO: 4). In addition, there are amino acid differences in position 5 of the CDR-H2 of 19B12 (i.e., SEQ ID NO: 4) and position 4 of the CDR-H2 of 3C5 (i.e., SEQ ID NO: 5). Taking into account the above-mentioned insertions, these positions correspond to each other. Finally, the difference between the CDR-L1 of 19B12 (i.e., SEQ ID NO: 7) and the CDR-L1 of 3C5 (i.e., SEQ ID NO: 8) is the amino acid at position 7.

[0179] Thus, the monoclonal 1,6-fucAFP antibody or antigen-binding fragment of the present disclosure may comprise

[0180] (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 an amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by an amino acid substitution at amino acid position 4; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and

[0181] (ii) a light chain variable domain (VL), which VL comprises: 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 an amino acid substitution at amino acid position 7; 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.

[0182] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0183] (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 modified by an amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by an amino acid substitution at amino acid position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0184] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 7 or 8 or a variant of SEQ ID NO: 7 or 8 modified by a single amino acid substitution at amino acid position 7; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0185] In an embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0186] (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 a conservative amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by a conservative amino acid substitution at amino acid position 4; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and

[0187] (ii) a light chain variable domain (VL), which VL comprises: 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 a conservative amino acid substitution at amino acid position 7; 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.

[0188] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0189] (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 modified by a conservative amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by a conservative amino acid substitution at amino acid position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0190] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 7 or 8 or a variant of SEQ ID NO: 7 or 8 modified by a conservative amino acid substitution at amino acid position 7; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0191] In an embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0192] (i) a heavy chain variable domain (VH), the 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 a highly conservative amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by a highly conservative amino acid substitution at amino acid position 4; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and

[0193] (ii) a light chain variable domain (VL), which VL comprises: 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 a highly conservative amino acid substitution at amino acid position 7; 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.

[0194] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0195] (i) a heavy chain variable domain (VH), the 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 modified by a highly conservative amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by a highly conservative amino acid substitution at amino acid position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0196] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 7 or 8 or a variant of SEQ ID NO: 7 or 8 modified by a highly conservative amino acid substitution at amino acid position 7; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0197] If not otherwise specified, the amino acid substitutions in any (e.g., all) of the variants of SEQ ID NOs: 3 to 10 may be any amino acid exchange. In specific embodiments, the amino acid substitutions in any (e.g., all) of the variants of SEQ ID NOs: 3 to 10 may be conservative amino acid exchanges. In even more specific embodiments, the amino acid substitutions in any (e.g., all) of the variants of SEQ ID NOs: 3 to 10 may be highly conservative amino acid exchanges.

[0198] In an embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0199] (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 an amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by an amino acid substitution at amino acid position 4; and a CDR-H3 having the amino acid sequence of SEQ ID NO: 6; and

[0200] (ii) a light chain variable domain (VL), the VL comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 7 or 8; 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,

[0201] wherein amino acid position 5 in the variant of SEQ ID NO: 4 is modified to serine, and wherein amino acid position 4 in the variant of SEQ ID NO: 5 is modified to asparagine.

[0202] In a more specific embodiment, the monoclonal antibody or antigen-binding fragment of the present disclosure comprises

[0203] (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 modified by an amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by an amino acid substitution at amino acid position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0204] (ii) a light chain variable domain (VL), the VL comprising 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;

[0205] wherein amino acid position 5 in the variant of SEQ ID NO: 4 is modified to serine, and wherein amino acid position 4 in the variant of SEQ ID NO: 5 is modified to asparagine.

[0206] The monoclonal antibodies or antigen-binding fragments according to the present invention may comprise the CDR sequences of monoclonal antibody 19B12 as identified herein.

[0207] Thus, the monoclonal antibodies or antigen-binding fragments of the present invention may comprise

[0208] (i) a heavy chain variable domain (VH), the 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; and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0209] (ii) a light chain variable domain (VL), which VL comprises CDR-L1, which consists of the amino acid sequence of SEQ ID NO:7; CDR-L2, which consists of the amino acid sequence of SEQ ID NO:9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO:10.

[0210] The monoclonal antibody or antigen-binding fragment according to the invention may comprise the CDR sequences of monoclonal antibody 3C5 as identified herein.

[0211] Thus, the monoclonal antibodies or antigen-binding fragments of the present disclosure may comprise

[0212] (i) a heavy chain variable domain (VH), the 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: 5; and CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; and

[0213] (ii) a light chain variable domain (VL), which VL comprises CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 8; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0214] Various embodiments are disclosed above describing the amino acid sequences as depicted in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9 and 10 and optionally variants thereof. Also provided herein are corresponding embodiments, wherein reference to “SEQ ID NO:3” is replaced by “CDR-H1 sequence contained in SEQ ID NO:11 or 12”, reference to “SEQ ID NO:4” is replaced by “CDR-H2 sequence contained in SEQ ID NO:11”, reference to “SEQ ID NO:5” is replaced by “CDR-H2 sequence contained in SEQ ID NO:12”, reference to “SEQ ID NO:6” is replaced by “CDR-H3 sequence contained in SEQ ID NO:11 or 12”, reference to “SEQ ID NO:7” is replaced by “CDR-L1 sequence contained in SEQ ID NO:11”, reference to “SEQ ID NO:8” is replaced by “CDR-L1 sequence contained in SEQ ID NO:12”, reference to “SEQ ID NO:9” is replaced by “CDR-L2 sequence contained in SEQ ID NO:11 or 12” and / or reference to “SEQ ID NO:10” is replaced by “SEQ ID NO:11 or 12 contained in the CDR-L1 sequence" is replaced.

[0215] In embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises

[0216] (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 to SEQ ID NO: 11 or 12; and

[0217] (ii) a light chain variable domain (VL) having 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 to SEQ ID NO: 13 or 14.

[0218] Therefore, the present invention provides 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 comprising the α-1,6-core-fucosylation (e.g., a glycopeptide of Formula I), wherein the monoclonal antibody or antigen-binding fragment comprises

[0219] (i) a heavy chain variable domain (VH) having an 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 thereto; and

[0220] (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.

[0221] As is apparent from the accompanying examples and figures, the two antibodies identified herein, 19B12 and 3C5, show significant sequence similarity in the CDRs and framework regions of VH and VL, but also have many amino acid substitutions. Based on the VH and VL sequences of these antibodies, other antibodies with sequence similarity to 19B12 and 3C5 can be generated with similar characteristics (e.g., specificity, affinity and other kinetic parameters for α-1,6-core-fucosylated AFP). For example, such variant antibodies can be generated by affinity maturation.

[0222] Therefore, in an embodiment, the antibody or antigen-binding fragment of the present invention may comprise

[0223] (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

[0224] (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.

[0225] In an embodiment, the antibody or antigen-binding fragment of the invention may comprise

[0226] (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 modified by an amino acid substitution at amino acid position 5 or a variant of SEQ ID NO: 5 modified by an amino acid substitution at amino acid position 4; and a CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 6; or an affinity matured variant of said VH; and

[0227] (ii) a light chain variable domain (VL), which VL comprises CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 7 or 8; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10; or an affinity matured variant of the VH.

[0228] By definition, affinity matured variants have at least the same affinity as 19B12 or 3C5, and exhibit at least the same binding preference (i.e., the difference in binding of different structures) as 19B12 or 3C5. Affinity matured variants may include amino acid substitutions, insertions and / or deletions. In an embodiment, affinity matured variants may include amino acid substitutions. In an embodiment, affinity matured variants show at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the corresponding parent VH or VL sequence (e.g., any one of SEQ ID NO 11 to 14).

[0229] It is known in the art that an antibody heavy chain or light chain variable domain comprises four framework domains in addition to three CDRs. Specifically, it is known that framework region 1 (FW1) represents the most N-terminal portion of the variable chain domain, and framework region 4 (FW4) represents the most C-terminal portion, wherein CDRs are interspersed between framework regions:

[0230] FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4

[0231] Although it is obvious from the context whether it refers to the framework region (FW) or the complementarity determining region (CDR) of the heavy chain or light chain variable domain, the indicators "H" or "L" are still used herein to distinguish FW from CDR. For example, the components FW and CDR of the heavy chain variable domain are referred to herein as the following schematic representations:

[0232] (FW-H1)-(CDR-H1)-(FW-H2)-(CDR-H2)-(FW-H3)-(CDR-H3)-(FW-H4)

[0233] Similarly, the component FWs and CDRs of the light chain variable domain are referred to herein as follows schematically:

[0234] (FW-L1)-(CDR-L1)-(FW-L2)-(CDR-L2)-(FW-L3)-(CDR-L3)-(FW-L4).

[0235] In an embodiment, the VH of the α-1,6-core-fucosylated AFP-specific antibody or antigen-binding fragment of the invention comprises a framework region (FW) and has the following structure:

[0236] FW-H1–CDR-H1–FW-H2–CDR-H2–FW-H3–CDR-H3–FW-H4.

[0237] FW-H1 may comprise or consist of the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0238] FW-H2 may comprise or consist of the amino acid sequence of SEQ ID NO: 16 or 17 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0239] FW-H3 may comprise or consist of the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0240] FW-H4 may comprise or consist of the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0241] In embodiments, the framework sequence of VH has at most 2 or at most 1 amino acid substitution, insertion and / or deletion events.

[0242] In an embodiment, the VL of the α-1,6-core-fucosylated AFP-specific antibody or antigen-binding fragment of the invention comprises a framework region (FW) and has the following structure:

[0243] FW-L1–CDR-L1–FW-L2–CDR-L2–FW-L3–CDR-L3–FW-L4.

[0244] FW-L1 may comprise or consist of the amino acid sequence of SEQ ID NO: 20 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0245] FW-L2 may comprise or 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%, even 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% thereof.

[0246] FW-L3 may comprise or consist of the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0247] FW-L4 may comprise or consist of the amino acid sequence of SEQ ID NO: 24 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0248] 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.

[0249] In an embodiment, the framework regions of VH and / or VL of the antibody or antigen-binding fragment of the present invention may comprise the framework sequences FW1, FW2, FW3 and FW4 of rabbit origin.

[0250] In an embodiment, the framework regions of VH and / or VL of the antibody or antigen-binding fragment of the invention are derived from the same (rabbit) germline as the framework regions of 19B12 or 3C5.

[0251] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention may be rabbit antibodies.

[0252] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention may have a detectable label attached thereto.

[0253] In embodiments, the monoclonal antibodies and antigen-binding fragments of the invention may have a capture label attached thereto.

[0254] The present inventors have discovered that the use of F containing 19B12 VThe signal-to-noise ratio of an immunoassay as described in the accompanying examples can be increased by more than 2-fold (as in conventional antibodies), for example, 8-fold (also referred to herein as p8).

[0255] Thus, in embodiments, the 1,6-fucAFP antibody of the invention may be a multivalent antibody comprising at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 F domains formed by any of the combinations of VH and VL domains as described above in the context of the heavy and light chains of the antibodies of the invention. V In embodiments, the 1,6fucAFP antibody of the present invention may comprise any one of the combinations of VH and VL domains as described herein at least 3, at least 4, at least 5, at least 6, at least 7, 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 may comprise at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 Fab domains comprising any one of the combinations of VH and VL domains as described herein. V In one embodiment, the multivalent antibody of the invention comprises 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 a variant thereof as defined above. V Domain (formed by VH and VL).

[0256] As used herein, a multivalent antibody refers to an antibody comprising at least three Fv domains. In preferred embodiments, the multivalent antibody comprises 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 Fv domain. V .

[0257] Exemplary but non-limiting embodiments of multivalent antibodies and methods for generating such antibodies are disclosed in WO2019 / 057816, which is incorporated herein by reference in its entirety. Specifically, all embodiments of the structural configurations of such multivalent antibodies and methods for generating such multivalent antibodies (also referred to as p3, p4, p5, p6, p7 or p8) are disclosed herein by reference.

[0258] In an embodiment, the multivalent antibody of the present invention may comprise a heavy chain comprising a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies, 8 copies in a specific embodiment) of a VH-CH1 domain (e.g., all comprising the (preferably identical) VH of the 1,6fucAFP antibody or antigen-binding fragment of the present invention), the domain being, for example, flanked by a linker sequence (e.g., one of the linker sequences described in WO2019 / 057816, which is incorporated herein by reference). Compared to conventional antibodies, the additional VH-CH1 domain may be placed upstream and / or downstream of the hinge-CH2-CH3 sequence. The light chain in such a multimeric antibody may be a conventional light chain consisting of a VL and a constant domain.

[0259] Alternative methods for generating multivalent antibodies involve chemical polymerization / cross-linking of antibodies or antigen-binding fragments.

[0260] Antibodies of the invention can be generated using the immunization and selection methods as described in the accompanying Examples.

[0261] Another suitable method for producing or isolating antibodies and antibody antigen-binding fragments of the present invention includes, but is not limited to, methods for selecting recombinant antibodies from peptide or protein libraries (e.g., but not limited to phage, ribosome, oligonucleotide, RNA, cDNA, or yeast display libraries) using target binding activity. For example, antibodies or antigen-binding fragments can be selected from such libraries by positive selection for specific binding to glycopeptides of formula I and negative selection for binding to glycopeptides of formula III and / or core-fucosylated polysaccharides of formula IV and / or peptides of SEQ ID NO: 2 or 25. Display libraries are well known in the art, for example, available from various commercial suppliers, including but not limited to Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsried / Planegg, Del.), Biovation (Aberdeen, Scotland, UK) and Bioinvent (Lund, Sweden). Similarly, selected clones can be processed according to conventional methods for subsequent recombination processing.

[0262] Variants of antibodies or antigen-binding fragments disclosed herein can be generated using conventional recombinant DNA techniques using sequences disclosed herein. For example, recombinant DNA techniques can be used to remove or modify DNA sequences encoding antibodies and / or antibody antigen-binding fragments disclosed herein (e.g., encoding heavy chain and / or light chain variable domains as defined above). For example, recombinant DNA techniques can be used to replace or remove portions of the coding sequence that are not necessary for maintaining specific and selective binding to the target antigen. Molecules expressed by such modified or truncated DNA molecules are also encompassed by the antibodies of the present invention.

[0263] The antibodies of the present invention can be recombinantly expressed. Therefore, 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 attached examples and elsewhere herein.

[0264] In a second aspect, the present invention provides a nucleic acid molecule or a polynucleotide group encoding a 1,6fucAFP antibody or 1,6fucAFP antigen-binding fragment disclosed herein. In particular, polynucleotides encoding 1,6fucAFP heavy chain and / or light chain variable domains as defined above are provided. As used herein, "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and similar terms include both genomic DNA and cDNA, and RNA capable of driving the expression of the antibody or antigen-binding fragment of the present invention. It is understood that the term "RNA" as used herein includes all forms of RNA, including mRNA, tRNA and rRNA, as well as genomic RNA, such as in the case of RNA of RNA viruses. Preferably, the embodiment recording "RNA" is for mRNA. The nucleic acid molecules / nucleic acid sequences of the present invention may be natural, synthetic or semi-synthetic. Therefore, the nucleic acid molecule may be, for example, a nucleic acid molecule that has been synthesized according to conventional protocols of organic chemistry, according to recombinant methods, or semi-synthetically produced (e.g., by combining chemical synthesis and recombinant methods). Those skilled in the art are familiar with the preparation and use of such nucleic acid molecules. A "polynucleotide set" refers to at least two polynucleotides encoding portions 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.

[0265] In the embodiments, a polynucleotide or set of polynucleotides encoding one of the VH and VL combinations disclosed in the context of the first aspect of the invention is provided. Thus, for each of the aspects and embodiments relating to a 1,6fucAFP antibody or antigen-binding fragment thereof as described herein, the corresponding polynucleotide encoding the corresponding antibody or antigen-binding fragment is provided mutatis mutandis.

[0266] In a specific embodiment, a polynucleotide or a group of polynucleotides encoding an antibody heavy chain variable domain VH of SEQ ID NO: 11 or 12 or a variant thereof as defined in the context of the first aspect of the invention and / or an antibody light chain variable domain of SEQ ID NO: 13 or 14 or a variant thereof as defined in the context of the first aspect of the invention is provided.

[0267] In another specific embodiment, a polynucleotide or a set of polynucleotides encoding the VH of SEQ ID NO: 11 and the VL of SEQ ID NO: 13 is provided.

[0268] In another specific embodiment, a polynucleotide or a set of polynucleotides encoding the VH of SEQ ID NO: 12 and the VL of SEQ ID NO: 14 is provided.

[0269] In some embodiments, the polynucleotide may comprise further sequences to ensure expression of not only the heavy and / or light chain variable domains, but also the remaining heavy and / or light chain constant regions, such that a full-length antibody (e.g., IgG) comprising the heavy and light chain variable domains of the invention is expressed.

[0270] In the third aspect, a vector comprising a polynucleotide or polynucleotide group according to the second aspect of the present invention is provided herein. In particular, a vector comprising a nucleic acid molecule encoding an antibody or antibody antigen-binding fragment of the present invention is provided. As used herein, the term "vector" refers to a circular or linear nucleic acid molecule that can be autonomously replicated 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 plasmids or contained in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) and phages. However, the art provides many suitable vectors, the selection of which depends on the desired function. The development and use of suitable vectors are well documented in the art; see, for example, Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001) and Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY (1989), (1994) described in the technology. The vector used in connection with the present invention comprises a nucleic acid sequence encoding a 1,6fucAFP antibody or antigen-binding fragment as disclosed herein. Thus, for each of the aspects and embodiments relating to a monoclonal antibody or antigen-binding fragment that specifically binds to α-1,6-core-fucosylated AFP as described herein, provided herein is a vector comprising a corresponding polynucleotide encoding the corresponding antibody or antigen-binding fragment.

[0271] As used herein, the term "vector comprising ..." is understood in the art that there are further nucleic acid sequences in the vector that are necessary and / or sufficient for the desired vector activity in the host cell, such as driving the replication of the vector (and thereby encoding the nucleic acid sequence) and / or for guiding the host cell to express the antibody or antigen-binding fragment of the present invention. Such further nucleic acid sequences include, but are not limited to, sequences that control vector replication and / or expression of the desired sequence in a specific cell system. For example, a vector may include a nucleic acid molecule encoding an antibody or antibody antigen-binding fragment of the present invention, which is operably connected to a regulatory sequence and / or under the control of a regulatory sequence. The term "regulatory sequence" refers to a DNA sequence that is necessary for achieving expression of a coding sequence that is operably connected thereto. The term "control sequence" is intended to include at least all components that may be equally necessary for expression, and may further include other advantageous components, for example, for allowing replication. As understood in the art, the properties of such regulatory and control sequences vary from host organism to host organism. For example, in prokaryotes, control sequences typically include promoters, ribosome binding sites, and terminators. In eukaryotes, control sequences generally include promoters, terminators, and in some cases enhancers, transactivators, and / or transcription factors.

[0272] The vector used in the present invention is preferably an expression vector. The expression vector is capable of directing the replication and expression of the nucleic acid molecule of the present invention in a host cell, and thus provides, for example, the expression of the heavy chain and / or light chain variable domains of the 1,6fucAFP monoclonal antibody or antigen-binding fragment of the present invention. In some embodiments, the vector may contain further sequences to ensure that not only the heavy chain and light chain variable domains are expressed, but also the remaining heavy chain and light chain constant regions are expressed, so that a full-length antibody (e.g., IgG) comprising the heavy chain and light chain variable domains of the present invention is expressed. Suitable expression vectors have been widely described in the literature, and those skilled in the art can easily determine the expression vector suitable for a particular cell system using conventional methods. Preferably, the vector disclosed herein comprises a recombinant polynucleotide (i.e., a nucleic acid sequence encoding a monoclonal antibody according to the present invention) and an expression control sequence operably connected. The vector as provided herein preferably further comprises a promoter. The vector described herein may also include a selection marker gene and a replication origin to ensure replication in the host; moreover, the vector provided herein may also include a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in host cells.

[0273] The nucleic acid molecules and / or vectors of the present invention can be designed to be transfected 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, nuclear transfection), non-chemical methods (electroporation, sonoporation, phototransfection, gene electrotransfer, hydrodynamic delivery, or transformation that occurs naturally after contacting cells with nucleic acid molecules of the present invention), particle-based methods (gene guns, magnetic transfection, puncture infection), phage-based methods, and viral methods. For example, expression vectors derived from viruses (e.g., retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, Semliki forest viruses, or bovine papilloma viruses) can be used to transfect nucleic acid molecules into a target cell population. In addition, baculovirus systems can also be used as vectors in eukaryotic expression systems for nucleic acid molecules of the present invention.

[0274] The term "prokaryotic organism" is intended to include all bacteria that can be transformed, transduced or transfected with DNA or DNA or RNA molecules to express the protein of the present invention. Prokaryotic hosts can include gram-negative bacteria and gram-positive bacteria, such as Escherichia coli, Serratia typhimurium, Serratia marcescens, Corynebacterium (Corynebacterium glutamicum), Pseudomonas (fluorescein Pseudomonas), Lactobacillus, Streptomyces, Salmonella and Bacillus subtilis. The term "eukaryotic" refers to yeast, higher plants, insects and mammalian cells. Non-limiting examples of mammalian host cells commonly 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 COS 7), CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.

[0275] According to a fourth aspect, the present invention relates to a host cell comprising a polynucleotide or a polynucleotide set according to the second aspect of the present invention or a vector according to the third aspect. The host cell can be configured so as to express the 1,6fucAFP antibody or antigen-binding fragment provided herein. 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 cells and eukaryotic cells are disclosed above in the context of the third aspect of the present invention. In a specific embodiment, the cell is a HEK cell. In another specific embodiment, the host cell is a CHO cell.

[0276] Preferably, the host cell according to the present invention is a eukaryotic cell (e.g., HEK or CHO). Although it is possible to express antibodies and antigen-binding fragments as disclosed herein in both prokaryotic and eukaryotic host cells, it is preferred to express the antibodies in eukaryotic cells, and most preferably in mammalian host cells, because such eukaryotic cells (particularly the most preferred mammalian cells) are more likely to express properly folded antibodies / antibody fragments containing appropriate post-translational modifications such that they are immunologically active.

[0277] When a recombinant expression vector encoding an antibody heavy chain and / or light chain variable domain as 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 of time sufficient to allow the antibody or antigen-binding fragment to be expressed in the host cell, or preferably, to allow the antibody or antigen-binding fragment to be secreted into the culture medium in which the host cell is grown. The antibody and / or antigen-binding fragment can be recovered from the culture medium using standard protein purification methods.

[0278] Therefore, in a fifth aspect, the present invention also provides a method for producing a 1,6-fucAFP antibody or a 1,6-fucAFP antibody antigen-binding fragment as disclosed herein, the method comprising culturing a host cell of the present invention expressing the antibody or antigen-binding fragment of the present invention under suitable conditions and isolating the produced 1,6-fucAFP antibody or antigen-binding fragment.

[0279] The transformed host cells can be grown in bioreactors and cultured according to techniques known in the art to achieve optimal cell growth. Antibodies and / or antibody antigen-binding fragments of the present invention can be subsequently separated from cell fractions or growth medium by any conventional method, such as, but not limited to, affinity chromatography (e.g., using fusion tags such as Strep-tag II or His6 tags), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC or immunoprecipitation.

[0280] In a sixth aspect, provided herein is a 1,6-fucAFP antibody or antigen-binding fragment obtained or obtainable by a method according to the fifth aspect of the invention.

[0281] In a seventh aspect, the present invention relates to a composition comprising a 1,6-fucAFP antibody or antigen-binding fragment of the present invention, a polynucleotide of the present invention, a vector of the present invention, or a host cell of the present 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 used in an in vitro diagnostic test to detect α-1,6-core-fucosylated AFP (i.e., a clinically relevant portion of AFP-L3). In a preferred embodiment, the diagnostic composition may be a reagent for an immunoassay for detecting α-1,6-core-fucosylated AFP (i.e., a clinically relevant portion of AFP-L3). The diagnostic composition is preferably configured so that it allows detection of α-1,6-core-fucosylated AFP (i.e., a clinically relevant portion of AFP-L3) in a sample obtained from a subject. The sample may be a body fluid. In a specific embodiment, the sample may be a blood sample (e.g., whole blood, serum, or plasma).

[0282] In an embodiment, the composition of the invention is a composition for detecting (preferably quantifying) alpha-1,6-core-fucosylated AFP (i.e., the clinically relevant portion of AFP-L3) in a sample in vitro, preferably using an immunoassay. In an embodiment, the immunoassay is a heterogeneous 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 an immunoassay of the invention.

[0283] The 1,6fucAFP antibodies and antibody antigen-binding domains of the present invention and methods of producing and using the same are not only provided as diagnostic tools, but are also envisioned to have applicability in treating and alleviating diseases and disease symptoms, as well as applicability in model systems for studying disease therapies. Accordingly, the present invention provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers and (i) a 1,6fucAFP antibody or 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 polynucleotide of (ii) and / or the vector of (iii) expressing the antibody or antigen-binding fragment of (i).

[0284] Pharmaceutical compositions disclosed herein are formulated to be suitable for administration to human or animal subjects. In the manufacture of pharmaceutical preparations, the antibodies or antigen-binding fragments of the present invention are mixed with pharmaceutical carriers, excipients and / or diluents. Of course, the carrier, excipient and / or diluent must be acceptable, which means that it is compatible with any other ingredients in the formulation and must not be harmful to the subject. Examples of suitable pharmaceutical carriers for use with antibody-based compositions are well known in the art and can be prepared by conventional methods.

[0285] In an eighth aspect, the present invention provides a 1,6fucAFP antibody or antigen-binding fragment according to the present invention or a composition according to the present invention for use in an in vitro immunoassay, in particular for detecting (and optionally quantifying) α-1,6-core-fucosylated AFP (or a partial sequence thereof comprising α-1,6-core-fucosylated AFP) in a sample. Since α-1,6-core-fucosylated AFP is a core component of AFP-L3, the immunoassay can also be referred to as an immunoassay for detecting (and optionally quantifying) AFP-L3 in a sample.

[0286] 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.

[0287] Non-limiting examples of immunoassays are enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), Western blot assay or immunoassays based on luminescent, fluorescent, chemiluminescent or electrochemiluminescent detection.

[0288] Further exemplary immunoassay formats for detection and / or quantification using 1,6-fucAFP antibodies or antigen-binding fragments (also different antibodies provided herein) are also described in EP 3 252 073, US2018 / 0110889, and Egashira Y et al. (Scientific Reports, 2019, 9: 12359), all of which are incorporated herein by reference in their entirety.

[0289] The principle of the immunoassay using the 1,6-fucAFP antibody or antigen-binding fragment (or a composition comprising it) according to the present invention is to incubate the 1,6-fucAFP antibody or antigen-binding fragment (or a composition comprising it) with a sample (suspected of containing or containing α-1,6-core-fucosylated AFP). If the target analyte (i.e., α-1,6-core-fucosylated AFP) is present, a detection complex comprising the 1,6-fucAFP antibody or antigen-binding fragment and α-1,6-core-fucosylated AFP is formed by antibody-antigen binding reaction / binding. In order to detect and / or quantify α-1,6-core-fucosylated AFP, the formation of the detection complex can be detected. Methods for detecting the formation of the detection complex are well known. For example, the detection may involve a detection label (e.g., attached to the antibody or antigen-binding fragment of the present invention).

[0290] Therefore, the use according to the eighth aspect of the present invention may include:

[0291] a) mixing a sample containing α-1,6-core-fucosylated AFP with the 1,6-

[0292] fucAFP antibody or antigen-binding fragment is incubated together to form a core containing α-1,6-

[0293] - a detection complex of fucosylated AFP and the 1,6-fucAFP antibody or antigen-binding fragment of the present invention; and

[0294] b) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex.

[0295] As shown in the appended examples, the signal in immunoassays can be substantially increased by pretreatment (eg, reducing pretreatment) of samples containing α-1,6-core-fucosylated AFP. Pretreatment appears to facilitate accessibility of the epitope of the antibodies or antigen-binding fragments of the invention.

[0296] Thus, the use according to the eighth aspect may comprise adding a pre-treatment agent according to the invention (as described elsewhere herein) to the sample, preferably prior to incubating the sample with the 1,6-fucAFP antibody or antigen-binding fragment of the invention.

[0297] The use may also include further diluting the sample after pretreatment, for example, by adding a certain volume to modify, preferably before or simultaneously adding the 1,6-fucAFP antibody or antigen-binding fragment of the invention to the sample. In other words, the concentration of the pretreatment agent may be reduced to a concentration acceptable for forming a detection complex. For example, the concentration of the components of the pretreatment agent may be diluted between 2 and 3 times.

[0298] In an embodiment, the pretreatment may be a reducing pretreatment, ie, may include adding a reducing agent (eg, a protein reducing agent) to the sample. In other words, the pretreatment reagent may contain or consist of a reducing agent.

[0299] A protein reducing agent is a reducing agent that is capable of reducing / destroying disulfide bonds within a protein / polypeptide chain or between protein / polypeptide chains.

[0300] Non-limiting examples of reducing agents or protein reducing agents for pretreatment are dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), β-mercaptoethanol and dithiobutylamine (DTBA). In an embodiment, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and dithiobutylamine (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.

[0301] In an embodiment, a protein reducing agent is used that has 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.

[0302] The pH during the pretreatment is selected so that the reducing compound has effective reducing ability. As is well known in the art, DTT, for example, has effective reducing activity only at pH 6.5 to 9.0 (preferably pH 7 to 8). TCEP, for example, has redox activity in the pH range of about 1.5 to about 8.5.

[0303] The concentration of the (protein) reducing agent may vary and depends on the exact reducing agent used. As shown in the accompanying DTT example, the concentration for effective pretreatment can be determined by comparing the immunoassay signal of samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of the (protein) reducing agent prior to performing an otherwise identical immunoassay.

[0304] Exemplarily, if DTT is used as a (protein) reducing agent, the final concentration of DTT in the sample during pretreatment may be 0.1mM to 100mM, 0.1mM to 60mM, 0.1mM to 50mM, or 0.1mM to 47.8mM. In an embodiment, the DTT concentration may be at least 0.2mM, at least 0.3mM, at least 0.5mM, or at least 1mM. In an embodiment, the DTT concentration may be at most 100mM, at most 60mM, at most 50mM, or at most 47.8mM. In an embodiment, the DTT concentration may be 2.2mM. In an embodiment, the DTT concentration may be 2.3mM. In an embodiment, the DTT final concentration of DTT in the mixture of pretreatment agent and sample may be 0.6mM to 107.8mM. In an embodiment, the DTT final concentration of DTT in the mixture of pretreatment agent and sample may be 0.7mM to 107.8mM.

[0305] The concentration of DTT in the pretreatment agent is higher than the concentration of DTT in the final mixture of the sample during the pretreatment. For example, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold concentrate); i.e., a stock solution.

[0306] The pretreatment agent for sample pretreatment can further comprise a chelating agent, such as a chelating agent combined with ions. It has been found that adding this type of chelating agent increases the stability of the pretreatment agent. In a particular embodiment, the chelating agent can be a chelating agent combined with ions. In a more specific embodiment, the chelating agent can be a chelating agent combined with divalent ions.

[0307] Non-limiting examples of chelating agents for binding to (divalent) ions are aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrins, polyamines, crown ethers, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0308] Other 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 triamide thiol, DADS, MAMA, DADT, N2S4 diamine tetrathiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propylamine oxime, tetraamine, cyclamine, or a combination thereof.

[0309] In an embodiment, the chelating agent is EDTA.

[0310] The concentration of chelating agent during sample pretreatment may vary and may depend on the exact chelating agent and reducing agent used.A useful concentration range can be determined by comparing the immunoassay signal of different samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of chelating agent.

[0311] For example, if EDTA is used, 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.

[0312] In an embodiment, the pretreatment agent further comprises a buffer substance (also referred to herein as a buffer). The buffer can be selected based on the pH required for the protein reducing agent to reduce disulfide bonds.

[0313] In embodiments, the pretreatment agent comprises or consists of a protein reducing agent and an ion (eg, divalent ion) chelator and optionally a buffer.

[0314] In an embodiment, the pretreatment agent comprises DTT and EDTA and optionally a buffer.

[0315] The pretreatment of the sample can be carried out for different times. For example, the pretreatment can be carried out for 1 min to 30 min, 2 min to 20 min, or 3 min to 10 min. In an embodiment, the pretreatment can be carried out for 9 min.

[0316] In an embodiment, the pretreatment may 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 min). Optionally, a chelating agent (e.g., EDTA) as described above may also be present. In an embodiment, the pretreatment may also include adding a buffer (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) to the sample, thereby adjusting the pH in the sample to above 7. After pretreatment, the concentration of DTT may be reduced so that the detection complex can still be formed.

[0317] In embodiments, the use may further comprise incubating the sample with an AFP-specific antibody that does not compete with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention for binding to AFP. In these embodiments, the detection complex further comprises the AFP-specific antibody, i.e., a sandwich structure comprising 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 1,6-fucAFP antibody or antigen-binding fragment of the present invention for binding to AFP. In embodiments using pretreatment, the AFP-specific antibody should be selected so that the antibody can still bind to AFP in the presence of a pretreatment agent.

[0318] A non-limiting example of an AFP-specific antibody that does not compete with the antibodies of the present invention for binding is the commercially available anti-AFP antibody Tu11 (Roche Diagnostics Deutschland GmbH, material number: 11492080103). Therefore, an antibody that does not compete with the antibodies of the present invention for binding to AFP may be an antibody or antigen-binding fragment thereof that has the same epitope as Tu11 or competes with the Tu11 antibody for binding to AFP. As demonstrated in the attached examples, the Tu11 antibody is also compatible with the pretreatment of samples according to the present invention.

[0319] In an embodiment, the use may include digesting the 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, wherein the glycopeptide comprises a partial AFP sequence containing Asn-251 of α-1,6-core-fucosylation of AFP (e.g., as described elsewhere herein). Optionally, the reagents used to generate the glycopeptide or peptide may be removed or inactivated prior to adding the antibody or antigen-binding fragment of the invention. The presence or level of α-1,6-core-fucosylated AFP in the sample may then be determined based on the detection of a detection complex comprising a partial AFP sequence containing Asn-251 of α-1,6-core-fucosylation of AFP and the antibody or antigen-binding fragment of the invention.

[0320] The immunoassay in which the 1,6fucAFP antibody or antigen-binding fragment according to the present invention is used may be a heterogeneous immunoassay.

[0321] Additionally or alternatively, the 1,6-fucAFP antibodies or antigen-binding fragments according to the invention may be used in sandwich immunoassays (eg, heterogeneous sandwich immunoassays).

[0322] In embodiments, the immunoassay may be an immunohistochemistry (IHC) assay.

[0323] In embodiments, immunoassays can be used to detect the glycopeptide of Formula I or a glycoprotein comprising the glycopeptide of Formula I.

[0324] 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 comprising 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. Therefore, the present invention also provides the use of 1,6fucAFP antibodies and antigen-binding fragments for distinguishing α-1,6-core-fucosylated AFP or a partial AFP sequence comprising 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.

[0325] 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 comprising the α-1,6-core-fucosylated AFP in a sample using the 1,6-fucAFP antibody or antigen-binding fragment of the present invention.

[0326] The embodiments disclosed in the context of the use according to the eighth aspect apply mutatis mutandis.

[0327] In an embodiment, the method according to the ninth aspect may include:

[0328] (i) binding the antibody or antigen-binding fragment of the present invention to the α-1,6-core-fucosylated AFP or a partial AFP sequence containing the α-1,6-core-fucosylation contained in the sample to form a detection complex; and

[0329] (ii) detecting the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation by detecting the detection complex, and thereby determining the presence and, optionally, the amount of the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation in the sample.

[0330] In an embodiment, the method according to the ninth aspect may include:

[0331] a) mixing a sample containing α-1,6-core-fucosylated AFP with the 1,6-

[0332] fucAFP antibody or antigen-binding fragment is incubated together to form a core containing α-1,6-

[0333] - a detection complex of fucosylated AFP and the 1,6-fucAFP antibody or antigen-binding fragment of the present invention; and

[0334] b) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex.

[0335] As shown in the accompanying examples, the signal in an immunoassay can be substantially increased by pretreatment (e.g., reducing pretreatment) of a sample containing α-1,6-core-fucosylated AFP. In the context of full-length α-1,6-core-fucosylated AFP, pretreatment appears to facilitate the accessibility of the epitope of the antibody or antigen-binding fragment of the invention.

[0336] Thus, the method according to the ninth aspect of the invention may comprise adding a pre-treatment agent according to the invention to the sample, preferably prior to incubating the sample with the 1,6-fucAFP antibody or antigen-binding fragment of the invention.

[0337] Therefore, the method according to the ninth aspect may include:

[0338] a) incubating a sample (eg, a sample comprising α-1,6-core-fucosylated AFP) in the presence of a pretreatment agent according to the present invention;

[0339] b) incubating the pretreated sample with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention, so 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 has been formed; and

[0340] c) detecting the presence or level of α-1,6-core-fucosylated AFP in the sample by detecting the detection complex.

[0341] The method may include further diluting the sample after pretreatment, for example, by adding a certain volume to modify, preferably before or simultaneously adding the antibody or antigen-binding fragment of the invention to the sample. In other words, the concentration of the pretreatment agent can be reduced to a concentration acceptable for forming a detection complex.

[0342] In an embodiment, the pretreatment may be a reducing pretreatment, ie, may include adding a reducing agent (eg, a protein reducing agent) to the sample. In other words, the pretreatment reagent may contain or consist of a reducing agent (eg, a protein reducing agent).

[0343] A protein reducing agent is a reducing agent that is capable of reducing / destroying disulfide bonds within a protein / polypeptide chain or between protein / polypeptide chains.

[0344] Non-limiting examples of reducing agents or protein reducing agents for pretreatment are dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), β-mercaptoethanol and dithiobutylamine (DTBA). In an embodiment, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and dithiobutylamine (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 dithiothreitol (DTT).

[0345] In an embodiment, a protein reducing agent is used that has 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.

[0346] The pH during the pretreatment is selected so that the reducing compound has effective reducing ability. As is well known in the art, DTT, for example, has effective reducing activity at pH values ​​of 6.5 to 9.0 (preferably pH 7 to 8). TCEP, for example, has redox activity in a pH range of about 1.5 to about 8.5.

[0347] The concentration of the (protein) reducing agent may vary and depends on the exact reducing agent used. As shown in the accompanying DTT example, the concentration for effective pretreatment can be determined by comparing the immunoassay signal of samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of the (protein) reducing agent prior to performing an otherwise identical immunoassay.

[0348] Exemplarily, if DTT is used as a (protein) reducing agent, the final concentration of DTT in the sample during pretreatment may be 0.1mM to 100mM, 0.1mM to 60mM, 0.1mM to 50mM, or 0.1mM to 47.8mM. In an embodiment, the DTT concentration may be at least 0.2mM, at least 0.3mM, at least 0.5mM, or at least 1mM. In an embodiment, the DTT concentration may be at most 100mM, at most 60mM, at most 50mM, or at most 47.8mM. In an embodiment, the DTT concentration may be 2.2mM. In an embodiment, the DTT concentration may be 2.3mM. In an embodiment, the DTT final concentration of DTT in the mixture of pretreatment agent and sample may be 0.6mM to 107.8mM. In an embodiment, the DTT final concentration of DTT in the mixture of pretreatment agent and sample may be 0.7mM to 107.8mM.

[0349] The concentration of DTT in the pretreatment agent is higher than the concentration of DTT in the final mixture of the sample during the pretreatment. For example, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold concentrate); i.e., a stock solution.

[0350] The pretreatment agent for sample pretreatment can further include a chelating agent, such as a chelating agent combined with an ion. It has been found that this type of chelating agent improves the stability of the pretreatment agent including a reducing agent such as DTT. In a specific embodiment, the chelating agent can be a chelating agent combined with an ion. In a more specific embodiment, the chelating agent can be a chelating agent combined with a divalent ion.

[0351] Non-limiting examples of chelating agents for binding to (divalent) ions are aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrins, polyamines, crown ethers, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0352] Other 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 triamide thiol, DADS, MAMA, DADT, N2S4 diamine tetrathiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propylamine oxime, tetraamine, cyclamine, or a combination thereof.

[0353] In an embodiment, the chelating agent is EDTA.

[0354] The concentration of chelating agent during sample pretreatment may vary and may depend on the exact chelating agent and reducing agent used.A useful concentration range can be determined by comparing the immunoassay signal of different samples containing detectable levels of α-1,6-core-fucosylated AFP using different concentrations of chelating agent.

[0355] For example, if EDTA is used, 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.

[0356] In an embodiment, the pretreatment agent further comprises a buffer substance (also referred to herein as a buffer). The buffer can be selected based on the pH required for the protein reducing agent to reduce disulfide bonds.

[0357] In embodiments, the pretreatment agent comprises or consists of a protein reducing agent and an ion (eg, divalent ion) chelator and optionally a buffer.

[0358] In an embodiment, the pretreatment agent comprises DTT and EDTA and optionally a buffer.

[0359] The pretreatment of the sample can be carried out for different times. For example, the pretreatment can be carried out for 1 min to 30 min, 2 min to 20 min, or 3 min to 10 min. In an embodiment, the pretreatment can be carried out for 9 min.

[0360] In an embodiment, 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 min). Optionally, a chelating agent (e.g., EDTA) as described above may also be present. In an embodiment, pretreatment may also include adding a buffer (e.g., Tris concentration of 10 mM to 200 mM, 30 mM to 150 mM, 50 mM to 130 mM or 100 mM) to the sample, thereby adjusting the pH in the sample to above 7. Such pH ensures the reduction potential of DTT. Preferably, the pH may be adjusted to a pH between 7 and 10, between 7 and 9, or between 7 and 8.

[0361] After pretreatment, the concentration of DTT can be reduced so that the detection complex can still form.

[0362] In an embodiment, the method of the ninth aspect may further include incubating the sample with an AFP-specific antibody that does not compete with the 1,6-fucAFP antibody or antigen-binding fragment of the present invention for binding to AFP. In these embodiments, the detection complex further comprises the AFP-specific antibody, i.e., a sandwich structure comprising 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 1,6-fucAFP antibody or antigen-binding fragment of the present invention for binding to AFP. In embodiments using pretreatment, the AFP-specific antibody should be selected so that the antibody can still bind to AFP in the presence of a pretreatment agent.

[0363] A non-limiting example of an AFP-specific antibody that does not compete with the antibodies of the present invention for binding is the commercially available anti-AFP antibody Tu11 (Roche Diagnostics Deutschland GmbH, material number: 11492080103). Therefore, an antibody that does not compete with the antibodies of the present invention for binding to AFP may be an antibody or antigen-binding fragment thereof that has the same epitope as Tu11 or competes with the Tu11 antibody for binding to AFP. As demonstrated in the attached examples, the Tu11 antibody is also compatible with the pretreatment of samples according to the present invention.

[0364] In an embodiment, the method of the ninth aspect may include digesting the 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, wherein the glycopeptide comprises a partial AFP sequence containing Asn-251 of α-1,6-core-fucosylated AFP (e.g., as described elsewhere herein). Optionally, the reagents used to generate the glycopeptide or peptide may be removed or inactivated prior to adding the antibody or antigen-binding fragment of the present invention. The presence or level of α-1,6-core-fucosylated AFP in the sample may then be determined based on the detection of a detection complex comprising a partial AFP sequence containing Asn-251 of α-1,6-core-fucosylated AFP and the antibody or antigen-binding fragment of the present invention.

[0365] In an embodiment, the method according to the ninth aspect may be a heterogeneous immunoassay. In an embodiment, the method according to the ninth aspect may be a sandwich immunoassay (e.g., a heterogeneous sandwich immunoassay). In an embodiment, the method according to the ninth aspect may be an immunohistochemistry (IHC) assay and the sample may be a tissue slide. In an embodiment, the method according to the ninth aspect is a humoral immunoassay, wherein the sample is a body fluid.

[0366] The sample used in the method according to the ninth aspect 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.

[0367] The method according to the ninth aspect is an in vitro method performed on a sample previously obtained from a subject.

[0368] As known in the art, AFP-L3 is a useful marker, preferably in combination with other markers, for detecting early hepatocellular carcinoma (HCC), and α-1,6-core-fucosylated AFP is a core component of AFP-L3.

[0369] Thus, provided herein is an in vitro method for aiding in the detection or detection of HCC, the method comprising:

[0370] 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;

[0371] b) The determined level of α-1,6-core-fucosylated AFP in the sample was compared with the

[0372] - a reference level of fucosylated AFP for comparison; and

[0373] c) assisting in detecting HCC or detecting HCC based on the comparison.

[0374] In particular, AFP-L3 and therefore α-1,6-core-fucosylated AFP are also often 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, involving sex, age, AFP-L3, AFP and DCP (also known as PIVKA-II).

[0375] Therefore, the present invention also provides an in vitro method for facilitating the detection or detection of HCC, the method comprising:

[0376] a) determining the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using a method according to the ninth aspect of the invention;

[0377] b) calculating a score for HCC detection taking into account the determined level of α-1,6-core-fucosylated AFP; and

[0378] c) aiding in the detection of HCC or detecting HCC based on the calculated score (eg, by comparison with a reference value of said score that is indicative of HCC).

[0379] The score for HCC detection can take into account the level of at least one additional biomarker for HCC detection (e.g., AFP or DCP). Thus, the method can further include determining or receiving the level of at least one additional HCC biomarker in the sample or another sample taken from the subject (e.g., at the same time).

[0380] The score for HCC detection may additionally or alternatively take into account clinical parameters (e.g., ultrasound data) or demographic information (e.g., age and / or gender) of the subject. Thus, the method may further include receiving the clinical parameters or the demographic information of the subject.

[0381] In a particular embodiment, the score for HCC detection may include the AFP level and DCP level in the sample, as well as the sex and age of the subject, i.e., may be a GALAD score. Thus, the method may further include determining or receiving the AFP level and DCP (PIVKA-II) level in the sample or another sample taken from the subject (e.g., simultaneously) and the age and sex of the receiving subject.

[0382] Also provided herein is a computer-implemented method for detecting HCC, the method comprising:

[0383] a) receiving the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject using a method according to the ninth aspect of the invention;

[0384] b) calculating a score for HCC detection taking into account the determined level of α-1,6-core-fucosylated AFP; and

[0385] c) aiding in the detection of HCC or detecting HCC based on the calculated score (eg, by comparison with a reference value of said score that is indicative of HCC).

[0386] The embodiments of the aforementioned in vitro method for detecting HCC using a fraction taking into account the level of α-1,6-core-fucosylated AFP in a sample obtained from a subject determined using the method according to the ninth aspect of the invention apply mutatis mutandis with the only exception that the levels of other biomarkers are received but not determined.

[0387] In a tenth aspect, the present invention provides a pretreatment reagent for use in an immunoassay for detecting α-1,6-core-fucosylated AFP (eg, using an antibody or antigen-binding fragment of the present invention), comprising a reducing agent.

[0388] The term pretreatment reagent is used interchangeably herein with the terms "pretreatment agent," "pretreating agent," and "pretreating reagent," and relates to a pretreatment composition applied to a sample (e.g., a blood sample such as serum or plasma) to promote binding of 1,6-fucAFP antibodies to α-1,6-core-fucosylated AFP contained in the sample.

[0389] In an embodiment, the reducing agent in the pretreatment reagent of the tenth aspect of the present disclosure may be a protein reducing agent. In other words, the pretreatment reagent may contain or consist of a protein reducing agent.

[0390] A protein reducing agent is a reducing agent that is capable of reducing / destroying disulfide bonds within a protein / polypeptide chain or between protein / polypeptide chains.

[0391] Non-limiting examples of reducing agents or protein reducing agents are dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), β-mercaptoethanol and dithiobutylamine (DTBA). In an embodiment, the reducing agent or protein reducing agent can be selected from dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP) and dithiobutylamine (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 dithiothreitol (DTT).

[0392] 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.

[0393] The pH of the pretreatment reagent can be selected within a wide range and can vary depending on the components included in the pretreatment reagent. As needed, the pH can be selected to make the reduction potential as high as possible, or the pH can be selected to maintain the stability (shelf life) of the reducing agent. In an embodiment, the pH can be selected so that the pH of the sample to which the pretreatment reagent is added is adjusted to a pH that allows the reducing activity of the reducing agent after being added to the sample.

[0394] Exemplarily, the reducing agent is DTT and the pH of the pretreatment agent is selected to be below pH 6.5 (e.g., pH 4 to 6.5, pH 5 to 6.5, pH 5 to 6.5, or pH 5.5 to 6.5). pH values ​​within this range stabilize DTT, i.e., allow for longer storage of the pretreatment agent.

[0395] The concentration of the (protein) reducing agent in the pretreatment agent can vary and depends on the exact reducing agent used and the factor by which the pretreatment agent is concentrated to the final use concentration. In addition, the stability of the reducing agent may depend on the exact concentration. The technician can select the concentration of the reducing agent in the pretreatment agent so that it promotes the binding of the 1,6fucAFP antibody or antigen-binding fragment to the α-1,6-core-fucosylated AFP in the sample (e.g., a blood sample such as plasma or serum). For example, the signal of an immunoassay using a control sample containing α-1,6-core-fucosylated AFP and using different concentrations of reducing agents 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.

[0396] Exemplarily, if DTT is used as a reducing agent, the concentration in the pretreatment agent can be selected so that a 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 more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In an embodiment, the concentration in the pretreatment agent can be selected so that a 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 more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In embodiments, the concentration of DTT in the pretreatment agent can be selected so that a 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 more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In embodiments, the concentration in the pretreatment agent can be selected so that a final concentration of DTT in the sample during pretreatment of 2.2 mM can be achieved (e.g., without diluting the sample more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In embodiments, the concentration in the pretreatment agent can be selected so that a final concentration of DTT in the sample during pretreatment of 2.3 mM can be achieved (e.g., without diluting the sample more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In embodiments, the concentration of DTT in the pretreatment agent can be selected so that a 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 more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent). In embodiments, the concentration of DTT in the pretreatment agent can be selected so that a 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 more than 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold by adding the pretreatment agent).

[0397] Exemplary concentrations of reducing agents (e.g., DTT) in the pretreatment agent may 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 embodiments, the concentration of reducing agents (e.g., DTT) in the pretreatment agent may be 1 mM to 10 mM (e.g., 10 mM). In embodiments, the concentration of reducing agents (e.g., DTT) in the pretreatment agent may be 3 mM to 110 mM.

[0398] In an embodiment, the pretreatment agent can be a 2-fold to 20-fold or 2-fold to 10-fold concentrate (e.g., a 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20-fold concentrate); i.e., a stock solution. In an embodiment, the pretreatment agent can be a 4 to 8-fold concentrate.

[0399] The pretreatment agent according to the tenth aspect of the present invention may further comprise a chelating agent, such as a chelating agent combined with an ion. It has been found that the addition of such a chelating agent improves the stability of the pretreatment agent including a reducing agent such as DTT. In a particular embodiment, the chelating agent may be a chelating agent combined with an ion. In a more specific embodiment, the chelating agent may be a chelating agent combined with a divalent ion.

[0400] Non-limiting examples of chelating agents for binding to (divalent) ions are aminopolycarboxylic acids such as ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA), or porphyrins, polyamines, crown ethers or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0401] Other 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 triamide thiol, DADS, MAMA, DADT, N2S4 diamine tetrathiol, N2P2 dithiol-bisphosphine, 6-hydrazinonicotinic acid, propylamine oxime, tetraamine, cyclamine, or a combination thereof.

[0402] In an embodiment, the chelating agent is EDTA.

[0403] The concentration of the chelating agent in the pretreatment agent can vary and depends on the exact chelating agent used and the factor by which the pretreatment agent is concentrated to the final use concentration. The technician can select the concentration of the chelating agent in the pretreatment agent so that it promotes the binding of the 1,6fucAFP antibody or antigen-binding fragment to the α-1,6-core-fucosylated AFP in the sample (e.g., a blood sample such as plasma or serum). For example, the signal of an immunoassay using a control sample containing α-1,6-core-fucosylated AFP and using different concentrations of chelating agents 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 attached examples.

[0404] For example, if EDTA is used as a 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 (eg, 4 mM).

[0405] In an embodiment, the pretreatment agent further comprises a buffer 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. For example, Tris, Hepes or citrate can be used.

[0406] In embodiments, the pretreatment agent comprises or consists of a protein reducing agent and an ion (eg, divalent ion) chelator and optionally a buffer.

[0407] In an embodiment, the pretreatment agent comprises DTT and EDTA and optional buffer. The pretreatment agent can be a 4 to 5-fold concentrate and can comprise 8 to 12 mM DTT (e.g., 10 mM) and 1 to 5 mM EDTA (e.g., 4.5 mM). The pH of the pretreatment agent can be pH 5.5 or lower (e.g., pH 4 to 5.5, or pH 5.5).

[0408] In a specific embodiment, a pretreatment reagent set is provided for use in an immunoassay for detecting α-1,6-core-fucosylated AFP (e.g., using an antibody or antigen-binding fragment of the invention). The pretreatment reagent set may comprise a first and a second pretreatment composition, each provided in a separate container. The first pretreatment composition may be any of the pretreatment agents defined in the context of the tenth aspect and comprises a reducing agent. The pH of the first pretreatment composition is preferably selected so that the stability of the components is high.

[0409] The second pretreatment composition may comprise a buffer, and may have a pH and buffer concentration that permits reducing activity of the reducing agent if both the first and second pretreatment compositions are added to the sample.

[0410] In a specific embodiment, the first pretreatment composition can include DTT and EDTA. The pretreatment agent can be 4 to 5 times concentrate and can include 8 to 12mM DTT (e.g., 10mM) and 1 to 5mM EDTA (e.g., 2mM). The pH of the pretreatment agent can be pH 5.5 or lower (e.g., pH 4 to 5.5, or pH 5.5). The second pretreatment composition can include a buffer and can have a pH of 6.5 to 9, such as 7.5 to 8.5. The buffer concentration can be 10mM to 150mM, 20mM to 100mM or 30mM to 80mM (e.g., 100mM). The buffer can be a buffer with a pKa of 7 to 9, such as 7.5 to 8.5. In an embodiment, the buffer can be Tris (e.g., pH 8.5).

[0411] In the eleventh aspect, the present invention provides a kit comprising the 1,6fucAFP antibody or antigen-binding fragment of the present invention. The kit can be a kit for detecting and / or quantifying α-1,6-core-fucosylated AFP or a partial AFP sequence comprising the α-1,6-core-fucosylated AFP in a sample (e.g., a blood sample, such as plasma or serum). The kit can be a kit for detecting and / or quantifying α-1,6-core-fucosylated AFP in a sample (e.g., a blood sample, such as plasma or serum).

[0412] In embodiments, the kit may be an immunoassay kit, such as a kit for a sandwich immunoassay (eg, a heterogeneous sandwich immunoassay).

[0413] In a specific embodiment, the kit according to the eleventh aspect of the invention further comprises a pretreatment agent (see above) or a set of pretreatment agents (see above) of the invention. In an embodiment, the antibody or antigen-binding fragment and the pretreatment agent are provided in separate containers.

[0414] As demonstrated in the appended examples, especially the combination of the 1,6fucAFP antibodies or antigen-binding fragments of the invention with the pretreatment agents of the invention allows the detection and quantification of native α-1,6-core-fucosylated AFP with a high signal using immunoassays such as those described in the context of the present invention.

[0415] The present invention also particularly relates to the following items:

[0416] 1. 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 comprising said α-1,6-core-fucosylation.

[0417] 2. The monoclonal antibody or antigen-binding fragment according to item 1, comprising

[0418] (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by one amino acid substitution;

[0419] CDR-H2 having an amino acid sequence of SEQ ID NO: 4 or 5 or a variant thereof modified by up to two amino acid substitutions; and CDR-H3 having an amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by one amino acid substitution.

[0420] sequence; and

[0421] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which has 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, which has the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and CDR-L3, which has the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution.

[0422] 3. The monoclonal antibody or antigen-binding fragment according to item 1 or 2, comprising

[0423] (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 thereof 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

[0424] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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; CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution.

[0425] 4. The monoclonal antibody or antigen-binding fragment according to item 2 or 3, wherein the sum 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.

[0426] 5. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 4, comprising

[0427] (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 having an amino acid sequence of SEQ ID NO: 3; a CDR-H2 having an 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 an amino acid sequence of SEQ ID NO: 6; and

[0428] (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.

[0429] 6. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 5, comprising

[0430] (i) a heavy chain variable domain (VH), the 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

[0431] (ii) a light chain variable domain (VL), which VL comprises: CDR-L1, which consists 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, which consists of the amino acid sequence of SEQ ID NO: 9; and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 10.

[0432] 7. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 6, wherein the amino acid substitution in the variant of SEQ ID NO: 4 or 5 comprises an amino acid substitution at position 5 of SEQ ID NO: 4 or an amino acid substitution at position 4 of SEQ ID NO: 5, and / or wherein the amino acid substitution in the variant of SEQ ID NO: 7 or 8 comprises an amino acid substitution at position 7 of SEQ ID NO: 7 or 8, respectively.

[0433] 8. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 7, wherein the amino acid substitution in the variant of SEQ ID NO: 4 or 5 comprises 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 wherein the amino acid substitution in the variant of SEQ ID NO: 7 or 8 comprises a conservative amino acid substitution at position 7 of SEQ ID NO: 7 or 8.

[0434] 9. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 7, wherein all of the amino acid substitutions are conservative or highly conservative amino acid substitutions.

[0435] 10. The monoclonal antibody or antigen-binding fragment according to any one of items 2 to 8, wherein the amino acid corresponding to the amino acid at position 5 of SEQ ID NO: 4 or at position 4 of SEQ ID NO: 5 in the variant of SEQ ID NO: 4 or 5 is serine or asparagine; and / or wherein the amino acid corresponding to amino acid position 7 of SEQ ID NO: 7 or 8 in the variant SEQ ID NO: 7 or 8 is serine or glycine.

[0436] 11. The monoclonal antibody or antigen-binding fragment according to any one of items 7, 8 and 10, wherein all amino acid substitutions except at position 5 of SEQ ID NO: 4 or position 4 of SEQ ID NO: 5 and / or position 7 of SEQ ID NO: 7 or 8 are conservative or highly conservative amino acid substitutions.

[0437] 12. The monoclonal antibody or antigen-binding fragment according to any one of items 8, 9 or 11, wherein the conservative substitution is a substitution of one amino acid with another amino acid selected from the same group thereof, wherein the amino acid group is:

[0438] a) non-polar hydrophobic amino acids, consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met;

[0439] b) Polar neutral amino acids, consisting of Ser, Thr, Asn and Gln;

[0440] c) positively charged basic amino acids consisting of Arg, Lys and His; and

[0441] d) Negatively charged acidic amino acids, composed of Asp and Glu

[0442] wherein if Cys is to be conservatively substituted, it is substituted with Ser or Ala, and

[0443] Wherein if Pro is to be conservatively substituted, it is substituted with Ala.

[0444] 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

[0445] a) replacing Ala with Val, Leu, Ile or Gly;

[0446] b) Substitution of Arg with Lys;

[0447] c) Substituting Asn with Gln;

[0448] d) Replace Asp with Glu;

[0449] e) replacing Cys with Ser;

[0450] f) replacing Gln with Asn;

[0451] g) Substituting Asp for Glu;

[0452] h) replacing Gly with Ala;

[0453] i) Substituting His with Arg;

[0454] j) substitution of Ile with Leu, Val or Ala;

[0455] k) substitution of Leu with Ile, Val or Ala;

[0456] l) replacing Lys with Arg;

[0457] m) substitution of Met with Leu, Ile or Val;

[0458] n) replacing Phe with Tyr or Trp;

[0459] o) Replace Pro with Ala;

[0460] p) replacing Ser with Thr;

[0461] q) replacing Thr with Ser;

[0462] r) replacing Trp with Phe or Tyr;

[0463] s) replacing Tyr with Phe or Trp; and

[0464] t) Substitution of Val with Leu, Ile or Ala.

[0465] 14. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 13, comprising

[0466] (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 to SEQ ID NO: 11 or 12; and

[0467] (ii) a light chain variable domain (VL) having 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 to SEQ ID NO: 13 or 14.

[0468] 15. The monoclonal antibody or antigen-binding fragment according to item 14, wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are as defined in any one of items 2 to 13.

[0469] 16. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 15, wherein the heavy chain variable domain (VH) comprises a framework region (FW) and has the following structure:

[0470] FW-H1–CDR-H1–FW-H2–CDR-H2–FW-H3–CDR-H3–FW-H4

[0471] wherein the FW-H1 has the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 60%, preferably at least 70%, even 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% thereof;

[0472] wherein the FW-H2 has the amino acid sequence of SEQ ID NO: 16 or 17 or a variant thereof having at least 60%, preferably at least 70%, even 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 98% and even more preferably 99% of SEQ ID NO: 16 or 17;

[0473] wherein the FW-H3 has the amino acid sequence of SEQ ID NO: 18 or a variant thereof having at least 60%, preferably at least 70%, even 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 98% and even more preferably 99% thereof; and

[0474] Wherein the FW-H4 has the amino acid sequence of SEQ ID NO: 19 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0475] 17. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 16, wherein the light chain variable domain (VL) comprises a framework region (FW) and has the following structure:

[0476] FW-L1–CDR-L1–FW-L2–CDR-L2–FW-L3–CDR-L3–FW-L4

[0477] wherein the FW-L1 has the amino acid sequence of SEQ ID NO: 20 or a variant thereof having at least 60%, preferably at least 70%, even 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% thereof;

[0478] wherein the FW-L2 has the amino acid sequence of SEQ ID NO: 21 or 22 or a variant thereof having at least 60%, preferably at least 70%, even 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 98% and even more preferably 99% of SEQ ID NO: 21 or 22;

[0479] wherein the FW-L3 has the amino acid sequence of SEQ ID NO: 23 or a variant thereof having at least 60%, preferably at least 70%, even 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 98% and even more preferably 99% thereof; and

[0480] Wherein the FW-L4 has the amino acid sequence of SEQ ID NO: 24 or a variant thereof having at least 60%, preferably at least 70%, even 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%.

[0481] 18. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 17, comprising

[0482] (i) a heavy chain variable domain (VH) having the amino acid sequence of SEQ ID NO: 11 or 12; and

[0483] (ii) a light chain variable domain (VL) having the amino acid sequence of SEQ ID NO: 13 or 14.

[0484] 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.

[0485] 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 comprising the α-1,6-core-fucosylation comprises or consists of the glycopeptide of formula I

[0486]

[0487] 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 comprising the α-1,6-core-fucosylation comprises or consists of the glycopeptide of formula II

[0488]

[0489] 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 between (i) the α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising the α-1,6-core-fucosylation and (ii) AFP or a partial sequence thereof lacking α-1,6-core-fucose residues.

[0490] 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.

[0491] 24. The monoclonal antibody or antigen-binding fragment according to item 22 or 23, wherein the AFP or partial sequence thereof lacking the α-1,6-core-fucose residue comprises or consists of a glycopeptide of formula III:

[0492]

[0493] 25. The monoclonal antibody or antigen-binding fragment according to any one of items 22 to 24, wherein the equilibrium dissociation constant (K) of the antibody or antigen-binding fragment for binding to (ii) the AFP lacking α-1,6-core-fucose residues or a partial sequence of AFP lacking the α-1,6-core-fucose residues is D ) is a K that binds to (i) the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising the α-1,6-core-fucosylated AFP 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, or at least 245-fold that of (i) and (ii), wherein the binding affinities for (i) and (ii) are determined under the same conditions.

[0494] 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 comprising the α-1,6-core-fucosylation and (ii) the α-1,6-core-fucosylated glycan of formula (IV),

[0495]

[0496] 27. The monoclonal antibody or antigen-binding fragment according to item 26, wherein the equilibrium dissociation constant (K) of the antibody or antigen-binding fragment binding to the α-1,6-core-fucosylated polysaccharide of (ii) formula (IV) is D ) is a K that binds to (i) the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising the α-1,6-core-fucosylated AFP 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, or at least 245-fold, of that of (i) and (ii), wherein the binding affinities for (i) and (ii) are determined under the same conditions.

[0497] 28. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 27, wherein the antibody or antigen-binding fragment distinguishes between (i) the α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising the α-1,6-core-fucosylation and (ii) the AFP peptide of SEQ ID NO: 2 or 25.

[0498] 29. The monoclonal antibody or antigen-binding fragment according to item 28, wherein the equilibrium dissociation constant (K) of the monoclonal antibody or antigen-binding fragment binding to (ii) the AFP peptide of SEQ ID NO: 2 or 25 is D ) is a K that binds to (i) the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising the α-1,6-core-fucosylated AFP 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 that of (i) and (ii), wherein the binding affinities for (i) and (ii) are determined under the same conditions.

[0499] 30. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 29, wherein the antibody or antigen-binding fragment distinguishes the glycopeptide according to formula I from both the glycopeptide of formula III and the core-fucosylated glycan of formula IV.

[0500] 31. The monoclonal antibody or antigen-binding fragment of any one of items 1 to 30, wherein the antibody or antigen-binding fragment distinguishes the glycopeptide according to formula I from 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.

[0501] 32. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 31, which has 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 Binding to the glycopeptide of Formula I (optionally, if K is measured by affinity in solution surface plasmon resonance spectroscopy) D 0.9 nM or less or 0.4 nM or less), optionally wherein K is measured at 37°C D .

[0502] 33. The monoclonal antibody or antigen-binding fragment antibody according to any one of items 1 to 32, wherein the association rate constant k of the glycopeptide of formula I is a At least 2.0*10 4 M -1 s -1 , in the embodiment at least 10 5 M-1 s -1 , in the embodiment at least 2.5*10 5 M -1 s -1 , in the embodiment at least 6.0*10 5 M -1 s -1 and in the embodiment is at least 1.0*10 6 M -1 s -1 , optionally wherein k is measured at 37°C a .

[0503] 34. The monoclonal antibody or antigen-binding fragment antibody according to any one of items 1 to 33, wherein the dissociation rate constant k of the glycopeptide of formula I is d At most 1.2*10 -2 s -1 , at most

[0504] 8.0*10 -3 s -1 , up to 7.3*10 -3 s -1 , up to 3.2*10 -3 s -1 or at most 1.5*10 -3 s -1 , optionally wherein k is measured at 37°C d .

[0505] 35. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 34 (if one or more K D and / or k a and / or k d ), where K D and / or k a and / or k d The values ​​were determined by surface plasmon resonance spectroscopy.

[0506] 36. The monoclonal antibody or antigen-binding fragment according to item 35, wherein the surface plasmon resonance spectroscopy comprises the monoclonal antibody or antigen-binding fragment captured on a C1 sensor chip and the injection of the corresponding glycopeptide or peptide (e.g., a glycopeptide of Formula I) as an analyte, optionally wherein 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 ) as system buffer and system buffer supplemented with 1 mg / ml carboxymethyl dextran as sample buffer.

[0507] 37. The monoclonal antibody or antigen-binding fragment according to item 35 or 36, wherein the K is determined at 37°C. D and / or k a and / or k d value.

[0508] 38. The monoclonal antibody or antigen-binding fragment according to any one of items 35 to 37, wherein the surface plasmon resonance spectroscopy is performed using a Biacore T200 or 8K instrument.

[0509] 39. The monoclonal antibody or antigen-binding fragment according to any one of items 35 to 38, wherein the K D and / or the k a The determination of comprises fitting the surface plasmon resonance data using a Langmuir fitting model, preferably using R max Fitting as a whole.

[0510] 40. The monoclonal antibody or antigen-binding fragment according to any one of item 35, wherein the K D Determined by affinity method in solution.

[0511] 41. The monoclonal antibody or antigen-binding fragment of item 40, wherein the affinity method in solution comprises competition of the antibody or antigen-binding fragment for binding to an analyte bound to the chip and the same analyte in solution.

[0512] 42. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 41, which is a rabbit IgG antibody comprising a heavy chain variable domain of SEQ ID NO: 11 and a light chain variable domain of SEQ ID NO: 13 for the glycopeptide of formula I. D 1 / 10, 1 / 8, 1 / 6, 1 / 4 or 1 / 2 times of K D binds to the glycopeptide of Formula I, wherein the K is measured under the same conditions and using the same method. D value.

[0513] 43. The monoclonal antibody or antigen-binding fragment according to any one of items 1 to 42, which is a rabbit IgG antibody comprising a heavy chain variable domain of SEQ ID NO: 12 and a light chain variable domain of SEQ ID NO: 14 against the glycopeptide of formula I. D 1 / 10, 1 / 8, 1 / 6, 1 / 4 or 1 / 2 times of K D binds to the glycopeptide of Formula I, wherein the K is measured under the same conditions and using the same method. D value.

[0514] 44. The 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) that has been pretreated with the pretreatment agent according to any one of items 74 to 78.

[0515] 45. The 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) that has been pretreated with the pretreatment agent of any one of items 74 to 78 better than the binding without said pretreatment.

[0516] 46. ​​A polynucleotide or a group of polynucleotides encoding:

[0517] (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

[0518] (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.

[0519] 47. A vector comprising the polynucleotide or the set of polynucleotides according to item 46.

[0520] 48. A host cell comprising the polynucleotide or polynucleotide set according to item 46 or the vector according to item 47.

[0521] 49. The host cell according to item 48, which is a prokaryotic cell or a eukaryotic cell.

[0522] 50. The host cell according to item 48, which is a eukaryotic cell, wherein the cell is a CHO cell.

[0523] 51. A method of producing the monoclonal antibody or antigen-binding fragment according to any one of items 1 to 45, comprising culturing the host cell according to any one of items 48 to 50 and isolating the antibody or antigen-binding fragment.

[0524] 52. An antibody according to any one of items 1 to 46 obtainable by the method according to item 51.

[0525] 53. A composition comprising the antibody or antigen-binding fragment according to any one of items 1 to 45 and 52, the polynucleotide or polynucleotide set according to item 46, the vector according to item 47 or the host cell according to any one of items 48 to 50.

[0526] 54. A composition comprising the antibody or antigen-binding fragment according to any one of items 1 to 45 and 52, which is a diagnostic composition.

[0527] 55. Use of the antibody or antigen-binding fragment according to any one of items 1 to 45 and 52, or the composition according to item 53 or 54, for an in vitro immunoassay, in particular an in vitro immunoassay for detecting α-1,6-core-fucosylated alpha-fetoprotein (AFP) or AFP-L3.

[0528] 56. The use according to item 55, wherein the immunoassay is a heterogeneous immunoassay.

[0529] 57. The use according to item 55, wherein the immunoassay is an immunohistochemistry (IHC) assay.

[0530] 58. The use according to any one of items 55 to 57, wherein the sample used for the immunoassay consists of or is a sample prepared from blood, plasma or serum.

[0531] 59. The use according to any one of items 55 to 58, which is used for detecting α-1,6-core-fucosylated AFP or a partial sequence of AFP comprising said α-1,6-core-fucosylation.

[0532] 60. The 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 comprising a glycopeptide of formula I.

[0533] 61. Use of the antibody according to any one of items 55 to 60, for distinguishing α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation from (i) AFP or a partial AFP sequence lacking α-1,6-core-fucosylation and / or (ii) α-1,6-core-fucosylated proteins other than AFP.

[0534] 62. An in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation in a sample using the antibody or antigen-binding fragment as defined in any one of items 1 to 45 and 52.

[0535] 63. A method according to item 62, which comprises (i) allowing the antibody or antigen-binding fragment as defined in any one of items 1 to 45 and 52 to bind to an α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation contained in a sample to form a detection complex, (ii) detecting the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation by detecting the detection complex, and thereby determining the presence and optional amount of the α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation in the sample.

[0536] 64. The method according to item 63, wherein the method is an IHC assay, wherein the sample is a tissue slide.

[0537] 65. A method according to item 63, wherein the method is a humoral immunoassay and wherein the sample is a body fluid.

[0538] 66. The method according to item 65, wherein the body fluid is a blood sample, semen or urine.

[0539] 67. The method according to item 65 or 66, wherein the body fluid is a blood sample, and the blood sample is whole blood, capillary blood, serum or plasma (preferably serum or plasma).

[0540] 68. A method according to any one of items 65 to 67, wherein the method comprises (i) pretreating the sample with a pretreating agent and (ii) incubating the pretreated sample with an antibody or antigen-binding fragment as defined in any one of items 1 to 45 and 52.

[0541] 69. A method according to item 68, wherein the pretreatment agent is a pretreatment agent as defined in any one of items 74 to 78.

[0542] 70. The method according to any one of items 63 to 69, wherein the method distinguishes between an α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation and an AFP lacking said α-1,6-core-fucosylation or a partial AFP sequence lacking said α-1,6-core-fucosylation.

[0543] 71. A method according to any one of items 65 to 70, wherein the method is a sandwich immunoassay and wherein the method comprises incubating the sample with an AFP-specific antibody or antigen-binding fragment which does not compete for binding with the antibody according to any one of items 1 to 45 and 52 and binds to AFP independent of α-1,6-core-fucosylation.

[0544] 72. The method according to item 71, wherein the AFP-specific antibody is anti-AFP (Tu-11).

[0545] 73. A method according to any one of items 63 to 72, wherein the method is used to aid in the detection or detection of hepatocellular carcinoma, in embodiments early stage hepatocellular carcinoma.

[0546] 74. A pretreatment reagent for treating a sample containing α-1,6-core-fucosylated AFP, the pretreatment reagent comprising a reducing agent, in particular a protein reducing agent.

[0547] 75. A 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 dithiobutylamine (DTBA).

[0548] 76. A pretreatment reagent according to item 74 or 75, wherein the reducing agent is DTT.

[0549] 77. The pretreatment agent according to any one of items 74 to 76, wherein the pretreatment agent further comprises a chelating agent, in embodiments an ion chelating agent, in particular a divalent ion chelating agent, in embodiments a chelating agent selected from the following: ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether or ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0550] 78. A pretreatment reagent according to any one of items 74 to 77, wherein the pretreatment reagent further comprises a buffer, such as Tris.

[0551] 79. A kit comprising an antibody or antigen-binding fragment as defined in any one of items 1 to 45 and 52.

[0552] 80. The kit according to item 79, which is an immunoassay kit.

[0553] 81. The kit according to item 79 or 80, wherein the kit is used for detecting or quantifying α-1,6-core-fucosylated AFP or a partial AFP sequence comprising the α-1,6-core-fucosylated AFP.

[0554] 82. A kit according to any one of items 79 to 81, wherein the kit comprises a pretreatment agent, such as a pretreatment agent as defined in any one of items 74 to 78.

[0555] 83. A kit according to item 82, wherein the antibody or antigen-binding fragment and the pretreatment reagent are provided in separate containers.

[0556] The following definitions and embodiments apply to all aspects and embodiments of the invention provided above and in the claims.

[0557] As is known in the art, human AFP is a glycoprotein having the amino acid sequence SEQ ID NO: 1 (or a naturally occurring variant thereof, as described in Uniprot ID P02771), which comprises a single N-glycosylation site corresponding to Asn-251 of Uniprot ID P02771 (version 209). The N-glycan at Asn-251 may comprise a core fucose residue (see Figure 1 As further understood in the art, the term "core fucosylation" within a glycan means that the fucosyl residue is an α-1,6-linked core GlcNac residue attached to Asn-251 of the AFP protein or a partial sequence thereof comprising Asn corresponding to Asn-251 (see Figure 1 ). The terms "core-fucosylation" and "α-1,6-core-fucosylation" are considered to be interchangeable. Thus, the term "having specificity for (or specifically binding to) core-fucosylated AFP and / or a partial sequence of AFP comprising core-fucosylation" is interchangeable with the term "having specificity for (or specifically binding to) α-1,6-core-fucosylated AFP and a partial sequence thereof comprising 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 antigen-binding fragments thereof.

[0558] Lectins can be used for the analysis of glycoproteins. By utilizing the selective binding ability of lectins to the sugar chain structure of glycoproteins, marker glycoprotein fractions with specific sugar chain structures can be separated and concentrated. In the case of AFP, lectins derived from Lentil agglutinin-A (LCA) have been widely used. By using LCA, AFP can be fractionated into three variants, L1, L2, and L3, of which AFP-L3 has the highest affinity for LCA. The AFP-L3 fraction is composed of AFPs that are N-glycosylated at Asn-251 with N-glycans containing α-1,6-core-fucosylation (i.e., AFPs with fucose bound to N-acetylglucosamine (GlcNAc), which are 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 lentil agglutinin (LCA) reactive fraction of alpha-fetoprotein (AFP-L3) is specifically increased in patients with HCC (Khien VV et al., The International Journal of Biological Markers. 2001; 16(2): 105-111).

[0559] As used herein, the terms "antibody", "antibodies" and similar terms refer to whole immunoglobulin molecules and encompass naturally occurring antibody 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 antigen-binding fragments and derivatives of all of the foregoing. If not explicitly mentioned, the terms "antibody", "antibodies" and similar terms as used herein also refer to antigen-binding fragments thereof. 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 retains the ability to specifically bind to a target antigen, i.e., alpha-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP comprising said alpha-1,6-core-fucosylation (e.g., a glycopeptide of Formula I or Formula II), as known in the art, including but not limited to antigen-binding fragments comprising an Fv domain (i.e., paired heavy and light chain variable domains), such as Fab, Fab', F(ab')2 and Fv fragments, and recombinant constructs, such as single-chain Fv domains known in the art as scFv. These terms also include antibody antigen-binding fragments comprising a single unpaired heavy or light chain variable domain known in the art, which variable domain retains the ability to specifically and selectively bind to an antigen as defined herein, including but not limited to single domain antibodies (also referred to in the art as sdAb, dAb and / or nanobodies) and camelid heavy chain-based V domains. H H domain.

[0560] In certain embodiments, the monoclonal antibody of the present invention may be a whole immunoglobulin, Fab, Fab', F(ab')2, Fv or scFv. In a specific embodiment, the monoclonal antibody of the present invention may be a Fab fragment.

[0561] In certain embodiments, the monoclonal antibodies of the invention may be multivalent antibodies.

[0562] As used herein, a "multivalent antibody" refers to an antibody comprising 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 comprises 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 Fv or Fab domain. V .

[0563] Exemplary but non-limiting embodiments of multivalent antibodies and methods for generating such antibodies are disclosed in WO2019 / 057816, which is incorporated herein by reference in its entirety. Specifically, all embodiments of the structural configurations of such multivalent antibodies and methods for generating such multivalent antibodies (also referred to as p3, p4, p5, p6, p7 or p8) are disclosed herein by reference.

[0564] In an embodiment, the multivalent antibody of the present invention may comprise a heavy chain comprising a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies, 8 copies in a specific embodiment) of a VH-CH1 domain (e.g., all comprising the (preferably identical) VH of the 1,6fucAFP antibody or antigen-binding fragment of the present invention), the domain being, for example, flanked by a linker sequence (e.g., one of the linker sequences described in WO2019 / 057816, which is incorporated herein by reference). Compared to conventional antibodies, the additional VH-CH1 domain may be placed upstream and / or downstream of the hinge-CH2-CH3 sequence. The light chain in such a multimeric antibody may be a conventional light chain consisting of a VL and a constant domain.

[0565] Alternative methods for generating multivalent antibodies involve chemical polymerization / cross-linking of antibodies or antigen-binding fragments.

[0566] Antibodies can be polyclonal or monoclonal. The antibodies of the present invention are monoclonal. The term "monoclonal" as used herein to refer to antibodies or their antigen-binding fragments refers to a population of antibody polypeptides or fragments produced by a single B cell clone, which contains only one antigen binding site specificity that can immunoreact with a specific epitope of an antigen. This is in contrast to "polyclonal" antibodies and compositions, which refer to populations of antibody polypeptides or antigen-binding fragments containing multiple antigen-binding site specificities. 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, wherein the antibody or antigen-binding fragment includes one or more additional domains, such as for separating and / or preparing recombinantly produced antibodies / fragments / constructs.

[0567] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs), such as complementarity determining regions (CDRs). (See, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated using VH or VL domains from antibodies that bind to the antigen, respectively, to screen for libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0568] As used herein, the term "hypervariable region" or "HVR" refers to individual regions of an antibody variable domain that are hypervariable in sequence and determine antigen binding specificity, such as the "complementarity determining region" ("CDR").

[0569] Typically, antibodies contain 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:

[0570] (a) hypervariable loops present 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));

[0571] (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

[0572] (c) Antigenic contact points present 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)).

[0573] Unless otherwise indicated, CDRs are determined according to the method described in Kabat et al. (supra). Those skilled in the art will appreciate that CDR names may also be determined according to the method described in Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system.

[0574] "Framework" or "FR" refers to the variable domain residues excluding the complementarity determining regions (CDRs). The FR of a variable domain is usually composed of the following four FR domains: FR1, FR2, FR3, and FR4. Therefore, CDR and FR sequences usually appear in the following sequence in VH (or VL): FR1-CDR-H1 (CDR-L1)-FR2-CDR-H2 (CDR-L2)-FR3-CDR-H3 (CDR-L3)-FR4.

[0575] As used herein, the phrase "specific binding" in the context of an antibody or antibody antigen-binding fragment means that the corresponding antigen binds to the antibody or antibody antigen-binding fragment via an antigen-antibody reaction. The term "specific binding" also means that the antibody or antigen-binding fragment binds to a specified structure in preference to other structures that may exhibit cross-reactivity. As also explained herein, the term distinction means that the antibodies and antigen-binding fragments of the present invention specifically bind to the target antigen (i.e., core-fucosylated AFP and / or its core-fucosylated partial sequence, most preferably the glycopeptide of formula I or II), but not to AFP / AFP partial sequences lacking core-fucose residues, and / or to core-fucosylated glycans in another case (such as a single core-fucosylated asparagine listed in formula IV).

[0576] As used herein, the term "distinguishes and / or" and similar terms with respect to two antigens, for example, an antibody distinguishes between antigen X and / or antigen Y, indicating 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. Therefore, the term "distinguishes" and similar terms as used herein refer to the antibody or antigen-binding fragment "not specifically binding to the non-target antigen" / "not significantly binding to the non-target antigen" (used interchangeably). As is well known in the art, the terms "specific binding" and "not significantly binding" indicate the degree to which an antibody distinguishes between two antigens. This is because it is well known that no antibody has absolute specificity, meaning that it will react with only one epitope regardless of the conditions. That is, in the presence of other (non-target) antigens, an antibody or antigen-binding domain can react to a certain 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 the relevant epitope. This difference in affinity is used to establish assay conditions under which the antibody or antigen-binding fragment binds almost exclusively to a specific epitope. In this regard, the binding (or non-binding) of the antibody or antigen-binding fragment to the antigen is not to be 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 this binding activity is at a significantly reduced level relative to the binding activity to core-fucosylated AFP or a core-fucosylated partial sequence of AFP (preferably a glycopeptide of formula I or a glycoprotein comprising a glycopeptide of formula I).

[0577] For example, the feature that distinguishes the target antigen from / from the non-target antigen can be that the affinity of the 1,6-fucAFP antibody or antibody antigen-binding fragment for the target antigen is at least 10 times, at least 20 times, at least 50 times, or at least 100 times, at least 150 times, at least 180 times, or at least 245 times greater than the affinity for the non-target antigen (i.e., the K for binding to the target antigen is greater than the K for binding to the non-target antigen). D The formulation of at least "XX" also encompasses K values ​​for non-target antigens. D Therefore, whether the antibody or antigen-binding fragment can distinguish target structures from non-target structures can be determined using the same methods (e.g., as described below for determining K D The preferred method) is to determine the K for the corresponding binding D value.

[0578] In an embodiment, an immunoassay can be used to assess the ability of an antibody to distinguish between a target antigen and / or a non-target antigen, in which the binding of the antibody or antigen binding fragment to be tested to the target structure is detected. Using such an immunoassay, an immunoassay signal obtained with a first sample containing a defined concentration of a target antigen can be compared with an immunoassay signal obtained with a second sample containing the same concentration of a non-target antigen. The immunoassay signal of the first sample is higher than the immunoassay signal of the second sample indicating the distinction between the target antigen and the non-target antigen. In an embodiment, if the immunoassay signal of the first sample is at least 5 times, at least 10 times, at least 20 times, at least 40 times, at least 50 times, or at least 100 times the immunoassay signal of the second sample, the test antibody or antigen binding fragment can distinguish between the target structure and the non-target structure. An exemplary but non-limiting immunoassay that can be used for such analysis is provided in Example 7. An exemplary concentration of the target antigen and the non-target antigen can be 12 nm.

[0579] As used herein in the context of amino acids, the term "substitution", "exchange" or "mutation" refers to the replacement of an amino acid with another amino acid. The term "substitution" obviously does not include the situation where an amino acid is deleted at a certain position and one (or more) amino acids are inserted at another position. As mentioned above, the present invention encompasses conservative or highly conservative amino acid substitutions as already defined herein above.

[0580] Amino acids are spelled or abbreviated herein using either the one-letter code or the three-letter code.

[0581] In the context of the present invention, it refers to a variant of a sequence (particularly a CDR). These variants generally comprise one or more amino acid substitutions. Obviously, a variant CDR is a functional variant, i.e., an amino acid sequence that may be different from a reference amino acid sequence, but the different sequence exhibits or maintains the same functional activity as the reference sequence in the case of the described heavy chain and / or light chain variable domains. Specifically, as used herein, the term same functional activity means that the antibody or antibody binding fragment of the present invention comprising one or more variant CDRs will maintain specific binding to α-1,6-core-fucosylated alpha-fetoprotein (AFP) or a partial sequence of AFP comprising the α-1,6-core-fucosylation (e.g., a glycopeptide of formula I or II) and the characteristics 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 polysaccharide of formula IV. In an embodiment, functional activity may also refer to the retention of the kinetic parameters for binding to the glycopeptide of formula I mentioned herein.

[0582] As used in the context of the present invention, "conservative amino acid substitution" refers to the substitution of one amino acid by another amino acid selected from the same physicochemical group thereof, wherein the physicochemical group of the amino acid is:

[0583] a) non-polar hydrophobic amino acids, consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp and Met;

[0584] b) Polar neutral amino acids, composed of Ser, Thr, Asn and Gln;

[0585] c) positively charged basic amino acids consisting of Arg, Lys and His; and

[0586] d) Negatively charged acidic amino acids, composed of Asp and Glu,

[0587] Wherein if Cys is to be conservatively substituted, it is substituted with Ser or Ala, and wherein if Pro is to be conservatively substituted, it is substituted with Ala.

[0588] As used in the context of the present invention, "highly conservative amino acid substitutions" refer to the following amino acid substitutions:

[0589] a) replacing Ala with Val, Leu, Ile or Gly;

[0590] b) Substitution of Arg with Lys;

[0591] c) Substituting Asn with Gln;

[0592] d) Replace Asp with Glu;

[0593] e) replacing Cys with Ser;

[0594] f) replacing Gln with Asn;

[0595] g) Substituting Asp for Glu;

[0596] h) replacing Gly with Ala;

[0597] i) Substituting His with Arg;

[0598] j) substitution of Ile with Leu, Val or Ala;

[0599] k) substitution of Leu with Ile, Val or Ala;

[0600] l) replacing Lys with Arg;

[0601] m) substitution of Met with Leu, Ile or Val;

[0602] n) replacing Phe with Tyr or Trp;

[0603] o) Replace Pro with Ala;

[0604] p) replacing Ser with Thr;

[0605] q) replacing Thr with Ser;

[0606] r) replacing Trp with Phe or Tyr;

[0607] s) replacing Tyr with Phe or Trp; and

[0608] t) Substitution of Val with Leu, Ile or Ala.

[0609] As used herein, the term "% sequence identity" in connection with the amino acid sequence of a polypeptide / peptide and / or nucleic acid sequence or nucleic acid molecule describes the number of matches of identical amino acid or nucleic acid residues of two or more aligned sequences compared to the number of residues constituting the total length of the compared sequences (or the total compared portion thereof). Using the alignment of two or more sequences or subsequences, the percentage of identical residues can be determined when the (sub) sequences are compared and aligned for maximum correspondence over a comparison window or over a specified region as measured using a sequence comparison algorithm known in the art, or when manually aligned and visually inspected. Non-limiting examples of algorithms for 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). Although the FASTA algorithm generally does not consider internal mismatched deletions or additions, i.e., gaps, in its calculations, it can be manually corrected to avoid overestimating % sequence identity. However, CLUSTALW does take into account sequence gaps in its identity calculations. BLAST and BLAST 2.0 algorithms are also available (Altschul et al., Nucl Acids Res., 25 (1977), 3389).

[0610] In the context of the present invention, glycopeptides (e.g., Formula I, Formula II, Formula III) and N-glycan structures (e.g., Formula IV) of AFP are disclosed. In the corresponding formulas described above, the type of connection is not shown. The preferred connection is Figure 1 Linkages shown in the exemplary glycan structures shown.

[0611] Therefore, in a specific embodiment, the glycopeptide of Formula I may have the following structure Ia

[0612]

[0613] Therefore, in a specific embodiment, the glycopeptide of formula II may have the following structure IIa

[0614]

[0615] Therefore, in a specific embodiment, the glycopeptide of formula III may have the following structure IIIa

[0616]

[0617] Thus, in a specific embodiment, the N-glycan of formula IV may have the following structure IVa

[0618]

[0619] As used herein, "nucleic acid molecules", "nucleic acid sequences", "polynucleotides" and similar terms include both genomic DNA and cDNA, and RNA that can drive the expression of the antibodies or antigen-binding fragments of the present invention. It is understood that the term "RNA" as used herein includes all forms of RNA, including mRNA, tRNA and rRNA, as well as genomic RNA, such as in the case of RNA of RNA viruses. Preferably, the embodiments recording "RNA" are for mRNA. The nucleic acid molecules / nucleic acid sequences of the present invention can be natural, synthetic or semi-synthetic. In an embodiment, the nucleic acids / nucleic acid sequences of the present invention can be isolated. Therefore, nucleic acid molecules can be, for example, nucleic acid molecules that have been synthesized according to conventional protocols of organic chemistry, according to recombinant methods, or semi-synthetically produced (e.g., by combining chemical synthesis and recombinant methods). Those skilled in the art are familiar with the preparation and use of such nucleic acid molecules.

[0620] As used herein, "immunoassay" is a recognized bioanalytical method in which the detection or quantification of an analyte depends on the reaction of the analyte and at least one analyte-specific binding agent, thereby forming an analyte: binding agent complex (also called detection complex). In the context of the present invention, at least one of the at least one analyte-specific binding agent is an antibody of the present invention. Specific embodiments of "sandwich" immunoassays can be used for analytes with more than one recognition epitope. Therefore, sandwich assays require at least two binding agents that are attached to non-overlapping epitopes on the analyte. In a "heterogeneous sandwich immunoassay", one binding agent has the function of an analyte-specific capture binding agent; this binding agent is immobilized on a solid phase (during the assay). The second analyte-specific binding agent is provided in a dissolved form in a liquid phase. Once the corresponding analyte binds to 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 called a "detection complex". In 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.

[0621] The term "heterogeneous" (as opposed to "homogeneous") refers to two basic and independent steps in the assay process. In the first step, a detection complex containing the label is formed and fixed, but unbound label still surrounds the complex. Before determining the label-dependent signal, the unbound label is removed from the fixed detection complex, thus representing the second step. In contrast, a homogeneous assay generates an analyte-dependent detectable signal by a one-step incubation and does not require a washing step.

[0622] In heterogeneous immunoassays, the solid phase is functionalized so that before contact with the analyte, the solid phase may have a functional capture binding agent (first binding agent) bound to its surface; or the surface of the solid phase is functionalized so that the first binding agent can be anchored after the first binding agent reacts with the analyte. In the latter case, the anchoring process must not interfere with the ability of the binding agent to specifically capture and bind to the analyte. The second binding agent present in the liquid phase is used to detect the bound analyte. Therefore, in a heterogeneous immunoassay, the analyte is allowed to bind to the first (capture) and second (detection) binding agents. A "detection complex" is thus formed, in which the analyte is sandwiched between the capture binding agent and the detection binding agent. In a typical embodiment, the detection binding agent is labeled before contact with the analyte; alternatively, after the analyte binds, the label is specifically attached to the detection binding agent. In the case of fixing the detection complex on a solid phase, the amount of detectable label on the solid phase corresponds to the amount of analyte sandwiched in the middle. After removing the unbound label, the fixed label indicating the presence of the analyte and the amount of the analyte can be detected.

[0623] As used herein, a "competitive immunoassay" preferably employs a single binding agent that interacts directly with the analyte. "Competitive heterogeneous immunoassays typically detect a detection marker signal that is inversely proportional to the amount of analyte in the sample.

[0624] As used herein, "detectable label" refers to a label that allows detection. According to one embodiment of the present invention, the detectable label is an enzyme or a label that emits light, in one embodiment, is fluorescent, luminescent, chemiluminescent, electrochemiluminescent or radioactive. In a preferred embodiment, the label is an electrochemiluminescent label, in one embodiment, is tris (2,2'-bipyridine) ruthenium (II)-complex (Ru (bpy)). Since the interference is caused by the three-dimensional structure of the label molecule and similar interfering molecules that attract autoantibodies, rather than by the signal emission mechanism of the label (such as, for example, light or radioactivity), all of the above labels can be used in the present invention.

[0625] As used herein, "capture label" refers to a label that can immobilize a capture agent (e.g., an antibody having a capture label attached thereto) on a surface (e.g., a magnetic particle such as a microbead). A non-limiting example is a member of a binding pair. A non-limiting example of a capture label is biotin or a derivative thereof, which can interact with streptavidin or a derivative thereof. Different capture labels are well known in the art.

[0626] The term "and / or" should be understood to mean either or both of the alternatives.

[0627] As used herein and unless otherwise specified, it should be understood that the term "about" is used synonymously with the term "approximately". Illustratively and unless otherwise specified, the use of the term "about" when used in conjunction with a stated value or range means slightly more or slightly less than the stated value or range, within ±15% of the stated value, within ±10% of the stated value, within ±5% of the stated value, or conveniently within ±2% of the stated value. Such values ​​are therefore encompassed by the scope of the claims reciting the term "about" or "approximately".

[0628] As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, a protein, a carbohydrate structure or glycolipid, a metabolite, an mRNA, a miRNA, a protein, a DNA (cDNA or genomic DNA), a DNA copy number, or an epigenetic change, for example, increased, decreased, or altered 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); altered nucleosome positioning, the expression or presence of a biomarker in or on a mammalian tissue or cell can be detected by standard methods (or methods disclosed herein), and the biomarker may be predictive, diagnostic, and / or prognostic for individual health or disease. Therefore, the more general term "marker" is sometimes used below when discussing more general terms and definitions. The term marker also includes a glycan structure or glycan, or a glycopeptide as analyzed in the present disclosure.

[0629] The term "in vitro method" is used to indicate that the method is performed outside a living organism, preferably in a body fluid, isolated tissue, organ or cell. An in vitro method may also be referred to as an ex vivo method.

[0630] Hepatocellular carcinoma (HCC) is the main histological type of primary liver cancer occurring worldwide, accounting for 70% to 85% of the total burden. It is known that basic liver diseases such as liver fibrosis and cirrhosis are the main risk factors for the development of HCC. For patients diagnosed early, HCC can be treated by resection, liver transplantation or radiofrequency local ablation. If this malignancy is diagnosed early, the 5-year survival rate of HCC patients may be as high as 70%. However, the 5-year survival rate of HCC patients decreases significantly with the later diagnosis of the disease, and if HCC is diagnosed in the late stage of the disease, the 5-year survival rate will drop to 15% (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).

[0631] The term "helps detect hepatocellular carcinoma (HCC)" is used to indicate that the method according to the present invention will assist / help medical professionals, including, for example, doctors, in assessing whether an individual suffers from HCC or is at risk of developing HCC. It should be understood that doctors can use several alternative methods (e.g., ultrasound, radiography, MRT or CT) and combine them with in vitro biomarker data (such as glycan structure data) to detect or exclude the presence of HCC. The final diagnosis of HCC is usually made by tissue biopsy or postoperative tissue samples. The term "helps detect HCC" includes that the method is used as a separate diagnostic tool or as one of multiple diagnostic tools. Not all (100%) patients with HCC have an amount of markers that is higher than a reference level, and not all healthy individuals have a level of markers that is lower than a reference level or a cut-off level. As those skilled in the art will appreciate, in many diseases, no biochemical marker has 100% specificity and 100% sensitivity at the same time. In contrast, the marker analyzed or the combination of markers comprising the marker gives a certain probability, for example, at a given level of specificity or a given level of sensitivity, that the individual whose sample has been analyzed has a certain clinical state, for example, suffering from HCC. Those skilled in the art are fully familiar with mathematical / statistical methods for calculating 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 indication of the presence or absence of HCC.

[0632] A convenient goal of quantifying the diagnostic accuracy of a laboratory test is to express its performance with a single number. The most common global metric is the area under the curve (AUC) of the ROC graph. The area under the ROC curve is a measure of the probability that a perceptual measurement allows a condition to be correctly identified (or one condition to be distinguished from another). Values ​​are typically between 1.0 (perfect separation of the two sets of test values) and 0.5 (no obvious distribution difference between the two sets of test values). The area depends not only on a specific part of the graph, such as, for example, the point closest to the diagonal or the sensitivity at 90% specificity, but also on the entire graph. This is a quantitative descriptive expression of the proximity of the ROC graph to the perfect graph (area = 1.0). In the context of the present invention, two different conditions can be whether the patient suffers from HCC or not.

[0633] As used herein, the term "subject" or "individual" refers to a single person. The subject can be a healthy subject or patient, for example, suffering from cirrhosis, being at risk of developing HCC, experiencing or having experienced one or more signs, symptoms or other indicators of HCC. It is intended to be included as a subject, any subject who participates in a clinical research trial but does not show any clinical signs of the disease, or a subject who participates in an epidemiological study, or a subject whose sample can be used as a control. In an embodiment, it can be known that the subject is at risk of developing HCC, for example, by long-term drinking, hepatitis B and / or hepatitis C infection, non-alcoholic fatty liver disease, Wilson's disease, hereditary hemochromatosis, α1-antitrypsin deficiency, primary biliary cirrhosis, autoimmune hepatitis and other risk factors. Other risk factors can include obesity and / or liver transplantation. In an embodiment, it is known that the subject is at risk of developing HCC, for example, by liver fibrosis, non-cirrhotic liver disease, NASH, chronic HBV and chronic HCV.

[0634] In one embodiment according to the present disclosure, the subject from which the sample to be studied has been obtained is a healthy subject and is screened for (the presence of) HCC as part of routine oncology monitoring.

[0635] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained is a subject at risk of developing HCC and is screened for (the presence of) HCC as part of routine oncology monitoring.

[0636] A subject may be at risk for developing HCC if the subject is known to have chronic liver disease, viral or non-viral hepatitis, and / or cirrhosis.

[0637] In one embodiment according to the present disclosure, the subject from whom the sample to be studied has been obtained suffers from chronic liver disease, viral or non-viral hepatitis, liver cirrhosis, and a differential diagnosis is performed for the presence or absence of HCC.

[0638] As used in the context of the present disclosure, a "sample" may be a liquid sample comprising or expected to comprise α-1,6-core-fucosylated AFP or a partial sequence thereof comprising α-1,6-core-fucosylation. The sample may in particular be a body fluid, such as but not limited to 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.

[0639] As used herein, the terms "amount" or "level" of an analyte in a sample relate to any absolute measure, corresponding to or proportional to the absolute amount or concentration of the analyte in the sample, or any relative measure, i.e. a measure expressing the amount or concentration of the analyte relative to a reference amount or concentration, respectively.

[0640] As used herein, the term "reference amount" (or "reference level") of an analyte (e.g., a glycan structure or glycopeptide) refers to an independently established predetermined amount of the analyte. As will be appreciated by those skilled in the art, a reference amount is predetermined and is set to meet conventional requirements for detecting HCC (e.g., early HCC) in terms of, for example, specificity and / or sensitivity. Therefore, a reference amount can be selected so that it indicates HCC (e.g., early HCC). The requirements for detecting HCC can vary, for example, depending on regulatory agencies. For example, it may be necessary to set the assay sensitivity or specificity to certain limits, such as 80%, 90%, 95% or 98%, respectively. These requirements can also be defined according to positive or negative predictive values. For any selected requirement, for example, in terms of sensitivity or specificity level, a reference range (if the evaluation value and the reduction value indicate an abnormal state) or a reference level or a cutoff level (if the evaluation value or the reduction value indicate an abnormal state) can be determined by those skilled in the art. Similarly and according to the same principle, a reference ratio can be determined.

[0641] As used herein, for example in the context of a reference value of a score, the term "reference value" refers to an independently established predetermined value of each parameter (e.g., score). As will be appreciated by those skilled in the art, a reference value is predetermined and is set to meet conventional requirements for detecting HCC (e.g., early HCC) in terms of, for example, specificity and / or sensitivity. Thus, a reference value may be selected such that it indicates HCC (e.g., early HCC). The above content regarding reference amounts applies mutatis mutandis.

[0642] The terms "glycan structure" or "glycan" are used interchangeably herein. In the present disclosure, the glycans of interest are N-glycans. Therefore, the terms glycan and N-glycan are used interchangeably herein. Glycans or glycan structures consist of various types of sugar moieties. The linkage is preferably as follows Figure 1 Shown in Figure 1. The N-glycan structure is attached to the side chain of the amino acid asparagine.

[0643] The term "glycopeptide" is used to refer to a peptide or a peptide fragment of a larger polypeptide comprising amino acids covalently attached to a glycan.

[0644] The word "comprise" and variations such as "comprising" and "including" will be understood to imply the inclusion of stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps.

[0645] As used in this specification and the appended claims, the singular forms "a," "an," "the," and "said" include plural referents unless the content clearly dictates otherwise.

[0646] Concentration, amount and other numerical data can be expressed or presented in "range" format in this article.It should be understood that such range format is used only for convenience and simplicity, and therefore should be flexibly interpreted as not only including the numerical value clearly listed as the range limit, but also including all individual numerical values ​​or sub-ranges covered by the range, just as each numerical value and sub-range are clearly listed.As an illustration, the numerical range "150mg to 600mg" should be interpreted as not only including the value 150mg to 600mg clearly listed, but also including individual values ​​and sub-ranges within the indicated range.Therefore, individual values ​​are included in this numerical range, such as 150, 160, 170, 180, 190, ... 580, 590, 600mg and sub-ranges, such as from 150 to 200, 150 to 250, 250 to 300, 350 to 600, etc. This same principle applies to the scope of only citing a numerical value.In addition, no matter how wide the range or feature is, such explanation is applicable.

[0647] When used in connection with a numerical value, the term "about" is meant to encompass the numerical 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.

[0648] In the above detailed description of the present invention, many individual elements, characterizing features, techniques and / or steps are disclosed. It is readily appreciated that each of these has benefits not only when considered or used alone, but also when considered and used in conjunction with one another. Therefore, in order to avoid excessive repetition and redundant paragraphs, this description avoids repeating every possible combination and arrangement. However, whether or not explicitly stated, it is understood that such combinations are fully within the scope of the presently disclosed subject matter.

[0649] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. References to the techniques employed herein are intended to refer to techniques commonly understood in the art, including changes in those techniques that are obvious to those skilled in the art or substitutions of equivalent techniques.

[0650] All amino acid sequences provided herein start from 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 herein correspond to those code abbreviations commonly used for amino acids.

[0651] In this specification, a large number of documents, including patent applications and manufacturer manuals, are cited. The disclosures of the documents are considered irrelevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, the citation of all referenced documents is as if each individual document is specifically and individually incorporated herein by reference.

[0652] In this article, elements of the present invention are described. These elements are listed as aspects together with specific embodiments, however, it should be understood that they can be combined in any way and in any number to create other aspects and embodiments. In particular, the embodiments disclosed in the context of one aspect are applied to other aspects after necessary modifications. The various described examples and preferred embodiments should not be interpreted as limiting the present invention to only the embodiments clearly described. This description should be understood to support and encompass embodiments that combine the embodiments clearly described with any number of disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of the elements described in this application should be considered to be disclosed by the specification of the application.

[0653] Sequence Description

[0654] In the context of the present disclosure the following amino acid sequences are mentioned.

[0655] SEQ ID NO: 1: AFP sequence (see UniProt: P02771; version 209); Asn-251 as N-glycosylation site is underlined

[0656]

[0657] SEQ ID NO: 2 Peptide sequence of AFP (AS243 to 261 of SEQ ID NO: 1); Asn corresponding to Asn-251 is underlined

[0658] LSQKFTKV N FTEIQKLVLD

[0659] SEQ ID NO:3CDR-H1 19B12 and 3C5

[0660] TYGMG

[0661] SEQ ID NO:4CDR-H2 19B12

[0662] IIGDNGSTYYANWA

[0663] SEQ ID NO:5CDR-H2 3C5

[0664] IIDSGSTYYANWA

[0665] SEQ ID NO:6CDR-H3 19B12 and 3C5

[0666] DRDPSSSGYYFKM

[0667] SEQ ID NO:7CDR-L1 19B12QASQSISSYLA

[0668] SEQ ID NO:8CDR-L1 3C5QASQSIGSYLA

[0669] SEQ ID NO:9CDR-L2 19B12 and 3C5

[0670] GASNLES

[0671] SEQ ID NO:10CDR-L3 19B12 and 3C5

[0672] QTAFYIFSSDNA

[0673] SEQ ID NO: 11 VH sequence 19B12 (CDRs are underlined)

[0674]

[0675] SEQ ID NO: 12 VH sequence 3C5 (CDRs are underlined)

[0676]

[0677] SEQ ID NO: 13 VL sequence 19B12 (CDRs are underlined)

[0678]

[0679] SEQ ID NO: 14 VL sequence 3C5 (CDRs are underlined)

[0680]

[0681] SEQ ID NO:15 FW1 of VH of 19B12 and 3C5

[0682] QSVEESGGRLVAPGTPLTLTCTVSGIDLS

[0683] SEQ ID NO: 16 FW2 of VH of 19B12

[0684] WVRQAPGKGLEWIG

[0685] SEQ ID NO: 17 FW2 of VH of 3C5

[0686] WVRQAPGKGLEYIG

[0687] SEQ ID NO:18 FW3 of VH of 19B12 and 3C5

[0688] KGRFTISKTSTTVDLKMTSLTTEDTATYFCAR

[0689] SEQ ID NO: 19 FW4 of VH of 19B12 and 3C5

[0690] WGPGTLVTVSL

[0691] SEQ ID NO:20 FW1 of VL of 19B12 and 3C5

[0692] ALVMTQTPSSVSAAVGGTVTINC

[0693] SEQ ID NO:21 FW2 of VL of 19B12

[0694] WYQQKPGQPPKLLIF

[0695] SEQ ID NO:22 FW2 of VL of 3C5

[0696] WYQQKPGQPPRLLIY

[0697] SEQ ID NO:23 FW3 of VL of 19B12 and 3C5

[0698] GVPSRFKGSGSGTEFTLTISDLECDDAATYYC

[0699] SEQ ID NO:24 FW4 of VL of 19B12 and 3C5

[0700] FGGGTEVVVK

[0701] SEQ ID NO:25 Peptide sequence of AFP (AS248-256 of AFP is shown in SEQ ID NO:1); Asn corresponding to Asn-251 is underlined

[0702] TKV N FTEIQ

[0703] SEQ ID NO: 26 Peptide sequence of AFP (AS243-256 of AFP is shown in SEQ ID NO: 1); Asn corresponding to Asn-251 is underlined.

[0704] LSQKFTKVN FTEIQ

[0705] SEQ ID NO: 27 Exemplary linkers in multivalent antibodies

[0706] GGGSGGGSGGGSGGGS BRIEF DESCRIPTION OF THE DRAWINGS

[0707] The following drawings are provided to aid in the understanding of the present invention.

[0708] Figure 1 : Schematic diagram showing the N-glycan structure of AFP at Asn-251 (N251), highlighting the α-1,6-core-fucosylation.

[0709] Figure 2 Exemplary antibody kinetics of interaction with fucosylated AFP(243-261)-G0F-Bi (dark grey) and non-fucosylated AFP(243-261)-G2-Bi (light grey) at 25°C are shown. A) Selected antibodies that specifically bind to the fucosylated AFP-L3-peptide with acceptable background. B) Deselected antibodies that did not bind, had mixed binding behavior or had no difference between fucosylated and non-fucosylated AFP-L3-peptide (also called AFP-L1).

[0710] Figure 3: Kinetic profiles of binding of antibodies 19B12, 3C5, 3D2, 3A3 and 6B6 to various peptides. A) Antibody interaction of fucosylated AFP (243-261)-G0F-Bi (BMO 35.000148). A concentration series ranging from 3.3 nM to 90 nM is shown, with 30 nM as a repeat (black). For antibodies 19B12 and 3C5, the Langmuir fit model, Rmax overall, RI=0 (light gray) are superimposed. B) Antibody interaction of Asn (G0F) -Bi (BMO 35.000150). A concentration series ranging from 3.3 nM to 270 nM is shown, with 30 nM as a repeat (black). C) Antibody interaction of non-fucosylated AFP (243-261) -G2-Bi (BMO 35.000151). A concentration series ranging from 3.3 nM to 270 nM is shown, with 30 nM as a repeat (black). For antibodies 19B12 and 3C5, the Langmuir fit model, Rmax overall, RI=0 (light gray) are superimposed. D) Antibody interaction of AFP(243-261)-(Fuc-GlcNAc)-Bi(BMO 35.100001). A concentration series ranging from 3.3 nM to 270 nM is shown, with 30 nM as a repeat (black). For antibodies 19B12 and 3C5, the Langmuir fit model, Rmax overall, RI=0 (light gray) are superimposed.

[0711] Figure 4 : Simulation of antibody 19B12 binding to non-fucosylated AFP (243-261) -G2-Bi. The overlay shows 2 concentration ranges: 7.3 μM to 10 nM for the simulation data set and 1 to 270 nM for the experimental data set. The depicted black line is the data for the experimental and simulation data at 270 nM. The calculated affinity is > 620 nM.

[0712] Figure 5 : Affinity data in solution of antibodies 19B12 and 3C5 binding to AFP(243-261)-G0F or AFP(248-256), n=2.

[0713] Figure 6 : When the samples were pretreated with 50 mM citrate, 20 mM EDTA, 110 mM DTT pH 4.9 (control: PBS + 0.05% Tween 20), the five antibodies 5mAb <afp-l3>rRb-xx-IgG-SulfoBPRu (xx: 6B6, 3A3, 3C5, 3D2, 19B12) Comparison of protein detection in the assay format (total AFP, AFP-L3, AFP-L1 tested at two concentrations of approximately 70 nM and 7 nM).

[0714] Figure 7 :Using 19B12-IgG(P8) Method comparison of AFP-L3 prototype a and Wako AFP-L3 (in ng / mL).

[0715] Figure 8 :Using 3C5-IgG(P8) Method comparison of AFP-L3 prototype b and Wako AFP-L3 (in ng / mL).

[0716] Fig. 9 : AFP-L3 detection in PT1 with or without DDT for samples with low AFP-L3 concentration (master calibrator and native samples).

[0717] Fig.10 : AFP-L3 detection in PT1 with or without DDT for samples with medium and high AFP-L3 concentrations (master calibrator and native samples).

[0718] Fig.11 : Measured in clinical groups ROC curve of AFP-L3.

[0719] Fig.12 : Alignment of the VH and VL sequences of 19B12 and 3C5. The CDR sequences according to Kabat are depicted in bold.

[0720] Examples

[0721] The following examples are provided to aid in understanding the present invention.

[0722] Example 1: Synthesis of peptides for immunization and screening

[0723] Synthesis of glycopeptides

[0724] Peptides are synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis in a peptide synthesizer (e.g., from Protein Technologies, Inc). For amino acid coupling, 5 equivalents of each amino acid derivative are used. The amino acid derivative is dissolved in dimethylformamide containing 1 equivalent of 1-hydroxy-7-azabenzotriazole (HOAt). The peptide is synthesized on Sieber amide resin. The coupling reaction is carried out in dimethylformamide (DMF) with 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) for 5 minutes relative to the resin loading. 20% piperidine in DMF is used to cleave the Fmoc group for 8 minutes after each synthesis step. For the synthesis of disaccharide-containing peptides, sugar amino acid building blocks of Fmoc-protected Asn (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) were employed and coupled by standard conditions as described above. For peptides containing complex biantennary glycans (G0F and GF), a phenylalanine-threonine dipeptide was used as an Fmoc-protected pseudoproline derivative and Asn was introduced as Fmoc-Asn(ODmap). After assembly of the peptides on the resin, cleavage of Asp(ODmab) was achieved by washing the resin with 2% hydrazine in DMF (5x 5 min) followed by treatment with 5 mM sodium hydroxide / methanol (1:1) in water for 1 h. Release of the peptides from the synthesis resin was achieved by incubation with 1% trifluoroacetic acid in dichloromethane (10x 3 min). The reaction solution was then 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.

[0725] By adding 1,3-propanedithiol (40eq) and DIPEA (30eq.) in methanol, the biantennary glycosyl azide of G0F and G2 was reduced to the corresponding amine. After stirring for 4h, the sugar was precipitated by adding cold diisopropyl ether. Sugar coupling with peptide was achieved using 2eq sugar amine (G0F or G2), 2eq HATU, 2eq HOAt, 8eq DIPEA in DMF / DMSO (1:1) overnight. Subsequently, the cleavage of the acid-labile protecting group was achieved at room temperature with 9.5ml trifluoroacetic acid, 0.25ml triisopropylsilane and 0.25ml water 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 acetic acid aqueous 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. Table 1 shows an overview of all peptides synthesized and used for immunization and screening.

[0726] *) Reference for the chemical synthesis of biantennary fucosylated N-glycosyl azides: J. Seifert, C. Unverzagt, Tetrahedron Lett. 1997, 38, 7857-7860.

[0727] Synthesis of biotinylated Asn(G0F)

[0728] Asp-OBzl, biotin-PEG12-NHS ester (1 eq.) and trimethylamine (8 eq.) were dissolved in DMF and stirred for 2.5 h. The crude product was purified by preparative reverse phase HPLC using a gradient of acetonitrile / water containing 0.1% trifluoroacetic acid.

[0729] The G0F-azide was reduced to the corresponding amine by adding 1,3-propanedithiol (40eq) and DIPEA (30eq.) in methanol. After stirring for 4h, the sugar was precipitated by adding cold diisopropyl ether. Sugar coupling with biotin-PEG12-Asp-OBzl was achieved using 0.5eq sugar amine, 1eq HATU, 1eq HOAt, 4eq DIPEA in DMF / DMSO (1:1) overnight. 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 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) is shown in Figure 1 Shown, α-1,6-core-fucosylation is highlighted.

[0730] Immunogen synthesis

[0731] 3-(Maleimido) propionic acid N-hydroxysuccinimide ester was added to a solution of keyhole limpet hemocyanin (KLH) in phosphate buffer (20 mM, pH 7.2). The reaction was incubated at room temperature for 5 hours and then dialyzed with phosphate buffer (0.1 M, pH 7.0). The cysteine ​​containing glycopeptide was dissolved in DMSO and added to a solution of maleimide-activated KLH containing 0.1 M ethylenediaminetetraacetic acid (EDTA). The solution was stirred at room temperature for 5 hours and then dialyzed against phosphate buffer (0.1 M, pH 7.0) to produce KLH-peptide conjugate.

[0732] Glycopeptides and peptides

[0733]

[0734] Table 1: Overview of synthetic peptides used for immunization and screening

[0735] abbreviation

[0736] Ac: N-terminal acetylation

[0737] Ahx: 6-aminohexanoic acid

[0738] βAla: β-alanine

[0739] Bi: Biotin

[0740] Fuc: L-fucose G0F: fucosylated biantennary N-glycan

[0741] G2: non-fucosylated biantennary N-glycans

[0742] GlcNAc: N-acetylglucosamine

[0743] IMG: immunogen

[0744] MP: 3-(Maleimido)propionic acid

[0745] -NH2: C-terminal carboxamide

[0746] PEG: polyethylene glycol

[0747] Structure of glycopeptides and peptides

[0748] BMO 35.000146：

[0749]

[0750] (The AFP peptide portion has SEQ ID NO: 26)

[0751] BMO 35.000148：

[0752]

[0753] (The AFP peptide portion has SEQ ID NO: 2)

[0754] BMO 35.000151：

[0755]

[0756] (The AFP peptide portion has SEQ ID NO: 2)

[0757] BMO 35.000315：

[0758]

[0759] (The AFP peptide portion has SEQ ID NO: 2)

[0760] BMO 35.000150：

[0761]

[0762] BMO 35.000373：

[0763] Ac-TKVNETEIQ-NH2

[0764] (The AFP peptide portion has SEQ ID NO: 25)

[0765] BMO 35.100001：

[0766]

[0767] (The AFP peptide portion has SEQ ID NO: 2)

[0768] BMO 35.100002：

[0769]

[0770] (The AFP peptide portion has SEQ ID NO: 26)

[0771] Example 2: Antibody Generation

[0772] immunity

[0773] 2x2 New Zealand White rabbits (NZW) aged 12 to 16 weeks were immunized with the AFP-L3 glycopeptide (AFP(243-256)-G0F-IMG). To enhance the immunogenicity of the peptide, it was coupled to keyhole limpet hemocyanin (KLH) as a carrier protein. During the first month, animals were immunized weekly. From the second month onwards, the immunization schedule was reduced to monthly. For the first immunization, 500 μg KLH-coupled AFP(243-256)-G0F-IMG were dissolved in 0,9% NaCl and emulsified in 2 ml complete Freund's adjuvant (CFA). For all subsequent immunizations, CFA was replaced by 1 mL incomplete Freund's adjuvant (IFA) emulsion.

[0774] Titer analysis

[0775] Titer analysis was performed using an ELISA protocol. Serum titration was performed using a biotinylated AFP (243-261)-GOF-Bi screening peptide as a positive control.

[0776] The biotinylated peptide was used for screening and was therefore fixed on the surface of a streptavidin-coated 96-well microplate by incubating 100 μl of a 31.25 ng / ml solution per well for 60 min at room temperature. 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) was collected from each rabbit on the 45th and 105th days after the start of the immunization activity. The serum from each rabbit was diluted with PBS containing 1% BSA at 1:300, 1:900, 1:2700, 1:8100, 1:24300, 1:72900, 1:218700 and 1:656100. 100 μl of each dilution was added to the plate previously prepared with the screening peptide and incubated at room temperature for 60 min. The bound antibodies were detected with HRP-labeled F(ab`)2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche). The titer for analyzed animals was set as the 50% signal reduction of the dilution curve.

[0777]

[0778] Table 2: Exemplary titers following immunization with AFP-L3 peptides coupled to KLH

[0779] As demonstrated by the results in Table 2, polyclonal sera from immunized animals bound to the AFP (243-261)-GOF-Bi screening peptide. However, the titer was extremely low, but this was expected since glycopeptides are immunogens with low immunogenicity and it is extremely difficult to develop antibodies that bind to such molecules. Furthermore, it is expected that only very small amounts of antibodies will specifically bind to the core-fucosylated peptide and show low cross-reactivity with the non-fucosylated AFP-L1 variant.

[0780] Enrichment and single cell sorting of antigen-reactive B cells

[0781] For enrichment of antigen-reactive B cells, 31.25ng / ml biotinylated AFP (243-261)-GOF-Bi peptides were pre-incubated with peripheral blood mononuclear cells (PBMC) pools from immune animals at room temperature for 15min. After the washing step, the antigen-reactive B cells loaded with peptides were incubated with streptavidin-coated beads (Miltenyi) at room temperature for 15min. Positive B cells were sorted using MACS columns (Miltenyi) and subsequently incubated, as described in Seeber et al., PLoS One 9 (2014), the 2nd issue, e86184, with the only exception that the sorting of positive B cells involves MACS columns (Miltenyi) instead of plate binding. After B cells were cultured in 96-well plates for 7 days, supernatants were collected for subsequent ELISA analysis, and cells were lysed for cloning. A total of 84 B cells were extracted and enriched from immune animals, about 21 times for each immune animal. A huge advantage of the antibody development approach described here is that animals can be bled multiple times and the maturation of antibodies can be followed over a period of time during the immunization event. The large number of blots per animal also demonstrates how difficult it is to identify clones with the desired specificity for AFP-L3 and low cross-reactivity with AFP-L1.

[0782] Antibody Screening

[0783] Subsequently, Hit-ELISA (i.e., ELISA testing binding to screening agents) was used to identify B cells expressing antibodies with the desired binding characteristics, i.e., binding to the AFP(243-261)-G0F-Bi peptide with core-fucosylation, but not to the glycopeptide AFP(243-261)-G2-Bi without core-fucosylation. The peptides were immobilized on the surface of a streptavidin-coated 384-well plate (Nunc) by incubating 100 μl of a 31,25 ng / ml solution per well for 60 min at room temperature, respectively. The plates were washed and 30 μl of rabbit B-cell culture supernatant was transferred to each well and incubated for 1 h at room temperature. To detect antibodies bound to the screening agents, HRP-labeled F(ab`)2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche) were added according to the manufacturer's instructions. Several clones were identified that bound to glycopeptides with core-fucosylation, but not to peptides without core-fucosylation (in a total of 84 B cell sorting experiments from 4 immunized rabbits) (Table 2). Some of the cloned V regions were cloned into mammalian expression vectors and subsequently expressed in 2 ml HEK293 cells (described in Seeber et al., PLoS One 9 (2014), Issue 2, e86184.). After one week of expression, the supernatant of transfected HEK293 cells containing rabbit IgG was then used for selection of a subset of antibodies that met the performance criteria for detailed kinetic analysis based on the initial SPRBiacore (see Table 3).

[0784]

[0785] Table 3: ELISA results for exemplary clones producing antibodies characterized by specific binding to positive screening agents but not to negative screening agents

[0786] Example 3: Kinetic screening and further kinetic characterization

[0787] Surface plasmon resonance (SPR) spectroscopy–assisted kinetic screening and detailed kinetic characterization were performed to select antibodies with superior kinetic profiles and target specificity.

[0788] Kinetic screening was performed at 25 °C on a GE Healthcare Biacore TM B4000 instrument. The Biacore CM5 series S sensor was installed into the instrument and the hydrodynamics and pretreatment were performed according to the manufacturer's instructions. The system buffer was HBS ET pH 7.4, 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% (w / v) Tween 20. The system buffer was supplemented with 1mg / mL CMD (carboxymethyl dextran, Sigma, catalog number 86524) and used as the sample buffer for preparing the dilution series.

[0789] Rabbit antibody capture system was immobilized on the sensor surface. The polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (Cat. No. 111-005-046; Jackson Immuno Research, Cat. No. 105332) was amine coupled using EDC / NHS chemistry according to the manufacturer's instructions.

[0790] 30 μg / mL goat anti-rabbit Fcγ (GARbFcγ) in 10 mM sodium acetate buffer pH 4.5 was pre-concentrated to spots 1, 2, 4 and 5 in flow cells 1, 2, 3 and 4 and covalently bound to the sensor surface at a density of 12000-13000 RU. Free activated carboxyl groups were saturated with 1 M ethanolamine pH 8.5.

[0791] Points 1 and 5 were used for interaction measurements, and points 2 and 4 were used as references. Each rabbit antibody from the primary cell supernatant was diluted 1:2 in 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. In order to achieve sufficiently high sensitivity, additional molecular mass loading for 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. The biotinylated peptide, streptavidin and amino-PEO-biotin (Thermo Fisher, catalog number 21346) were mixed in a ratio of 1:10:5 and incubated at room temperature (RT) for 2 hours. The molecular weight of SA-grafted AFP (243-261)-G0F-Bi and SA-grafted AFP (243-261)-G2-Bi was calculated to be 64 kDa. The SA-grafted peptide was injected as an analyte at 150 nM at 30 μL / min into the corresponding surface-displayed anti-AFP-L3 antibody. The association phase and dissociation phase were monitored for 5 minutes.

[0792] Rabbit colonies were regenerated from the sensor surface by injecting 10 mM glycine pH 1.5 at 20 μL / min for 20 sec, followed by 2 injections of 10 mM glycine pH 1.7 for 60 sec. TM Kinetics were monitored with B4000 Control SW V1.1 and evaluated with Evaluation SW V1.1. The following reporting points were monitored: Late Binding (BL) shortly before the end of analyte injection and Late Stability (SL) shortly before the end of the dissociation phase. BL and SL data were used to characterize antibody / antigen binding stability. In addition, the dissociation rate constant k was calculated according to the Langmuir 1:1 model. d [s -1 ]. According to the formula ln(2) / 60*k d . Calculate the half-life of antigen / antibody complex stability (minutes). Calculate the molar ratio representing the binding stoichiometry using the following formula:

[0793] MW(antibody) / MW(antigen)*BL(antigen) / CL(antibody)

[0794] Specific antibody kinetics were identified from multiple antibodies, where the antibodies distinguished between fucosylated and non-fucosylated AFP-L3 peptides (see Figure 2 ).

[0795] Kinetic characterization

[0796] Using BIAcore from GE Healthcare TM Detailed kinetic studies were performed on a T200 instrument at 37°C using a Series S C1-sensor at 37°C for binding of the following AFP-L3 peptides:

[0797] ·AFP(243-261)-G0F-Bi

[0798] ·Asn(G0F)-Bi

[0799] ·AFP(243-261)-G2-Bi

[0800] ·AFP(243-261)-(Fuc-GlcNAc)-Bi

[0801] Five rabbit mAbs identified through kinetic screening <afp-l3>19B12, 3C5, 3D2, 3A3 and 6B6 were subjected to kinetic characterization.

[0802] The rabbit antibody capture system was immobilized on flow cells 1-4 at 700 to 800 RU as described. Flow cell 1 served as a reference, flow cells 2, 3 and 4 were used for interaction measurements. 150 nM antibodies 1 to 5 were injected at 10 μL / min for 30 seconds. The capture level (CL) was monitored in resonance units RU. A peptide analyte concentration series of 3.3 nM to 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 2 injections of 10 mM glycine pH 2.25 for 60 seconds. According to BIAcore TM T200 Evaluation SW 3.2 uses the Langmuir 1:1 fitting model to determine the kinetic rate constant and dissociation equilibrium constant K D Secondly, a Langmuir 1:1 fit was applied to Scrubber-SW V2.0c.

[0803] The kinetic profiles of peptide-binding antibodies 19B12, 3C5, 3D2, 3A3, and 6B6 are shown in Figure 3 The binding kinetics of antibody 19B12 and AFP(243-261)-G0F-Bi are shown in Figure 2. 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. Affinity is K D =2.5±0.08% nM (see Table 4 and Figure 4 By visual inspection, the kinetic profile of antibody 3C5 approached saturation for the highest concentration of antibody shown, 90 nM. The interaction showed complex binding behavior; therefore, the constants stated represent apparent values ​​(see Tables 4 and Figure 3 ).

[0804] By visual inspection, antibodies 3D2 and 3A3 showed slower complex formation when binding AFP(243-261)-G0F-Bi, not approaching antibody saturation at the highest concentration. The interaction did not obey Langmuir's law (see Figure 3 By visual inspection, antibody 6B6 showed the weakest interaction with fucosylated AFP(243-261)-G0F-Bi (see Figure 3 ).

[0805] These analyses revealed that clones 19B12 and 3C5 showed the best performance in terms of their specificity and affinity. Both clones were specific for core-fucosylation and also bound to the AFP peptide backbone. The latter was important, as clones that only bind core-fucose will bind to any other core-fucosylated proteins (e.g., IgG-type molecules) in the sample.

[0806]

[0807] Table 4: Binding constants of antibodies 19B12 and 3C5

[0808] The binding kinetics of the non-fucosylated AFP(243-261)-G2-Bi of antibody 19B12 could not be determined. Based on the determined rate constants for the measured concentration range, simulations for concentrations ranging from 7.3 μM to 10 nM inferred a weak affinity of >620 nM for non-fucosylated AFP(243-261)-G2-Bi. The theoretical response maximum R for the simulated data was max 9.3RU is calculated based on the experimental capture level (CL) of 155RU and the analyte molecular mass of 4.3kDa. Figure 4 In Figure 2, an overlay of both the simulated concentration range and the experimental concentration range is shown. The black line resembles the same concentration of 270 nM for both data sets.

[0809] Example 4: Affinity analysis in solution

[0810] Dissociation equilibrium constant K D Determine via the affinity method (AiS) in solution.According to the supplier's instructions of CAP-Kit (Cytiva), capture peptide AFP (243-261)-G0F-Bi on the CAP chip sensor surface.Hatch 10nM antibody 19B12 and 3C5 and the non-biotinylated AFP (243-261)-G0F of different concentrations between 120nM and 0.01nM or the mixture of the non-biotinylated AFP (248-256) of different concentrations between 200μM and 0.1nM until reaching equilibrium.Monitor the binding events of the mixture and the AFP (243-261)-G0F-Bi displayed on the surface.Along with the increase of the peptide concentration as a competitor, the 'free' antibody in the solution will be reduced.Then the free antibody concentration determined by the competition experiment is plotted against the peptide competitor concentration.Regenerate using the guanidinium / NaOH solution supplied by Cytiva.Analyze two independent series for each interaction. The data were evaluated using the in-solution affinity model from the BiacoreEvaluation software.

[0811]

[0812] Table 5: Affinity in solution of antibodies 19B12 and 3C5 binding to peptides AFP(243-261)-G0F and AFP(248-256)

[0813] AiS-based K of antibody 19B12 binding to peptide AFP(243-261)-G0F D 0.9±0.1nM, R 2 0.99702RU, B tot 9.8±0.1 nM is in the same range as the concentration-dependent kinetics described previously herein and thus validates the experimental approach (see Table 5). In addition, the affinity approach in solution showed the excellent specificity of antibodies 19B12 and 3C5 for fucosylated AFP(243-261)-G0F through their complete competition.

[0814] For antibody 3C5, K D 0.4±0.1nM, R 2 0.98917RU, B tot 9.7±0.1 nM determined AiS (see Table 5). K based on AiS D 5.7±0.5μM, R 2 0.98766RU, B tot 10.1±0.1nM or 12.6±2.9μM, R 2 0.94759RU, B tot 11.5±0.4 nM demonstrated that antibodies 19B12 and 3C5 bound to peptide AFP (248-256) with weak affinity.

[0815] Example 5: Cloning of antibodies and antibody formats, expression of antibodies, purification and labeling of antibodies

[0816] Cloning and expression of IgG (P8) multivalent IgG antibody

[0817] Multivalent anti-AFP-L3 antibodies were cloned and expressed as described in WO 2019 / 057816 A1. In detail, in order to generate multivalent anti-AFP-L3 antibodies, multiple VH-CH1 sequences flanking linker sequences (e.g., (G3S)4; SEQ ID NO: 27) were added upstream and downstream of the hinge-CH2-CH3 coding sequence to generate heavy chains encoding multiple VH-CH1 domains and cloned into an expression vector. The heavy chain vector is co-expressed with a light chain expression vector encoding a standard light chain consisting of a VL and a constant domain. Recombinant expression is performed transiently in human embryonic kidney (HEK) cells, or transiently or non-transiently in CHO cells. Transformed cells secrete multivalent monospecific anti-AFP-L3 antibodies into the serum-free culture supernatant from which they are isolated. The final multivalent P8 antibody format contains 8 different Fab portions, all of which have the same sequence (i.e., 19B12 or 3C5).

[0818] Antibody purification and ruthenium conjugation

[0819] Monoclonal multivalent anti-AFP-L3 antibodies of clones 19B12 and 3C5 were produced in sufficient yields and without significant losses during purification using protein An affinity chromatography of culture supernatants using MabSelect SuRe (Cytiva) according to the supplier's instructions. Purified multivalent recombinant anti-AFP-L3 antibodies were reacted with the sulfonated form of the labeling reagent terpyridine ruthenium (also known as "sBPRu") using standard NHS ester coupling chemistry. Under these conditions, the ruthenium label was covalently attached to the functional group of the lysine amino acid residues in the antibody backbone.

[0820] Purification, fragmentation and conjugation of mAbTU11 with biotin (generating mAb <afp> M-TU11-F(ab< / afp> ' )2-Bi)

[0821] 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 fragment of TU11 was finally conjugated to a biotin-PEG24-NHS tag via surface exposed lysine using standard NHS ester coupling chemistry. The F(ab')2-Bi-PEG24 conjugate was further purified using affinity chromatography using mSA (streptavidin mutein) to remove unconjugated F(ab')2 fragments.

[0822] Expression and purification of anti-AFP-L3 antibody

[0823] 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.

[0824] Purification and conjugation of anti-rabbit Fc antibodies

[0825] Polyclonal antibodies against rabbit Fc were generated by immunizing sheep with purified rabbit Fc. The polyclonal anti-rabbit Fc antibodies were further purified via positive immunoadsorption using rabbit Fc immunosorbent and labeled via surface exposed lysines using the sulfonated form of terpyridine ruthenium (also known as "sBPRu") using standard NHS ester coupling chemistry.

[0826] Example 6: Purification of AFP-L3 and L1 and protein labeling

[0827] Purification of AFP-L1 and AFP-L3

[0828] AFP purified from human hepatoma cell lines was purchased from BioRad (Cat. No. 13752600) or Scripps (Cat. No. 90492-0050). Affinity chromatography was performed on AFP using Lentil Agglutinin (LCA) (Vector Laboratories, Cat. No. AL-103). AFP run on an LCA column containing running buffer (50mM TRIS-HCl pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM CaCl2) can be separated into 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 200mM α-methyl mannoside and 200mM α-methyl glucoside to the running buffer.

[0829] Biotinylation of AFP

[0830] Purified AFP was biotinylated via surface exposed lysines with a biotin-DDS-NHS label using standard NHS ester coupling chemistry. Unreacted label was subsequently removed by dialysis.

[0831] Example 7: In immunological assays ( Comparison of selected antibody clones in the assay format with respect to binding to the AFP-L3 analyte

[0832] Five different antibody clones selected based on kinetic screening have been tested. <afp-l3>AFP-L3 specificity of rRb-xx-IgG (xx: 6B6, 3A3, 3C5, 3D2, 19B12) and its instrument e601 is based on The utility of generating signal kinetics in immunoassays.

[0833] Briefly, the biotinylated peptide or protein (sample) was incubated with the tested 1,6-fucAFP antibodies (6B6, 3A3, 3C5, 3D2, 19B12) and ruthenium-containing anti-rabbit Fc antibodies (see Example 6) for 9 min. Subsequently, 40 μl Beads (coated with streptavidin; "SA-beads") were added and the mixture was incubated for 9 min so that the detection complex formed in the first step became bound to the solid phase via the interaction of biotin and streptavidin. After incubation, the reaction mixture was aspirated into a measuring cell and The beads are captured magnetically onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0834] Test protocol and sandwich principle

[0835] 1st incubation: 40μl sample + 60μl reagent 1 + 60μl reagent 2, incubate for 9 minutes

[0836] 2nd incubation: +40μl SA beads. After adding streptavidin-coated microparticles, the complex binds to the solid phase via the interaction between biotin and streptavidin and incubates for 9min.

[0837] After incubation, the reaction mixture is aspirated into a measuring cell where the microparticles are magnetically captured onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0838] Reagent 1: mAb <afp-l3>rRb-xx-IgG (1μg / mL) (xx: 6B6, 3A3, 3C5, 3D2, 19B12)

[0839] Reagent 2: pAb <rrb-fcg>-S-IgG(IS)-sulfoRu(Sux)(0.5μg / mL)

[0840] Assay buffer: 40 mM NaPi pH 7.5 / 150 mM NaCl / 0.1% MIT / 0.1% Hydroxypyridone / 0.1% Polidocanol / 0.1% PAK-R-IgG / 2% RPLA 4

[0841] sample:

[0842] The following biotinylated peptides diluted in PBS / 0.05% Tween-20 were tested (2 concentration levels):

[0843] 1. AFP-(243-261)-G0F-Bi: with core-fucosylation

[0844] 2. AFP-(243-261)-G2-Bi: without core-fucose (AFP-L1 variant)

[0845] 3. AFP-(243-261)-(Fuc-GlcNAc)-Bi disaccharide L3

[0846] 4. AFP(243-256)-(GlcNAc-GlcNAc)-Bi: disaccharide L1

[0847] In addition, the following biotinylated proteins were tested:

[0848] 5. Total alpha-fetoprotein (AFP), including AFP-L3 fraction, derived from human hepatoma cell line

[0849] Lentil agglutinin A (LCA) affinity chromatography was performed with total AFP.

[0850] Two fractions were isolated:

[0851] a) Alpha-fetoprotein (AFP-L3) is a fucosylated variant of AFP that reacts with Lentil agglutinin A

[0852] b) Alpha-fetoprotein (AFP-L1) is a non-fucosylated variant of AFP that should not react with Phaseolus vulgaris agglutinin A

[0853] As shown in Table 6, all tested antibodies bound 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 results of the kinetic analysis, indicating that these two clones were the best binders.

[0854]

[0855]

[0856] Table 6: Initial comparison of five mAb candidates against binding motifs on peptides and proteins Counting measurements. All five antibodies bound to the peptides AFP-(243-261)-G0F-Bi and AFP-(243-261)-(Fuc-GlcNAc)-Bi).

[0857] At the peptide level, the selected antibodies could distinguish between core-fucosylated and non-fucosylated variants. At the level of the native antigen, very low signals were observed, indicating that this epitope may not be easily accessible. However, for antibodies 19B12 and 3C5, the AFP-L3 fraction produced higher signals than the AFP-L1 fraction, confirming their specificity also at the level of the native antigen.

[0858] Example 8: Preprocessing for improving AFP-L3 detection on Elecsys

[0859] Since the native AFP-L3 antigen showed very low signal compared to the peptide structure, we speculated that the epitope region might not be easily accessible in native AFP-L3 for the selected anti-peptide antibodies. To improve accessibility, we tested various pretreatment conditions that were applied to samples containing AFP-L3 for a predetermined pretreatment time with the goal of making the epitope more accessible to the antibody.

[0860] Several pretreatment conditions were initially tested and it became apparent that in particular pretreatment with protein reducing agents such as DTT or TCEP could significantly improve the signal.

[0861] To determine whether a protein reducing agent such as DTT is sufficient to promote the binding of antibodies 19B12 and 3C5 and whether this is related to the To determine how the other antibodies tested in Example 7 compared, the following experiments were performed.

[0862] Test plan:

[0863] First step: preprocessing

[0864] Manual sample pretreatment was performed as follows: 10 μl protein sample (protein concentration 250 μg / mL or 25 μg / ml in stock solution) + 490 μl pretreatment solution A or B (see below); the mixture was incubated at room temperature for 30 min.

[0865] The following biotinylated proteins have been used as samples:

[0866] 1. Total alpha-fetoprotein (AFP), including AFP-L3 fraction, derived from human hepatoma cell line

[0867] 2. Alpha-fetoprotein (AFP-L3), a fucosylated variant of AFP that reacts with Lentil agglutinin A

[0868] 3. Alpha-fetoprotein (AFP-L1) is a non-fucosylated variant of AFP that should not react with Phaseolus vulgaris agglutinin A

[0869] Two different pre-treatment solutions have been tested for each sample:

[0870] 50 mM citrate, 20 mM EDTA, 110 mM DTT pH 4.9 (pretreatment solution A)

[0871] And negative control: PBS + 0.05% Tween 20 (pretreatment solution B)

[0872] The final concentration of protein used in the sample pretreatment incubations was 5000 ng / mL or 500 ng / mL (this corresponds to protein concentrations of approximately 70 nM and 7 nM in the sample pretreatment incubations). The concentration of DTT in the sample pretreatment incubations was 107 mM.

[0873] After manual pretreatment, the assay was performed on a cobas e601 analyzer using the following reagents under the following incubation conditions:

[0874] Step 2: Adding ruthenium-labeled AFP-L3 antibody

[0875] 40 μl pre-treated sample (from the first step) + 60 μl reagent 1 + 60 μl reagent 2, incubate for 9 min

[0876] Already used The following reagents :

[0877] Reagent 1: mAb <afp-l3>rRb-xx-IgG-SulfoBPRu (1 μg / mL) (five different clones have been tested: xx: 6B6, 3A3, 3C5, 3D2, 19B12)

[0878] Reagent 2: 40mM NaPi pH 7.5 / 150mM NaCl / preservative / 0.1% polidocanol

[0879] Step 3: The mixture of step 2: + 40 μl SA-beads (microparticles coated with streptavidin). After adding SA-beads, the complex binds to the solid phase via the interaction of biotin and streptavidin and incubates for 9 min.

[0880] After incubation, the reaction mixture is aspirated into a measuring cell where the microparticles are magnetically captured onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0881] The results are depicted in Figure 6 middle.

[0882] Summary of results:

[0883] The samples pretreated with the pretreatment solution containing DTT produced much higher signals than the samples pretreated with the control solution (without DTT). This indicates that the recognition of AFP-L3 was improved by using the pretreatment solution (containing a reducing agent) to achieve better signal detection.

[0884] - Clones 3C5 and 19B12 delivered the highest signals, confirming that these antibodies also had better binding to pre-treated AFP-L3.

[0885] AFP and AFP-L3 proteins (both containing core-fucosylated AFP) are more

[0886] The L1 (non-core-fucosylated) protein delivered a much higher signal. This confirmed the specificity of the clone used, as previously reported in Biacore and shown at the peptide level.

[0887] Example 9: Preprocessing Optimization

[0888] In Example 8, 50 mM citrate, 20 mM EDTA, 110 mM DTT pH 4.9 was used for pretreatment. As is known in the art, DTT has a much higher redox potential at alkaline or neutral pH, but is much more stable at acidic pH (DTT is more stable in buffers with pH < 5.5). Therefore, although the pretreatment solution in Example 8 showed good stability, a relatively high concentration of DTT was used.

[0889] Since DTT shows high reducing activity at pH>7, we hypothesized that lower DTT concentrations could be used if the pH in the sample pretreatment incubations was within this pH range.

[0890] In order to achieve both reducing activity and adequate reagent stability at much lower DTT concentrations, one pretreatment solution was divided into 2 different bottles: PT1 and PT2.

[0891] PT1: 7.4mM DTT, 2mM EDTA pH 5.5, with stable DTT storage conditions

[0892] PT2: Two different PT2 preparations have been tested: 100 mM Tris pH 8.5 or 200 mM NaOH pH>13

[0893] Using this setup, a pH > 7.5 could be achieved in the sample pretreatment incubation (sample+PT1+PT2).

[0894] These pretreatment conditions were tested using 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 AFP-L3) was mixed with PT1 and PT2 solutions and incubated for 9 min. In step 2, R1 containing biotinylated AFP-specific antibody TU11 as F(ab')2 and R2 containing antibody 19B12 as ruthenium-containing multivalent antibody (P8 format) were added and the mixture was incubated for 9 min. Finally, in step 3, magnetic streptavidin microbeads were added and the resulting mixture was incubated again for 9 min.

[0895] Reagents used:

[0896] R1: mAb <afp>M-TU11-F(ab`)2-Bi(Linker) 2.3μg / ml

[0897] R2: mAb <afp-l3>rRb-19B12-IgG(P8)-SulfoBPRu 15μg / ml

[0898] Test plan:

[0899] 30μl sample + 13μl PT1 + 23μl PT2, incubate for 9 minutes

[0900] +51μl R1+53μl R2, incubate for 9 minutes

[0901] + 29 μl SA-beads, incubate for 9 min

[0902] After incubation, the reaction mixture is aspirated into a measuring cell where the microparticles are magnetically captured onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0903] result

[0904] AFP-L3 (ng / mL) PT2: 100 mM Tris pH 8.5 PT2: 200 mM NaOH pH>13 0.0000 717 789 809 134185 3255

[0905] Table 7: Comparison of the two PT2 conditions on human sera with low or high AFP-L3 protein concentrations Count measurement.

[0906] Both pretreatments produced a measurable signal, but using Tris pH 8.5 showed much higher performance than using NaOH (which produced a higher pH) as PT2.

[0907] The experiments concluded that TRis buffer is more suitable for PT2 than more irritating alkaline solutions (e.g., NaOH).

[0908] Furthermore, we noted in this experiment that the P8 format increased the signal relative to a similar experiment using IgG instead of the multivalent P8 format of the AFP-L3 antibody.

[0909] Example 10: Assessing the Importance of the Reducing Agent DTT in the Pretreatment Solution Using the 19B12 Clone

[0910] Examples 8 and 9 used the chelating agent EDTA in addition to DTT in the pretreatment. To gain a deeper understanding of the importance of DTT for pretreatment activity, different conditions were tested using native human serum and spiked samples (master calibrator sets MK1 to MK7, human serum spiked with AFP-L3 antigen at increasing concentrations).

[0911] The following two PT1 compositions were tested to confirm the role of DTT in pretreatment:

[0912] PT1 version 1: 10 mM DTT, 2 mM EDTA, pH 4.7: contains DTT

[0913] PT1 version 2: 2 mM EDTA, pH 4.7: without DTT

[0914] PT2: 150 mM TRIS pH 8.5

[0915] Test plan:

[0916] The assay principle used is the same as in Example 9 (i.e. sandwich immunoassay). Step 1: 30 μl sample + 13 μl PT1 (version 1 or 2) + 23 μl PT2, incubate for 9 min

[0917] Step 2: +51μl R1+53μl R2, incubate for 9 minutes

[0918] Step 3: Add 29 μl SA-beads, incubate for 9 min

[0919] R1: Biotinylated monoclonal anti-AFP antibody mAb <afp>M-TU11-F(ab')2-PEG24-Bi 3.4mg / L; TRIS buffer 200mM, pH 7.5; preservative

[0920] R2: monoclonal anti-AFP-L3 antibody mAb labeled with ruthenium complex <afp-l3>rRb-19B12-IgG(P8)-SulfoBPRu 22.0mg / L; TRIS buffer 200mM, pH 7.5; preservative

[0921] After incubation, the reaction mixture is aspirated into a measuring cell where the microparticles are magnetically captured onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0922] Fig. 9 and 10 The results shown in show the key role of reducing agents such as DTT for better detection of AFP-L3 in both native and spiked samples.

[0923] Example 11: Variation of DTT concentration (mM) and Tris buffer concentration (mM) in PT2

[0924] The purpose of this experiment was to evaluate different concentrations of DTT in PT1 and different concentrations of Tris in PT2.

[0925] Again a sandwich immunoassay format was used.

[0926] Pretreatment conditions for analysis :

[0927] Different concentrations in PT1 were tested:

[0928] PT1: pH 5.5, DTT + EDTA 2 mM varying from 0 to 10 mM

[0929] Two different concentrations of Tris have been tested in PT2:

[0930] PT2: pH 9.0, Tris buffer (50 or 200 mM)

[0931] For reagents 1 and 2, constant conditions have been used:

[0932] Reagent 1: mAb <afp>M-TU11-F(ab`)2-Bi(PEG24)(2.5μg / mL)

[0933] Reagent 2: mAb <afp-l3>rRb-19B12-IgG(P8)-SulfoBPRu(15μg / mL)

[0934] Samples measured:

[0935] Human serum (="HS") spiked with AFP-L3 protein, pH set to 8

[0936] Test plan:

[0937] First incubation: 39 μl sample + 16 μl PT1 + 16 μl PT2, incubate for 9 min

[0938] Second incubation: mixture of first incubation + 50μl R1 + 52μl R2, incubate for 9 minutes

[0939] Third incubation: Second incubation of mixture + 30 μl SA-beads, incubate for 9 min

[0940] After incubation, the reaction mixture is aspirated into a measuring cell where the microparticles are magnetically captured onto the electrode surface. Unbound material is then removed. Application of a voltage to the electrodes then induces electrochemiluminescent emission that is measured by a photomultiplier tube.

[0941] The DTT concentrations tested are summarized in Table 8 below.

[0942]

[0943] Table 8: Variation of DTT concentration.

[0944] result:

[0945] Table 9 summarizes the signals measured for different test conditions:

[0946]

[0947] Table 9: Comparison of different conditions [DTT concentration in PT1 and Tris concentration in PT2] for human serum (= HS) with high AFP-L3 protein concentration (different pH adjusted) Counting measurement

[0948] in conclusion:

[0949] As shown in Table 9, different DTT concentrations can be used for pretreatment. Concentrations as low as 0.676 mM in the pretreatment sample mixture were sufficient to enhance the signal compared to no DTT. This suggests that even lower concentrations of DTT can be used.

[0950] In this example, DTT concentrations ranging from 1 mM to 10 mM have been tested in PT1. In previous examples, particularly in Example 8, a higher DTT concentration of 110 mM has been used in the stock solution. This demonstrates that a wide range of DTT concentrations can be used to improve the signal, although to varying degrees depending on the concentration and pH of the pre-treatment sample incubation.

[0951] EDTA was found to stabilize DTT in PT1, even when used at 2 mM.

[0952] Example 12: Prototype Comparison of AFP-L3 assay with Wako instrument

[0953] Using two different prototypes The AFP-L3 assay was measured on a clinical panel (1000 clinical samples containing HCC cases and controls) and compared with μTAS Wako AFP-L3 (Fujifilm Wako Pure Chemical Corporation). The prototype assays were identical with the only exception of whether 19B12 or 3C5 P8 type multivalent antibodies were used.

[0954] The assay setup was as follows:

[0955]

[0956] Table 10: Assay protocol

[0957] Reagents used:

[0958] PT1: 6mM DTT, 2mM EDTA, pH 5.5

[0959] PT2: 100 mM TRIS pH 8.5

[0960] Two assay prototypes were evaluated, as shown in Table 11.

[0961]

[0962] Table 11: Prototype determinations a and b

[0963] In addition, the samples were measured by μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation) according to the manufacturer's instructions to determine the content of AFP-L3 (in %) and the total concentration of AFP (in ng / mL). The Wako AFP-L3 concentration (in ng / mL) was calculated from the μTASWako values ​​obtained (% AFP-L3 and total AFP concentration (in ng / mL)). As described in Example 11, prototypes a and b were used to directly obtain AFP-L3 value (in ng / mL).

[0964] Both assay prototypes have shown very similar results and show very good correlation with μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation), see Figure 7 and 8 .

[0965] The results of this method comparison confirmed that the prototype showed good correlation with the marketed and clinically validated μTASWako AFP-L3 (Fujifilm Wako Pure Chemical Corporation). This also confirmed that AFP-L3 was substantially detected by the assay of the present invention even without the use of LCA lectin.

[0966] Example 13: Assay Performance in a Clinical Panel

[0967] To further confirm the use of antibodies 19B12 and 3C5 Clinical value of the AFP-L3 prototype assay A clinical panel was analyzed and the diagnostic performance for detecting early HCC was assessed. The panel included 96 patients with liver disease, 38 with HCC, and 58 with liver disease but without diagnosed HCC (controls).

[0968] A prototype based on 19B12 is used as an example.

[0969] Test plan:

[0970] Sandwich immunoassay

[0971] Step 1: 30 μl sample + 13 μl PT1 + 23 μl PT2, incubate for 9 min

[0972] Step 2: +51μl R1+53μl R2, incubate for 9 minutes

[0973] Step 3: Add 29 μl SA-beads, incubate for 9 min

[0974] Reagents

[0975] PT1; 10mM DTT, 2mM EDTA, pH<5.5

[0976] PT2: 150 mM TRIS pH 8.5

[0977] R1: Biotinylated monoclonal anti-AFP antibody TU11; TRIS buffer 200 mM, pH 7.5; preservative

[0978] R2: monoclonal anti-AFP-L3 antibody 19B12 labeled with ruthenium complex as multivalent P8 antibody; TRIS buffer 200 mM, pH 7.5; preservative

[0979] ROC curve Fig.11 The AUC (area under the curve) of the 19B12-based AFP-L3 assay was 91.4% (CI: 85.8-97), demonstrating that the assay in this panel has very good performance for detecting HCC.

[0980] standard Deadline Sensitivity Specificity 95% Specifications 5.27 ng / mL 63.2 94.8 90% Specifications 3.28 ng / mL 71.1 89.7

[0981] Table 12: Predefined specificity criteria as measured in Panel A Clinical performance (sensitivity and specificity) of the AFP-L3 assay < / afp> < / afp> < / afp>

Claims

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 comprising said α-1,6-core-fucosylation, wherein said monoclonal antibody or antigen-binding fragment comprises: (i) a heavy chain variable domain (VH), the VH comprising: a CDR-H1 having the amino acid sequence of SEQ ID NO: 3 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 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 a 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: a CDR-L1 having an 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 having an amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by one amino acid substitution; and a CDR-L3 having an amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by one amino acid substitution.

2. The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the partial sequence of AFP comprising the α-1,6-core-fucosylation comprises or consists of the glycopeptide of formula I, 3. The monoclonal antibody or antigen-binding fragment according to claim 1 or 2, wherein the monoclonal antibody or antigen-binding fragment specifically binds to the glycopeptide of formula I and the glycopeptide of formula II shown in claim 2, 4. The monoclonal antibody or antigen-binding fragment according to claim 3, wherein the K for binding to the glycopeptide of formula II D and K for binding to the glycopeptide of Formula I D The ratio between them is at least 2.

5. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein (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 to SEQ ID NO: 11 or 12; and (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 to SEQ ID NO: 13 or 14.

6. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 4, wherein (i) the heavy chain variable domain (VH) has the amino acid sequence of SEQ ID NO: 11 or 12; and (ii) the light chain variable domain (VL) has the amino acid sequence of SEQ ID NO: 13 or 14.

7. The monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 6, which has a K of 100 nM or less, 20 nM or less, 10 nM or less, or 3.1 nM or less. D in combination with a glycopeptide of formula I as defined in claim 2, optionally wherein the K is measured at 37°C D .

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

9. A polynucleotide or a group of polynucleotides encoding: (i) a heavy chain or a heavy chain variable domain of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8, and / or (ii) a light chain or a light chain variable domain of the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8.

10. A vector comprising the polynucleotide or a group of polynucleotides according to claim 9.

11. A host cell comprising the polynucleotide or a group of polynucleotides according to claim 9 or the vector according to claim 10, and expressing the antibody or antigen-binding fragment according to any one of claims 1 to 8.

12. A method for producing the monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 8, the method comprising culturing the host cell according to claim 11 and isolating the antibody or antigen-binding fragment.

13. A composition comprising the antibody or antigen-binding fragment according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10 or the host cell according to claim 11.

14. Use of the antibody or antigen-binding fragment according to any one of claims 1 to 8 or the composition according to claim 13 for an in vitro immunoassay, in particular for detecting alpha-1,6-core-fucosylated alpha-fetoprotein (AFP).

15. An in vitro immunoassay method for detecting α-1,6-core-fucosylated AFP or a partial AFP sequence comprising said α-1,6-core-fucosylation in a sample using the antibody or antigen-binding fragment as defined in any one of claims 1 to 8, said in vitro immunoassay method optionally comprising pre-treating the sample with a pre-treatment reagent according to claim 16.

16. A pretreatment reagent for treating a sample containing α-1,6-core-fucosylated AFP, the pretreatment reagent comprising a reducing agent, in particular a protein reducing agent.

17. A kit comprising the antibody or antigen-binding fragment as defined in any one of claims 1 to 8 and optionally a pretreatment reagent according to claim 16.

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