Antibodies specific to α-1,6-core-fucosylated PSA and its fucosylated fragments.

Abstract

The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to α-1,6-core-fucosylated prostate-specific antigen (PSA) and subsequences thereof containing α-1,6-core-fucose residues. The antibodies and antigen-binding fragments significantly distinguish between core-fucosylated PSA or core-fucosylated PSA subsequences and other glycosylated PSA species and subsequences thereof lacking core-fucose residues, including aglycosylated PSA, as well as core-fucosylated glycans in other contexts. The present invention further relates to nucleic acid molecules encoding the light chain variable region or heavy chain variable region of the antibodies of the present invention, and vectors comprising such nucleic acid molecules. The present invention also relates to host cells comprising the vectors of the present invention, and methods for producing the antibodies or antigen-binding fragments of the present invention, comprising culturing the host cells of the present invention under appropriate conditions and isolating the produced antibodies. Furthermore, the present invention relates to antibodies obtainable by the methods of the present invention, and to compositions comprising at least one antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, the host cell of the present invention, or an antibody produced by the methods of the present invention. The present invention also relates to the use of the antibodies or antigen-binding fragments of the present invention for detecting and distinguishing core-fucosylated PSA or core-fucosylated subsequences thereof in biological samples.

Description

[Technical Field]

[0001] 1. Field of Invention The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to α-1,6-core-fucosylated prostate-specific antigen (PSA) and subsequences thereof containing α-1,6-core-fucose residues. The antibodies and antigen-binding fragments significantly distinguish core-fucosylated PSA or core-fucosylated PSA subsequences from other glycosylated PSA species and subsequences thereof, including aglycosylated PSA lacking core-fucose residues, and, in other contexts, core-fucosylated glycans. The present invention further relates to nucleic acid molecules encoding the light chain variable region or heavy chain variable region of the antibody of the present invention, and vectors containing the nucleic acid molecules. The present invention also relates to host cells containing the vector of the present invention, and to methods for producing the antibody or antigen-binding fragment of the present invention, comprising culturing the host cells of the present invention under appropriate conditions and isolating the produced antibody. Furthermore, the present invention relates to antibodies that can be obtained by the method of the present invention, and to compositions comprising at least one of the antibody or antigen-binding fragment of the present invention, the nucleic acid molecule of the present invention, the vector of the present invention, the host cells of the present invention, or the antibody produced by the method of the present invention. The present invention also relates to the use of antibodies or antigen-binding fragments of the present invention for detecting and identifying core-fucosylated PSA or core-fucosylated subsequences thereof in biological samples. [Background technology]

[0002] 2.Background Prostate cancer (PCa) is the fifth leading cause of cancer death in men worldwide, with an estimated 359,000 deaths in 2018; www.globocan.iarc.fr (Non-Patent Literature 1) (Accessed April 2020). The incidence of PCa has increased by more than 40% in the last decade, mainly due to improved detection rates from annual screening for prostate-specific antigen (PSA) in men beginning at age 50 and increased life expectancy in the population. Human PSA, also known as kallikrein-3 (KLK3), Uniprot ID P07288, is a glycoprotein with a single N-glycosylation site in Asn-69 containing α-1,6 core fucose. It is the most important protein in the management of men suspected of or diagnosed with prostate cancer (PCa). In clinical practice, PSA blood levels of 10 ng / ml or higher indicate a risk of prostate cancer, and prostate biopsy is usually recommended; Prcic et al., Acta Inform Med 24 (2016), 156-61 (Non-patent Literature 2). PSA analysis is commonly used in the conventional histopathological diagnosis after biopsy, and immunohistochemical (IHC) PSA staining is widely used to confirm the prostate origin of metastatic carcinomas, for example, to differentiate urothelial carcinoma from prostate cancer; Epstein et al., Am J Surg Path, 38 (2014), e6-e19 (Non-patent Literature 3), and Bostwick, Am J Clin Pathol. 102 (1994, 4 Suppl 1), 31-37 (Non-patent Literature 4). The usefulness of PSA in the diagnosis of prostate cancer has also been demonstrated by Goldstein et al., Am J Clin Path 117 (2002), 471-477 (Non-Patent Literature 5).

[0003] It is the biomarker recommended and most commonly used for PCa diagnosis, but PSA has many limitations in both serum and tissue-based assays, mainly as a result of its low specificity for PCa. Specifically, PSA is not a prostate cancer-specific biomarker because its blood levels do not efficiently distinguish between PCa and other prostate diseases, such as benign prostatic hyperplasia (BPH) or prostatitis; Hudson et al., J Urol, 142 (1989), 1011-1017 (Non-Patent Document 6). Furthermore, the serum level of PSA is known to be affected by multiple factors unrelated to prostate diseases, including age, comorbidities, ejaculation, catheterization, and some drug therapies; Hatekeyama et al., Int J Clin Oncol 22 (2017), 214-221 (Non-Patent Document 7). The PSA discrimination between PCa and benign prostate diseases is particularly inefficient in the so-called "gray area" in the range of 4-10 ng / ml (Barry, N Engl J Med 360 (2009), 1351-1354 (Non-Patent Document 8)), and in the discrimination between slow-progressing PCa and rapid-progressing PCa. Lamy et al., Eur Urol Focus 4 (2018), 790-803, epub Mar 7, 2017 (Non-Patent Document 9).

[0004] In immunohistochemistry, cellular PSA expression can be substantially reduced in poorly differentiated PCa, leading to false-negative results; therefore, PSA staining has similar limitations. In addition, PSA immunostaining cannot be used to assess the pathogenicity of cancer as defined by the Glisson classification system based on morphological appearance; Epstein et al., The American Journal of Surgical Pathology 40 (2016), 244-252 (Non-patent Literature 10). Furthermore, PSA can be ectopically expressed in other cancers, such as ovarian endometrial cancer, or breast cancer in women and men; Bonk et al., Oncotarget 52 (2019), 5439-5453 (Non-patent Literature 11); Alanen et al., Pathology-Research and Practice 192 (1996), 233-237 (Non-patent Literature 12); Kraus et al., Diagn Pathol 5 (2010) (Non-patent Literature 13).

[0005] Despite these constraints, PSA-based prostate cancer screening has led to a reduction in advanced disease and disease-specific mortality. However, the trade-off is overdiagnosis of cases that would not have caused clinical outcomes during a person's lifetime even if left untreated. This overdiagnosis has led to overtreatment with significant risks as side effects from biopsies or negative outcomes from treatment; Loeb et al., Eur Urol 65(2014),1046-1055(Non-patent Document 14). According to the US Preventive Medicine Special Committee (Final recommendation Statement: Screening for Prostate Cancer and Final Evidence Review: Screening for Prostate Cancer, www.uspreventiveservicestaskforce.org (Non-patent Document 15)(accessed in May 2018), and Sandhu and Andriole, J Natl Cancer Inst Monogr 45(2012),146-151(Non-patent Document 16)), 20-50% of men diagnosed as positive have indolent, non-threatening PCa that does not particularly clinically manifest or result in cancer-related death over the patient's lifetime. This data clearly shows the main problem of overdiagnosis (i.e., detection of cancers that do not particularly clinically manifest or result in cancer-related death over the patient's lifetime) and the need for improved, more specific tools for PCa diagnosis and assessment of cancer aggressiveness.

[0006] Currently, several research groups hypothesize that detecting altered glycosylation can enhance the diagnostic capabilities of PSA; Drake et al., Adv Cancer Res 126 (2015), 345-382 (Non-Patent Literature 17). Evaluations of altered fucosylation levels of PSA in serum from cancer patients using various lectin-based and mass spectrometry (MS)-based approaches have been described; Dwek et al., Clinica Chimica Acta 411 (2010), 1935-1939 (Non-Patent Literature 18); Llop et al., Theranostics 6 (2016), 1190-1204 (Non-Patent Literature 19); Zhao et al., Anal Chem 83 (2011), 8802-8809 (Non-Patent Literature 20). However, published results show diverse changes in glycosylation patterns for PSA with various fucose bindings. For example, Fukushima et al., Glycobiology 20 (2010), 452-460 (Non-Patent Literature 21) reported that in a cohort of 40 samples, peripheral α-1,2-fucosylated total PSA levels were higher in the serum of PCa patients than in BPH patients with a probability of over 95%. The peripheral α-1,2-fucosylated form of free PSA was also shown to increase in PCa patients compared to BPH patients in the serum of cancer patients with 92% specificity and 69% sensitivity; Dwek et al., Clinica Chimica Acta 411 (2010), 1935-1939 (Non-Patent Literature 18). In contrast, a significant reduction in α-1,6-core-fucosylated total PSA with 90% sensitivity and 95% specificity was found in high-risk PCa, which differentiated BPH and low-risk PCa from high-risk PCa patients in a 73 serum sample cohort; Llop et al., Theranostics 6(2016), 1190-1204 (Non-patent Literature 19). Information on differences in the glycosylation status of PSA between PCa and normal prostate tissue has not been reported to date.

[0007] While glycosylated forms of PSA, particularly core-fucosylated PSA, i.e., those containing α-1,6-core-fucosylation, have been proposed as promising biomarkers to complement or replace total PSA testing, to date there is no reliable tool for their specific detection, especially for distinguishing them from other glycosylated forms of PSA, i.e., for selective binding to core-fucosylated PSA than other PSA forms (whether glycosylated or not). In particular, the detection of PSA glycoforms containing α-1,6-core-fucose is currently performed using lectin or mass spectrometry-based methods; Kuzmanov et al., BMC Med 11 (2013), Article 31 (Non-Patent Literature 22); Tan et al., J Proteome Res 14 (2015), 1968-1978 (Non-Patent Literature 23); Yin et al., J Proteome Res 13 (2014), 2887-2896 (Non-Patent Literature 24); Liang et al., Glycobiology 25 (2015), 331-340 (Non-Patent Literature 25). However, a limitation of lectin-based approaches is that they are reactive only to sugar residues and not to protein moieties. Furthermore, lectins have low specificity for distinguishing between related glycan structures, and their reactivity is mostly based on binding activity rather than affinity. Therefore, current approaches lack the specificity, sensitivity, and / or ease of use necessary for the quantitative determination of glycosylated antigens, particularly core-fucosylated PSA; Kuzmanov et al., BMC Med 11 (2013), Article 31 (Non-Patent Literature 22).

[0008] Despite the increasing importance of detecting specific glycosylation patterns of proteins such as PSA in cancer diagnosis, antibodies for use in immunoassays that recognize and identify specific glycospecies of target proteins (i.e., often the same protein and very similar glycospecies of other proteins) are extremely rare. While not theoretically bound, the lack of suitable antibodies is likely due to known problems with the immunogenicity of glycan structures, which are often highly conserved in the animals used for immunization; Egashira et al., Scientific Reports, 9(2019), 12359 (Non-patent Literature 26). Furthermore, antibodies against carbohydrates often have very low affinity and may lack sufficient specificity; Vadim Dudkin et al., 2008. J Am Chem Soc 130 (2008), 13598-13607 (Non-patent Literature 27); and Egashira et al., Scientific Reports, 9 (2019), 12359 (Non-patent Literature 28). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] www.globocan.iarc.fr [Non-Patent Document 2] Prcic et al.,Acta Inform Med 24(2016),156-61 [Non-Patent Document 3] Epstein et al., Am J Surg Path, 38(2014), e6-e19 [Non-Patent Document 4] Bostwick, Am J Clin Pathol.102(1994,4 Suppl 1), 31-37 [Non-Patent Document 5] Goldstein et al.,Am J Clin Path 117(2002),471-477

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[0010] 3. Overview Despite known problems in the art relating to the use of glycostructures as immunogens and the development of antibodies that are specific to and therefore able to identify glycospecies, the inventors have, as described above, surprisingly developed antibodies characterized by their specific binding to α-1,6-core-fucosylated PSA (also referred herein as core-fucosylated PSA and / or 1,6-fucPSA) and its subsequences containing α-1,6-core-fucosylation. The developed antibody is the first antibody in its class to specifically bind to α-1,6-core-fucosylated PSA. The antibody disclosed herein is characterized in particular by its activity to distinguish core-fucosylated PSA / core-fucosylated subsequences from PSA / PSA subsequences lacking core-fucose residues (including aglycosylated PSA / PSA subsequences). Furthermore, the antibody distinguishes core-fucosylated PSA / core-fucosylated PSA subsequences from PSA core-fucosylated glycans in other contexts, such as a single core-fucosylated asparagine residue or an amino acid sequence unrelated to core-fucosylation, as described herein. The antibody exhibits remarkably high specificity and affinity in kinetic analysis using α-1,6-core-fucosylated PSA peptides. Analysis of the identified antibody has also enabled the development of a consensus structure that can result in specific and selective binding to α-1,6-core-fucosylated PSA as defined herein. Thus, the antibody and its use as a diagnostic and prognostic tool in the evaluation of prostate cancer and prostate cancer treatment are provided.

[0011] The antibodies and their antigen-binding fragments of the present invention specifically bind to core-fucosylated prostate-specific antigen (PSA) and subsequences of PSA containing core-fucosylation, as demonstrated by exemplary specific members of the family. As known in the art, PSA is a glycoprotein having the amino acid sequence of SEQ ID NO: 21 containing a single N-glycosylation site corresponding to Asn-69 of Uniprot ID P 07288, and the glycan may contain core fucose residues. As will be further understood in the art, the term “core-fucosylation” within the glycan indicates that a fucose residue is α-1,6-linked to a core GlcNac residue bound to Asn-69 of the PSA protein, or a subsequence of that Asn corresponding to Asn-69. The terms “core-fucosylation” and “α-1,6-core-fucosylation” are recognized as interchangeable. Therefore, the term “specific to (or specifically binding to) core-fucosylated prostate-specific antigen (PSA) and / or a subsequence of PSA containing core-fucosylation” is interchangeable with the term “specific to (or specifically binding to) α-1,6-core-fucosylated PSA and a subsequence of that containing α-1,6-core-fucosylation.” The antibody and antibody-antigen binding fragment of the present invention are also interchangeably referred to herein as anti-1,6-fucPSA antibody and antibody-antigen binding fragment.

[0012] The anti-1,6-fucPSA antibody and antigen-binding fragments bind core-fucosylated PSA and subsequences of PSA containing core-fucosylation, but do not bind glycans lacking core-fucose residues, nor do they bind core-fucosylated residues as irrelevant (non-targeted) elements, such as irrelevant peptides as both are disclosed herein, or isolated core-fucosylated glycans (i.e., bound to a single asparagine residue). Thus, the antibody recognizes an epitope of core-fucosylated PSA that includes both (1) core-fucose residues and (2) at least a portion of a PSA amino acid sequence containing core-fucosylation. In preferred embodiments, the terms “subsequences of PSA” and similar terms as used herein refer to an amino acid sequence containing or consisting of SEQ ID NO: 18. Therefore, the antibodies and antigen-binding fragments provided herein specifically bind to PSA and PSA subsequences only if they further contain core fucose residues (i.e., core fucose residues bound via N-glycosylation at Asn-69 of the PSA / PSA subsequence). Furthermore, the antibodies and antigen-binding fragments provided herein specifically bind to core fucose residues (or glycans containing core-fucose residues) only if they are bound to PSA or PSA subsequences via N-glycosylation at Asn-69. Thus, the antibodies and antibody-antigen-binding fragments distinguish core fucosylated PSA and core-fucosylated PSA subsequences as defined herein from PSA and PSA subsequences containing glycans lacking α-1,6-core-fucose residues, including PSA and PSA subsequences lacking both glycosylation (i.e., aglycosylated PSA and its aglycosylated subsequence). Furthermore, the anti-1,6-fucPSA antibody and antigen-binding fragments distinguish core-fucosylated PSA and core-fucosylated PSA subsequences (preferably subsequences containing or consisting of SEQ ID NO: 18) from other contexts, such as core-fucosylated glycans of PSA in isolated forms.As defined below, the term “recognize” indicates that the anti-1,6-fucPSA antibody and antibody-antigen binding fragment bind to a specific antigen target (i.e., core-fucosylated PSA and its core-fucosylated subsequence) with higher activity and / or specificity than binding to other antigens, such as PSA / PSA subsequences lacking core-fucose residues and / or, for example, core-fucosylated glycans in isolated forms. For example, as detailed below herein, the feature of recognizing a target antigen from / to a non-target antigen in certain embodiments is characterized by the anti-1,6-fucPSA antibody or antibody-antigen binding fragment having an affinity for the target antigen that is at least 10 times, at least 20 times, preferably at least 50 times, and more preferably at least 100 times better than its affinity for the non-target antigen. In this regard, it is preferable that the affinity of the antibody or antigen-binding fragment to the target antigen and the non-target antigen is determined as the KD.

[0013] Therefore, the anti-1,6-fucPSA antibodies and their antigen-binding fragments of the present invention are of formula I TIFF0007901069000001.tif38128 (wherein Z represents 0, 1, or 2 or more sugar residues) It specifically binds to glycopeptides of formula I or glycoproteins containing glycopeptides of formula I.

[0014] The anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also distinguish (i.e., do not specifically bind) the target antigen (i.e., the glycopeptide of formula I, or a glycoprotein containing the glycopeptide of formula I) from PSA and PSA subsequences lacking core fucose residues. Therefore, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention do not specifically bind to the glycopeptide of formula II or the glycoprotein containing the glycopeptide of formula II TIFF0007901069000002.tif37128 (wherein Z represents 0, 1, or 2 or more sugar residues) It identifies target antigens from glycoproteins containing these proteins.

[0015] In certain embodiments, the anti-1,6-fucPSA antibody and antibody-conjugated fragment also target the target antigen (i.e., the glycopeptide of formula I, or a glycoprotein containing the glycopeptide of formula I) in other situations, for example, in an isolated form, for example, formula III TIFF0007901069000003.tif37128 (wherein Z represents 0, 1, or 2 or more sugar residues) It identifies (i.e., does not specifically bind) PSA from core-fucosylated glycans in the case of a single glycosylated asparagine residue containing the α-1,6-core-fucose residue shown.

[0016] In certain embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention specifically bind to the glycopeptide of formula I (or a glycoprotein containing the glycopeptide of formula I) and distinguish from (i.e., do not specifically bind to) the glycopeptide of formula II (or a glycoprotein containing the glycopeptide of formula II) and the core-fucosylated glycan of formula III.

[0017] As used herein, the term sugar residue is understood to mean a monosaccharide known in the art. For all of the above formulas I, II, and III in which Z represents one or more sugar residues, the residues may be independently selected from mannose, GlcNac, fucose, galactose, and sialic acid. Furthermore, if Z represents two or more sugar residues, it may include unbranched or branched glycan moieties, which may be, for example, biantennae, triantennae, or tetraantennae.

[0018] In formula I, Z represents two or more sugar residues, and formula Ia TIFF0007901069000004.tif70128 (wherein Z1 and Z2 independently represent 0, 1, or 2 or more sugar residues) It is preferable that the biantennae branched glycan moiety is included as shown. Therefore, it is preferable that the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention specifically bind to a glycopeptide of formula Ia or a glycoprotein containing a glycopeptide of formula Ia.

[0019] In these preferred embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also contain a preferred target antigen (i.e., a glycopeptide of formula Ia, or a glycoprotein containing a glycopeptide of formula Ia), particularly Z being two or more sugar residues and formula IIa TIFF0007901069000005.tif72128 (wherein Z1 and Z2 independently represent 0, 1, or 2 or more sugar residues) It identifies (i.e., does not specifically bind) from glycopeptides of formula II or glycoproteins containing formula II, including biantennae branched glycan moieties as shown. Therefore, in certain embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also identify preferred target antigens from glycopeptides of formula IIa or glycoproteins containing formula IIa.

[0020] In certain preferred embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also target a preferred target antigen (i.e., a glycopeptide of formula Ia, or a glycoprotein containing a glycopeptide of formula Ia) in other situations, for example, in an isolated form, for example, in particular, Z is of formula IIIa TIFF0007901069000006.tif72128 (wherein Z1 and Z2 independently represent 0, 1, or 2 or more sugar residues) It distinguishes (i.e., does not specifically bind) the core-fucosylated glycan of PSA in the case of a single glycosylated asparagine residue containing the α-1,6-core-fucose residue shown in formula III, which includes two or more sugar residues and a biantennae branched glycan moiety as shown in formula III.

[0021] In further specific preferred embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention specifically bind to the glycopeptide of formula Ia (or glycoproteins containing the glycopeptide of formula Ia) and distinguish from (i.e., do not specifically bind to) the glycopeptide of formula IIa (or glycoproteins containing the glycopeptide of formula IIa) and the core-fucosylated glycan of formula IIIa.

[0022] For all of the above formulas Ia, IIa, and IIIa where Z1 and / or Z2 represent one or more sugar residues, the residues may be independently selected from mannose, GlcNac, fucose, galactose, and sialic acid. Furthermore, if Z1 and / or Z2 represent two or more sugar residues, it may include an unbranched or branched glycan moiety.

[0023] Regarding equation I, Z is equal to equation Ib It is most preferable to represent the sugar portion as shown in TIFF0007901069000007.tif65128.

[0024] The glycopeptide of formula Ib is also referred to herein interchangeably as "PSA(67-79)-G0F". Therefore, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention most preferably bind specifically to the glycopeptide of formula Ib or a glycoprotein containing the glycopeptide of formula Ib. The preferred binding of the glycoprotein of formula Ib (PSA(67-79)-G0F) within a glycan is that of formula Ic It is available at TIFF0007901069000008.tif65128.

[0025] In these most preferred embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also contain the most preferred target antigen (i.e., a glycopeptide of formula Ib, or a glycoprotein containing a glycopeptide of formula Ib), in particular, Z is formula IIb. Identifies (i.e., does not specifically bind) glycopeptides of formula II or glycoproteins containing formula II, including glycans as shown in TIFF0007901069000009.tif70128.

[0026] The glycopeptide of formula Ib is also referred to herein interchangeably as "PSA(67-79)-G2". Accordingly, in certain embodiments, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention also identify the most preferred target antigen from the glycopeptide of formula IIb or the glycoprotein containing formula IIb. The preferred binding of the glycoprotein of formula IIb (PSA(67-79)-G2) within a glycan is formula IIc It will be available at TIFF0007901069000010.tif70128.

[0027] In a particular most preferred embodiment, the anti-1,6-fucPSA antibody and antibody-conjugated fragment of the present invention also conjugate the most preferred target antigen (i.e., the glycopeptide of formula Ib, or a glycoprotein containing the glycopeptide of formula Ib) in other situations, for example, in isolated forms, for example, in particular, Z is IIIb Identify (i.e., not specifically bind) core-fucosylated glycans of PSA in the case of a single glycosylated asparagine residue containing the α-1,6-core-fucose residue shown in formula III, including glycans as shown in TIFF0007901069000011.tif64128.

[0028] The preferred bond in the glycan of fucosylated asparagine of formula IIIb is formula IIIc It is available at TIFF0007901069000012.tif66128.

[0029] In a particular most preferred embodiment, the anti-1,6-fucPSA antibody and antibody-binding fragment of the present invention specifically bind to the glycopeptide of formula Ib (or a glycoprotein containing the glycopeptide of formula Ib) and distinguish from (i.e., do not specifically bind to) the glycopeptide of formula IIb (or a glycoprotein containing the glycopeptide of formula IIb) and the core-fucosylated glycan of formula IIIb.

[0030] As described above, the "c" versions of formulas I–III provide preferred binding within the glycans of the glycopeptides / fucosylated asparagines of formulas Ib–IIIb, respectively. Therefore, any reference to any of the glycopeptides / fucosylated asparagines of formulas Ib–IIIb throughout this specification is also understood to implicitly enumerate the molecules shown in formulas Ic–IIIc, respectively, as preferred versions of these molecules.

[0031] As disclosed herein, anti-1,6-fucPSA antibodies and antigen-binding fragments distinguish core-fucosylated PSA and its core-fucosylated subsequence from PSA glycoproteins lacking α-1,6-core-fucosylation and / or glycosylated subsequences of PSA. Therefore, it is most preferable that anti-1,6-fucPSA antibodies and antigen-binding fragments do not significantly bind to the glycopeptide of formula IIb or glycoproteins containing the glycopeptide of formula IIb, i.e., glycopeptide PSA(67-79)-G2.

[0032] Furthermore, in certain embodiments, the anti-1,6-fucPSA antibody and antigen-binding fragment distinguish core-fucosylated PSA and its core-fucosylated subsequence from the core-fucosylated glycan of PSA in a different context, for example, in the context of formula III. Therefore, it is additionally or alternatively preferable that the anti-1,6-fucPSA antibody and antigen-binding fragment do not significantly bind to the core-fucosylated glycan of formula IIIb. It is most preferable that the antibody and antigen-binding fragment of the present invention do not significantly bind to both the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb.

[0033] As noted, and not limited to specific descriptions, the anti-1,6-fucPSA antibody and antigen-binding fragment are thought to bind to an epitope containing features from both the α-1,6-core-fucose residue and the PSA fragment of SEQ ID NO: 18. Therefore, the anti-1,6-fucPSA antibody and antigen-binding fragment may bind to PSA or PSA subsequences containing core-fucosylated glycans that are not endogenously expressed by human cells, provided that these two features of the epitope are present. Accordingly, the anti-1,6-fucPSA antibody and antigen-binding fragment provided herein may bind to glycopeptides of formula IV or glycoproteins containing glycopeptides of formula IV. It specifically binds to TIFF0007901069000013.tif22128.

[0034] The preferred bond within the glycan of formula IV is formula IVa It will be available at TIFF0007901069000014.tif22128.

[0035] The anti-1,6-fucPSA antibody or its antibody-antigen-binding fragment specifically binds to a target antigen, such as core-fucosylated PSA or its core-fucosylated subsequence (preferably a subsequence containing or consisting of SEQ ID NO: 18), only if the PSA or subsequence contains α-1,6-core-fucosylation, as shown in formulas I and / or IV. In this regard, the anti-1,6-fucPSA antibody or its antibody-antigen-binding fragment preferably specifically binds to a glycopeptide of formula Ia or a glycoprotein containing the glycopeptide of formula Ia, and most preferably specifically binds to a glycopeptide of formula Ib or a glycoprotein containing the glycopeptide of formula Ib. In these most preferred embodiments, the preferred binding in the glycopeptide of formula Ib or the glycopeptide containing formula Ib in the glycan is shown in formula Ic. When used herein, the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention specifically binds to its target antigen, and its key-to-dissociation (KD) to the antigen is 30 nM or less, preferably 20 nM or less, and most preferably 15 nM or less. The anti-1,6-fucPSA antibody or its antigen-binding fragment preferably binds to the glycopeptide of formula Ib with a KD of 30 nM or less, more preferably 20 nM or less, and most preferably 15 nM or less. In this preferred embodiment, the binding of the glycan in the glycopeptide of formula Ib is even more preferably as shown in formula Ic. As is understood in the art, the KD value is inversely proportional to the binding activity. Therefore, for example, an antibody or antibody-binding fragment that specifically binds to its antigen with a KD of 30 nM or less is understood in the art to bind to its antigen with a KD of at least 30 nM and / or with a KD of 30 nM or better. In a preferred embodiment, the assay conditions used to determine the antibody binding affinity are standardized such that the determined KD of an exemplary antibody 3B10 for the glycopeptide of formula Ib (having a glycan containing the preferred binding shown in formula Ic) (i.e., including SEQ ID NOs. 61 and 62, respectively), preferably a rabbit antibody, is 11 nM + the standard error of the particular assay.

[0036] As used herein, the terms “to identify from / against” and similar terms relating to two antigens, for example, “an antibody identifies antigen X from / against antigen Y,” indicate that the antibody or antigen-binding fragment specifically binds to the target antigen X (i.e., the glycopeptide of formula Ia or a glycoprotein containing the glycopeptide of formula Ia, most preferably the glycopeptide of formula Ib or a glycoprotein containing the glycopeptide of formula Ib), but not specifically binds to the non-target antigen Y (e.g., the glycopeptide of formula IIa or a glycoprotein containing the glycopeptide of formula IIa, most preferably the glycopeptide of formula IIb or a glycoprotein containing the glycopeptide of formula IIb, and / or other situations, for example, in isolated form, for example, the core-fucosylated glycan of PSA in the case of a single glycosylated asparagine residue containing the α-1,6-core-fucose residue shown in formula III, particularly Z containing the glycan shown in formula IIIb). Therefore, as used herein, the terms “identify” and similar terms mean that the antibody or antigen-binding fragment “does not specifically bind” to / “does not significantly bind” to a non-target antigen (these terms are used interchangeably). It is well known in the art that the terms “specifically bind” and “not significantly bind” describe the degree to which an antibody identifies two antigens. This is because antibodies are known not to have absolute specificity, meaning they react with only one epitope under any conditions. That is, in the presence of other (non-target) antigens, the antibody or antigen-binding domain can react to some extent with similar epitopes on these other (non-target) antigens. However, the affinity of a monoclonal antibody or monoclonal antigen-binding fragment for its target epitope / antigen is significantly greater than its affinity for the relevant epitope. This affinity difference is used to establish assay conditions in which the antibody or antigen-binding fragment binds almost exclusively to a particular epitope. In this regard, the binding (or unbinding) of an antibody or antigen-binding fragment to an antigen is not understood as absolute.In other words, the anti-1,6-fucPSA antibody and / or antigen-binding fragment may exhibit some (residual) binding activity to other (non-)targets, but at a significantly lower level compared to binding activity to core-fucosylated PSA or core-fucosylated subsequences of PSA, preferably glycopeptides of formula Ib or glycoproteins containing glycopeptides of formula Ib. The feature of discriminating the target antigen from / to the non-target antigen may be characterized by the anti-1,6-fucPSA antibody or antibody-antigen-binding fragment having an affinity for the target antigen, e.g., a KD value, that is at least 10-fold, at least 20-fold, preferably at least 50-fold, and more preferably at least 100-fold better than its affinity for the non-target antigen. In some embodiments, the 1,6-fucPSA antibody and / or antigen-binding fragment may not exhibit detectable binding to the non-target antigen. In such cases, "distinguishing from / to the non-target antigen" indicates that the anti-1,6-fucPSA antibody or antibody-antigen-binding fragment has a binding affinity at least 100-fold greater than the lowest detectable binding for a particular assay.

[0037] In relation to the embodiments described above, it is most preferable that the target antigen is a glycopeptide of formula Ib and the non-target antigen is a glycopeptide of formula IIb. Therefore, it is most preferable that the anti-1,6-fucPSA antibody or its antibody-antigen binding fragment distinguishes the glycopeptide of formula Ib from the glycopeptide of formula IIb, meaning that the anti-1,6-fucPSA antibody or its antibody-antigen binding fragment has an affinity for the glycopeptide of formula Ib, e.g., a KD value, that is at least 10 times, at least 20 times, preferably at least 50 times, and more preferably at least 100 times better than the affinity for the glycopeptide of formula IIb. In these most preferred embodiments, the preferred bindings in the glycans of formula Ib and formula IIb are as shown in formulas Ic and IIc, respectively.

[0038] In addition to the most preferred embodiment described above, as shown, it is most preferred to bind to the target antigen with an affinity 100-fold better than the affinity for the non-target antigen. Thus, in connection with the most preferred embodiment, i.e., where the target antigen is a glycopeptide of formula Ib and the non-target antigen is a glycopeptide of formula IIb, the anti-1,6fucPSA antibody or an antigen-binding fragment thereof most preferably has an affinity, e.g., a KD value, for the glycopeptide of formula Ib that is at least 100-fold better than the affinity for the glycopeptide of formula IIb. In these most preferred embodiments, the preferred linkages in the glycans in the glycopeptide of formula Ib and the glycopeptide of formula IIb are shown in formula Ic and formula IIc, respectively.

[0039] It is even most preferred that the comparison to establish the characteristics of "specifically binding" and "not specifically binding" with respect to the target and non-target antigens is determined using the same experimental protocol and the same experimental conditions (e.g., the same binding assay, concentration / density of the antibody or antigen-binding fragment, antigen concentration / density / flow rate, etc.).

[0040] Alternatively or additionally, the anti-1,6fucPSA antibody or an antigen-binding fragment thereof of the present invention has an association rate (k a M -1 s -1 or more, more preferably 2×10 5 M -1 s -1 or more, and most preferably 5×10 5 M -1 s -1 or more for specifically binding to its target antigen. In this connection, it is most preferred that the target antigen is a glycopeptide of formula Ib, and in the most preferred embodiment, it is preferred that the linkage in the glycan is shown in formula Ic. The association activity of the anti-1,6fucPSA antibody and antigen-binding fragment demonstrates rapid binding and thus response. The rapid association rate minimizes, for example, the incubation period required for a diagnostic assay and improves throughput and efficiency.

[0041] ​​The binding characteristics of anti-1,6-fucPSA antibodies and antigen-binding fragments can be established by any suitable method known in the Art and / or described herein, which allows for the quantification of binding parameters and, in particular, their quantitative comparison. Methods for analyzing the binding specificity and binding parameters of antibodies or antibody-antigen-binding fragments are described, for example, in Harlow & Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and Harlow & Lane (1999) Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Non-limiting examples of suitable studies include binding studies using structurally and / or functionally closely related molecules, and blocking / competition studies. These studies can be carried out, for example, by methods such as FACS analysis, flow cytometry titration (FACS titration), surface plasmon resonance (SPR, e.g., by BIAcore®), isothermal titration calorimetry (ITC), fluorescence titration, or by radiolabeled ligand binding assays. Further methods include, for example, Western blotting, ELISA (including competitive ELISA), RIA, ECL, IRMA, and physiological assays such as cytotoxicity assays. The specificity and selectivity of the antibodies and antibody-antigen binding fragments of the present invention are preferably determined by measuring antibody affinity, for example, by determining the dissociation constant (KD). If the KD is determined, it is even more preferably measured using surface plasmon resonance spectroscopy (SPR, for example using BIAcore®). Therefore, the specificity and selectivity of the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention against core-fucosylated PSA can preferably be determined by evaluating the specificity to the glycopeptide of formula Ib compared to the glycopeptide of formula IIb and / or glycosylated asparagine of formula IIIb using surface plasmon resonance spectroscopy (SPR, for example using BIAcore®).

[0042] SPR analysis can be performed using methods known in the art (e.g., according to the instructions of an SPR instrument manufacturer) or any suitable conditions described herein to enable quantitative or relative determination of antibody (or antigen-binding fragment) affinity. In non-limiting embodiments, the SPR analysis is performed using a Biacore 8k instrument. Furthermore, the SPR determination of antibody (or antigen-binding fragment) affinity can be performed (using a Biacore 8k or other SPR instrument) preferably at a temperature of 37°C using the following buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w / v Tween 20®, with 1 mg / ml carboxymethyl dextran added. In this embodiment, the SPR measurement may include capturing a monoclonal antibody or antigen-binding fragment on a CM5 sensor chip and a glycopeptide of formula Ib as the analyte. In a preferred embodiment, the SPR parameter is standardized such that the determined KD of an antibody, preferably a rabbit antibody, containing VH and VL of exemplary antibody 3B10 for the glycopeptide of formula Ib (having a glycan containing the preferred binding shown in formula Ic) (i.e., containing SEQ ID NOs. 61 and 62, respectively), is 11 nM + standard error of the particular assay. The capture level must be adjusted so that the molar ratio (MR) is 1 or 2. The MR is calculated as follows: MR = (Analyte Binding Late (RU) / Antibody Capture Level (RU)) × (MW (Antibody) / MW (Analyte)). Measurement of the binding activity of the antibody or antigen-binding fragment, e.g., KD (or relative KD) and / or k a The determination is made using the Langmuir fitting model, preferably R MAX This may include fitting surface plasmon resonance data locally.

[0043] The overall structure of antibodies is well known in the art and comprises two heavy chains and two light chains linked by disulfide bonds. The heavy and light chains contain one or more constant domains and one variable domain. Antigen binding specificity is the function of the antibody Fv domain, which includes paired light chain variable domains and heavy chain variable domains. However, as is known in the art, antigen binding can also be maintained and demonstrated by a single, i.e., unpaired heavy or light chain variable domain. Therefore, antibody-antigen binding fragments disclosed herein include, for example, single-domain antibodies (sdAb, dAb, and / or nanobodies, also known in the art) and / or V based on the heavy chain of a camelid animal. H Like the H domain, it may contain only a single heavy chain and / or light chain variable domain.

[0044] Antigen-binding specificity is determined by the portion of the antibody Fv domain that contacts the ligand, known as the complementarity-determining region (CDR), i.e., the heavy chain and light chain variable domain portions. The CDR is the most variable portion of the molecule and contributes to the antigen-binding diversity of these molecules. As is well understood, three CDR regions, CDR1, CDR2, and CDR3, are present in each heavy chain variable domain and light chain variable domain, embedded between four framework regions (FWs) according to the general pattern framework FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. As used herein, the term "CDR-HX," where X is the number 1, 2, or 3, refers to the CDR1, CDR2, or CDR3 region of the heavy chain variable domain, respectively. The term "CDR-LX," where X is the number 1, 2, or 3, refers to the CDR1, CDR2, or CDR3 region of the light chain variable domain, respectively. Similarly, the term "FW-HX" (or "FW-LX"), where X is the number 1, 2, 3, or 4, refers to framework regions 1, 2, 3, or 4 of the heavy-chain (or light-chain) variable domain, respectively.

[0045] The boundaries and lengths of individual CDRs are based on various classification and numbering systems known in the art, for example, but not limited to, Kabat et al., "Sequences of Proteins of Immunological Interest" 5 th This follows the Kabat system and the Chothia system as referred to in Edition, U.S. Department of Health and Human Services, 1992; and Chothia et al. (J.Mol.Biol.196(1987), 901, respectively. Unless otherwise indicated, the CDR domain and FW domain as referred to herein are defined according to Kabat.

[0046] As used herein, the term "contains" indicates that additional sequences / components may be included in addition to the sequences and / or components specifically described.

[0047] In embodiments of the present invention in which the antibody and antigen-binding fragments include amino acid sequences beyond the described amino acid sequences, further amino acids may be present at either the N-terminus, C-terminus, or both. These further sequences may include, for example, sequences introduced for purification or detection, as is known in the art. Furthermore, if individual sequences "include" the described sequences, they may also include further amino acids at either the N-terminus, C-terminus, or both.

[0048] As shown in the examples, the inventors have developed an antibody that recognizes an epitope specific to core-fucosylated PSA and core-fucosylated subsequences of PSA, particularly one formed from features of both core-fucose residues and surrounding peptide sequences containing or consisting of, for example, SEQ ID NO: 18. The exemplary embodiments of the present invention (i.e., the antibody) are consistent in their high specificity to the target antigen, rapid association rate, and discriminative / selective binding to core-fucosylated PSA / core-fucosylated PSA subsequences for non-target antigens, such as non-fucosylated PSA / PSA subsequences and / or core-fucosylated glycans such as formula IIIb. Through analysis of the exemplary embodiments of the present invention, the inventors were further able to identify exemplary consensus sequences of antibody CDRs and variable domains that can confer the identified functional features of the anti-1,6fucPSA antibody and antigen-binding fragments of the present invention. The analysis proceeded based on the understanding in the art that specific CDR / variable domain residues are primarily responsible for antibody-binding activity, with the influence of the remaining residues being less significant. Therefore, it is known that amino acid residues within the CDR region and / or variable domain region can be replaced without necessarily resulting in a (significant) loss of function. In other words, it is known in the art that specific amino acid residues in the CDR and / or variable region can be replaced, and sequence variants that maintain desired functional properties can be easily identified.

[0049] Therefore, the anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention comprises a monoclonal antibody and its antigen-binding fragment, which include the following: (HV1) A variant of CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or modified by a single amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; a variant of CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or modified by up to two amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; a variant of CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or modified by up to two amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; comprising an antibody heavy chain variable domain (VH); and / or A variable antibody light chain domain (VL) comprising: (LV1) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0050] The exemplary anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention as defined above comprises a monoclonal antibody and its antigen-binding fragment, which include the following: (HV2) A variant of the antibody heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and / or (LV2) A variant of the antibody light chain comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single conservative amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0051] All exemplary anti-1,6-fucPSA antibodies and antigen-binding fragments of the present invention as defined above include monoclonal antibodies and their antigen-binding fragments: (HV3) A variant of the antibody heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and / or (LV3) A variant of the antibody light chain comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0052] All exemplary anti-1,6-fucPSA antibodies and antigen-binding fragments of the present invention as defined above also include monoclonal antibodies and their antigen-binding fragments comprising: A heavy chain variable domain (VH) comprising: (HV4) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and / or (LV4) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by a single conservative amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0053] All exemplary anti-1,6-fucPSA antibodies and antigen-binding fragments of the present invention as defined above also include monoclonal antibodies and their antigen-binding fragments comprising: (HV5) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and / or (LV5) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0054] All exemplary anti-1,6-fucPSA antibodies and antigen-binding fragments of the present invention as defined above further comprise monoclonal antibodies and antigen-binding fragments comprising: (HV6) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and / or CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and / or (LV6) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15.

[0055] The anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention preferably include the heavy-chain and light-chain variable domain pairs of (HV1) and (LV1), (HV2) and (LV2), (HV3) and (LV3), (HV4) and (LV4), (HV5) and (LV5), or (HV6) and (LV6). Furthermore, the variant CDRs referenced above and throughout this disclosure represent functional variants, i.e., variants having an amino acid sequence that is different from the reference amino acid sequence but in which the different sequence exhibits or maintains the same functional activity as the reference sequence. Specifically, exemplary heavy-chain and / or light-chain variable domains of the present invention, including one or more variant CDRs as shown throughout this disclosure, exhibit specific and discriminative binding to core-fucosylated PSA or its fucosylated subsequences as described herein. In certain embodiments of the antibody activity described herein, the antibody or antigen-binding fragment comprises heavy and / or light chain variable domains having one or more variant CDRs disclosed herein, the antibody or antigen-binding fragment specifically binds to a target antigen (preferably a glycopeptide of formula Ia or a glycoprotein containing a glycopeptide of formula Ia, most preferably a glycopeptide of formula Ib or a glycoprotein containing a glycopeptide of formula Ib) and distinguishes the target antigen from / to a non-target antigen (most preferably a glycopeptide of formula IIb) and / or a core-fucosylated glycan of formula IIIb (i.e., does not specifically bind).

[0056] The amino acid sequences of the variant CDRs listed above in HV1-HV6 and LV1-LV6 are defined by a reference amino acid sequence having one or more amino acid substitutions. As used herein, the term “substitution” refers to replacing one amino acid with another. Thus, the total number of amino acids remains the same. Deletion of an amino acid at one position and introduction of one (or more) amino acids at different positions are not explicitly included in the term “substitution.”

[0057] As described above, for example with respect to HV2-HV6 and LV2-LV6, amino acid substitutions can be conserved or highly conserved residue substitutions. The term "conservative amino acid substitution" is well known in the art and refers to the substitution of an amino acid with a different amino acid having similar biophysical properties. As used herein, amino acids having similar biophysical properties are grouped as follows: (a) Nonpolar hydrophobic amino acids consisting of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), and methionine (Met); (b) Polar neutral amino acids consisting of serine (Ser), threonine (Thr), asparagine (Asn), and glutamine (Gln); (c) Positively charged basic amino acids consisting of arginine (Arg), lysine (Lys), and histidine (His); and (d) An acidic amino acid having an electrical load, consisting of aspartic acid (Asp) and glutamic acid (Glu).

[0058] Therefore, a conservative substitution is the substitution of a residue with another from the same group, namely (i) a nonpolar hydrophobic amino acid of group (a) with another amino acid of group (a); (ii) a polar neutral amino acid of group (b) with another amino acid of group (b); a positively charged basic amino acid of group (c) with another amino acid of group (c); and / or a positively charged acidic amino acid of group (d) with another amino acid of group (d). It is understood that the amino acids Cys and Pro are not included in the above grouping and / or list of conservative substitutions because, as is well known in the art, these residues are not suitable as common substituents. As used herein, if a residue Cys or Pro is substituted, it is a conservative substitution of Cys with Ser or Ala, and of Pro with Ala.

[0059] As used herein, highly conserved amino acid substitutions consist of the following possible substitutions: i) Substitution of Ala by Val, Leu, Ile, or Gly; ii) Substitution of Arg by Lys; iii) Substitution of Asn with Gln; iv) Replacement of Asp with Glu; v) Substitution of Cys by Ser; vi) Substitution of Gln with Asn; vii) Substitution of Glu by Asp; viii) Substitution of Gly by Ala; ix) Substitution of His by Arg; x) Substitution of Ile with Leu, Val, or Ala; xi) Substitution of Leu with Ile, Val, or Ala; xii) Substitution of Lys by Arg; xiii) Substitution of Met with Leu, Ile, or Val; xiv) Substitution of Phe with Tyr or Trp; xv) Pro replacement by Ala; xvi) Substitution of Ser by Thr; xvii) Substitution of Thr by Ser; xviii) Substitution of Trp with Phe or Tyr; xix) Substitution of Tyr by Phe or Trp; xx) Substitution of Val with Leu, Ile, or Ala.

[0060] The monoclonal antibodies and antigen-binding fragments disclosed herein comprise a heavy chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 90%, or preferably at least 93% sequence identity with SEQ ID NO: 19; and / or (preferably and) a light chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or preferably at least 96% sequence identity with SEQ ID NO: 20, wherein the antibody or antigen-binding fragment is characterized by specific and discriminant binding to core-fucosylated PSA, a core-fucosylated subsequence of PSA (preferably a sequence comprising or consisting thereof), more preferably a glycopeptide of formula Ib as described herein. In certain embodiments, the monoclonal antibodies and antigen-binding fragments disclosed herein comprise a heavy chain variable domain having an amino acid sequence having at least 93%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 19; and / or (preferably and) a light chain variable domain having an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20, wherein the antibody or antigen-binding fragment is characterized by specific and selective binding to core-fucosylated PSA, a core-fucosylated subsequence of PSA (preferably a subsequence comprising or consisting thereof of SEQ ID NO: 18), or most preferably, a glycopeptide of formula Ib as described herein. In certain embodiments, the heavy and light chain variable domains described in this paragraph include CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in the heavy and light chain pairs (HV1) and (LV1), (HV2) and (LV2), (HV3) and (LV3), (HV4) and (LV4), (HV5) and (LV5), or (HV6) and (LV6) disclosed above.

[0061] The anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention may be manufactured by any technique described herein and / or known in the art. The term “recombinant antibody” includes all antibodies prepared, expressed, produced or isolated by recombinant techniques, e.g., antibodies expressed using a recombinant expression vector transfected into host cells, antibodies isolated from a recombinant combinatorial human antibody library, or antibodies prepared, expressed, produced or isolated by any other means, including splicing a human immunoglobulin gene sequence with another DNA sequence. Accordingly, polynucleotides encoding the antibodies and antibody-antigen-binding fragments of the present invention and / or their heavy chain and / or light chain variable domains, as well as vectors containing such polynucleotides, are provided. The vector does not necessarily have to be an expression vector, but may be a vector that allows for the regeneration of the vector, and thus the replication of the polynucleotide sequence of the present invention, by, for example, culturing a host cell containing the vector. The vector may also be suitable for enabling the recombination operation of the polynucleotide sequence of the present invention, as known in the art. In preferred embodiments, the vector is an expression vector comprising a polynucleotide encoding the antibody and / or antibody-antigen-binding fragment disclosed herein (e.g., comprising a polynucleotide encoding the anti-1,6-fucPSA heavy chain and / or light chain variable domain), which, when introduced into a suitable prokaryotic or eukaryotic cell according to standard methods known in the art, results in the expression of the antibody or its antigen-binding domain of the present invention. When such an expression vector encoding the antibody heavy chain and / or light chain variable domain is introduced into a suitable host cell, the antibody or antibody fragment is produced by culturing the host cell for a period of time sufficient to cause the host cell to express the antibody or, more preferably, to secrete the antibody or antigen-binding fragment into the culture medium in which the host cell is growing. The host cell of the present invention may be a directly manipulated cell, i.e., a cell directly transfected with the vector or polynucleotide disclosed herein, or may be a daughter cell or progeny of a directly transfected cell.Accordingly, a method is provided for producing anti-1,6-fucPSA antibodies and antibody-antigen-binding fragments by culturing host cells containing polynucleotides encoding the antibody or antibody-antigen-binding fragment (e.g., contained in an expression vector). The method further comprises recovering and isolating the expressed antibody or antigen-binding fragment from the culture (e.g., from the cell fraction and / or culture medium) using standard protein purification methods. Thus, the present invention also provides antibodies and antigen-binding fragments that can be obtained by the methods disclosed herein.

[0062] Expression from host cells and / or their progeny is achieved by introducing one or more expression vectors encoding the heavy and / or light chains (or their variable domains) of an antibody into host cells using standard techniques. The introduction of such expression vectors is well known in the art and is referred to herein as transfection, encompassing a wide variety of standard techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells. Non-limiting examples of suitable transfection methods include electroporation, calcium phosphate precipitation, and DEAE-dextran transfection.

[0063] While the antibodies of the present invention can be expressed in either prokaryotic or eukaryotic host cells, eukaryotic cells (especially mammalian cells) are more likely than prokaryotic cells to assemble and secrete properly folded, immunologically active antibodies. Therefore, expression of the antibodies in eukaryotic cells is preferred, and expression in mammalian host cells is most preferred. Non-limiting examples of mammalian host cells for expressing the monoclonal antibodies or their antigen-binding fragments of the present invention include Chinese hamster ovary cells (CHO cells), NS0 myeloma cells, COS cells, and SP2 cells. It is preferable to use CHO cells as the host cells.

[0064] Recombinant antibodies and recombinant antibody antigen-binding domains may have variable and constant regions (if any) derived from germline immunoglobulin sequences of a species, which are isolated after standard immunization and selection procedures known in the art, and may include, for example, rabbit germline immunoglobulin sequences. However, antibody sequences may be subjected to in vitro mutagenesis, where, in particular, CDR sequences are combined with FW sequences of another species, e.g., human, as known in the process of humanization. Therefore, the amino acid sequences of variable heavy and light chain domains disclosed herein may be derived from and related to germline heavy or light chain sequences, but may not naturally exist in vivo within any endogenous antibody germline repertoire.

[0065] The anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention are envisioned as diagnostic tools for further distinguishing core-fucosylated PSA proteins / peptides from / against sequences lacking core-fucose residues and / or core-fucosylated glycans (most preferably glycans of formula IIIb, in this most preferred embodiment having the binding shown in formula IIIc) in assays involving the detection of α-1,6-core-fucosylated PSA or its α-1,6-core-fucosylated subsequences. Accordingly, diagnostic compositions are provided comprising host cells containing the anti-1,6-fucPSA antibody and antigen-binding fragment disclosed herein, polynucleotides encoding heavy chain and / or light chain variable domains of such antibodies and fragments (e.g., in the context of a vector), and vectors and / or polynucleotide sequences encoding such heavy chain and / or light chain variable domains. The antibodies, antigen-binding fragments, and diagnostic compositions disclosed herein are suitable for any antibody-based diagnostic assays known and / or described herein (e.g., immunoassays including immunohistochemical assays), particularly ex vivo and in vitro diagnostic assays and assay systems known in the art. For example, methods such as immunohistochemical staining of biological samples obtained from patients (e.g., including tissues or cells), or measurement of the amount of core-fucosylated PSA (or its core-fucosylated fragments) in specific tissues may be valuable. The compositions provided herein are also suitable for use in immunoassays, whether they can be used in liquid phase or conjugated to a solid phase support. Examples of immunoassays or immunohistochemical assays in which the antibodies of the present invention can be used are immunoassays or immunohistochemical assays in either direct or indirect format, and may be a single step in a multi-step (e.g., heterologous) assay. Examples of such assays include enzyme-linked immunosolvent assays (ELISA), enzyme immunoassays (EIA), radioimmunoassays (RIA), or immunoassays based on the detection of luminescence, fluorescence, chemiluminescence, or electrochemiluminescence.In certain embodiments, the antibodies, antibody-antigen conjugates, and / or diagnostic compositions disclosed herein may be used in methods for detecting α-1,6-core-fucosylated PSA, its subsequences containing the α-1,6-core-fucosylated portion, the glycopeptide of formula Ib, and / or glycoproteins containing the glycopeptide of formula Ib. Alternatively or additionally, the antibodies, antigen conjugates, and / or diagnostic compositions disclosed herein may be used in methods for distinguishing α-1,6-core-fucosylated PSA or α-1,6-core-fucosylated subsequences of PSA from / to PSA or PSA subsequences (including aglycosylated PSA or aglycosylated PSA subsequences) lacking the α-1,6-core-fucosylated portion.

[0066] The immunoassays described herein (including immunohistochemical assays) can be performed on any suitable biological sample known in the art and / or described herein. Since the assays described herein are for the specific and selective detection of core-fucosylated PSA and its core-fucosylated fragments, and in particular for the identification of PSA lacking the core-fucose portion and its subsequences, the sample is a biological sample from an object expected to contain or proven to contain PSA. Examples of biological samples suitable for the uses and methods disclosed herein include, but are not limited to, tissue samples and bodily fluid samples. Non-limiting examples of tissue samples include samples of prostate tissue and extraprostate tissue, such as tumor tissue (which may or may not be of prostate origin). Samples may be formalin-fixed paraffin-embedded (FFPE) samples or frozen samples, and may or may not be pre-treated before the diagnostic immunoassays disclosed herein. Non-limiting examples of such pre-treatment include antigen retrieval. In certain embodiments, the tissue sample is prepared as a tissue slide for immunohistochemical analysis. In particular, tissue sections mounted on microscope slides (e.g., glass microscope slides) can be used.

[0067] Non-limiting samples of bodily fluids suitable for diagnostic analysis by the uses and methods disclosed herein include blood (e.g., whole blood, plasma, or serum), semen, ejaculated semen, and urine, such as post-digital rectal examination (DRE) urine. Bodily fluid samples may or may not be pre-treated before the diagnostic immunoassays disclosed herein. In certain embodiments, bodily fluid samples are prepared for immunoanalysis as tissue slides. In particular, tissue sections mounted on microscope slides (e.g., glass microscope slides) may be used.

[0068] The term "composition," when referring, for example, to a diagnostic composition used in accordance with the present invention, relates to a composition comprising at least one of the antibodies or antigen-binding fragments, polynucleotides, vectors, and / or host cells disclosed herein. It may optionally include further molecules that can stabilize, modulate, and / or enhance the functions of the compounds of the present invention by altering their characteristics. The composition may be in solid or liquid form, and in particular may be in the form of a powder, tablet, or solution.

[0069] The components of the composition can be packaged in a container or a set of containers, such as sealed ampoules or vials, as aqueous solutions or as lyophilized formulations for reconstitution. Solutions for use are prepared by reconstituting the lyophilized compound using, for example, water for injection for therapeutic purposes or other desired solvents for diagnostic purposes, such as buffer solutions. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. The various components of the composition can be packaged as a kit with instructions for use. Thus, a kit is also provided that contains, for example, one or more compositions disclosed herein, including the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention.

[0070] The present invention also relates to a method comprising the use of the anti-1,6-fucPSA antibody and antigen-binding fragment disclosed herein as primary antibodies in an immunohistochemical or immunocytochemical staining procedure for microscopic analysis. Accordingly, the present invention relates to a method for preparing a histochemical or cytochemical sample for microscopic analysis, comprising the use of the anti-1,6-fucPSA antibody and antigen-binding fragment of the present invention. In exemplary embodiments, the immunohistochemical or immunocytochemical staining includes: (a) Contacting a sample, for example, a blood or tissue sample disclosed herein, with an anti-1,6-fucPSA antibody or antigen-binding fragment disclosed herein, under conditions sufficient to promote the specific binding of the antibody or antigen-binding fragment to the glycopeptide of formula Ib, but not to promote, for example, reduced or inhibited, the binding of the antibody or antigen-binding fragment to the glycopeptide of formula IIb and core-fucosylated asparagine of formula IIIb (i.e., using the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention as a primary antibody as understood in the art, under conditions sufficient to promote the identification of the glycopeptide of formula Ib from the glycopeptide of formula IIb and core-fucosylated glycan of formula IIIb); and (b) Remove the unbound primary antibody (i.e., the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention) from the sample.

[0071] In exemplary methods, including the use of the antibody and antigen-binding fragment of the present invention as a primary antibody in histochemical or cytochemical analysis, the primary antibody (i.e., the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention) can be conjugated to a detectable portion. Furthermore, the method may include a further step (c) comprising contacting a sample with a set of detection reagents suitable for depositing a detectable portion in close proximity to the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention (i.e., the primary antibody) conjugated to the sample, the detectable portion may be a plastid, fluorophore, phosphorescent molecule, luminescent molecule, or mass tag. In exemplary embodiments involving the use of a detectable reagent or portion, any further optional step (c) may include (in all cases (i) to (v) below, "primary antibody" refers to the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention): (i) The secondary antibody is bound to the primary antibody, and the secondary antibody is labeled in a detectable manner; (ii) A secondary antibody is bound to a primary antibody, a tertiary antibody is bound to a secondary antibody, and the tertiary antibody, or both the secondary and tertiary antibodies, are labeled for detection; (ii) A secondary antibody with an epitope tag is bound to the primary antibody, and a tertiary antibody specifically labeled for the epitope tag is bound to the secondary antibody; (iv) A secondary antibody conjugated to an enzyme is bound to a primary antibody, the signal transduction conjugate is reacted with the enzyme, the signal transduction conjugate includes an epitope tag and a potential reactive moiety, the enzyme catalyzes the transformation of the potential reactive moiety into a reactive species that binds to the sample, a tertiary antibody is bound to the epitope tag of the signal transduction conjugate that binds to the sample, the enzymes of the secondary and tertiary antibodies are the same, the enzyme is reacted with an additional reagent to result in the deposition of a detectable moiety on the sample; or (v) A secondary antibody conjugated to an epitope tag is bound to a primary antibody, a tertiary antibody conjugated to an enzyme is bound to the epitope tag, the sample is brought into contact with the signal transduction conjugate containing the epitope tag and a potential reactive moiety under conditions in which the enzyme catalyzes the conversion of the potential reactive moiety to a reactive species that binds to the sample, an additional tertiary antibody is bound to the epitope tag of the signal transduction conjugate that binds to the sample, and the enzyme is reacted with an additional reagent to result in the deposition of a detectable moiety on the sample. [Invention 1001] A monoclonal antibody or antigen-binding fragment thereof that is specific to α-1,6-core-fucosylated prostate-specific antigen (PSA) or a subsequence thereof containing the α-1,6-core-fucosylated portion, wherein the subsequence comprises or consists of SEQ ID NO: 18. [Invention 1002] The α-1,6-core-fucosylated prostate-specific antigen (PSA), or the partial sequence containing the α-1,6-core-fucosylated form, (i) Glycopeptide of formula Ib TIFF0007901069000015.tif65128 or (ii) Glycopeptide of formula IV TIFF0007901069000016.tif25128 A monoclonal antibody or antigen-binding fragment thereof according to the present invention 1001, comprising or consisting of the above. [Invention 1003] The α-1,6-core-fucosylated PSA, or a partial sequence of the PSA containing the α-1,6-core-fucosylated portion, (i) PSA or a partial sequence thereof lacking α-1,6-core-fucose residues; and / or (ii) α-1,6-coa-fucosylated glycan of formula (IIIb) TIFF0007901069000017.tif70128 A monoclonal antibody or antigen-binding fragment of the present invention 1001 or 1002 that identifies the following. [Invention 1004] The partial sequence of PSA lacking the aforementioned α-1,6-core-fucose residue is (i) Sequence ID 18; or (ii) Glycopeptide of formula IIb TIFF0007901069000018.tif77128 A monoclonal antibody or antigen-binding fragment of the present invention 1003, comprising or consisting of the above. [Invention 1005] (i) The α-1,6-core-fucosylated PSA or a partial fragment thereof containing the α-1,6-core-fucosylated PSA. The binding affinity of the antibody or fragment to the said antibody is (iia) PSA lacking the α-1,6-core-fucose residue or a partial sequence of PSA lacking the α-1,6-core-fucose residue; and / or (iib) Core-fucosylated glycan of formula IIIb above Its binding affinity to is at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than that of the other, Here, the binding to (i) and ((iia) and / or (iib)) is performed under the same conditions, using a monoclonal antibody or antigen-binding fragment of the present invention 1003 or 1004. [Invention 1006] The monoclonal antibody or antigen-binding fragment of the present invention 1005, wherein the binding affinity is determined as KD, and the antibody or fragment binds to the glycopeptide of formula Ib with a KD of 30 nM or less, 20 nM or less, or 11 nM or less. [Invention 1007] (i) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 3 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 4 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5 or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15; (ii) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1 or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 8 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 9 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10 or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11 or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15; or (iii) A heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 12 or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 13 or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 14 or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 15 or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 16 or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 17 or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. A monoclonal antibody or antigen-binding fragment antibody according to any of the present invention 1001 to 1006, including the above. [Invention 1008] (i) The conservative antibody amino acid substitution is an amino acid substitution by another amino acid selected from the same group, and the group of amino acids is a) Nonpolar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, and Met; b) Polar neutral amino acids consisting of Ser, Thr, Asn, and Gln; c) A positively charged basic amino acid consisting of Arg, Lys, and His; and d) A negatively charged acidic amino acid consisting of Asp and Glu And, Cys is replaced with Ser or Ala when conservatively substituted, and Pro is replaced with Ala when conservatively substituted. or (ii) The highly conserved amino acid substitutions are a) Substitution of Ala with Val, Leu, Ile, or Gly; b) Substitution of Arg by Lys; c) Replacement of Asn with Gln; d) Replacement of Asp with Glu; e) Substitution of Cys by Ser; f) Substitution of Gln by Asn; g) Substitution of Glu by Asp; h) Substitution of Gly by Ala; i) Substitution of His by Arg; j) Substitution of Ile with Leu, Val, or Ala; k) Substitution of Leu with Ile, Val, or Ala; l) Substitution of Lys with Arg; m) Substitution of Met with Leu, Ile, or Val; n) Substitution of Phe with Tyr or Trp; o) Replacement of Pro with Ala; p) Substitution of Ser by Thr; q) Substitution of Thr by Ser; r) Substitution of Trp with Phe or Tyr; s) Substitution of Tyr by Phe or Trp; and t) Substitution of Val with Leu, Ile, or Ala A monoclonal antibody or antigen-binding fragment of the present invention 1007, selected from the above. [Invention 1009] (i) Heavy chain variable domains having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or preferably at least 93% sequence identity with respect to SEQ ID NO: 19; and (ii) Light chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or preferably at least 96% sequence identity with SEQ ID NO: 20 A monoclonal antibody or antigen-binding fragment according to any of the invention 1001 to 1008, comprising: [Invention 1010] (i) The heavy chain or heavy chain variable domain of any monoclonal antibody or antigen-binding fragment of any of the inventions 1001 to 1009, and / or (ii) The light chain or light chain variable domain of any monoclonal antibody or antigen-binding fragment according to any of the invention items 1001 to 1009. A polynucleotide that codes for [something]. [Invention 1011] A vector comprising the polynucleotide of the present invention 1010. [Invention 1012] A host cell containing the polynucleotide of Invention 1010 or the vector of Invention 1011. [Invention 1013] A method for producing a monoclonal antibody or antigen-binding fragment according to any of the present invention 1001 to 1009, comprising culturing a host cell according to the present invention 1012, and isolating the antibody or antigen-binding fragment. [Invention 1014] A composition comprising any antibody according to Invention 1001 to 1009 or an antibody obtainable by the method of Invention 1013, a polynucleotide according to Invention 1010, a vector according to Invention 1011, or a host cell according to Invention 1012. [Invention 1015] In a sample that is a tissue slide or body fluid, (i) To detect α-1,6-core-fucosylated PSA or a partial sequence thereof containing the α-1,6-core-fucosylated form; and / or (ii) In order to distinguish α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion from PSA or a subsequence thereof lacking the α-1,6-core-fucosylated portion. The composition of the present invention 1014, which is a diagnostic composition for use in an in vitro assay. [Invention 1016] A kit comprising an antibody according to any of the present invention 1001 to 1009 or an antibody that can be obtained by the method of the present invention 1013. [Invention 1017] A method for preparing histochemical or cytochemical samples for microscopic analysis, (i) an antibody or antigen-binding fragment according to any of the present invention 1001 to 1009, or an antibody or antigen-binding fragment that can be obtained by the method of the present invention 1013. (ii) A composition of the present invention 1014, or (iii) Kit of Invention 1016 This includes performing immunohistochemical or immunocytochemical staining using the following method: A method wherein the antibody or antibody fragment, or a component of the antibody or antibody fragment, is a primary antibody. [Brief explanation of the drawing]

[0072] [Figure 1]This is a schematic diagram showing the N-glycan structure of PSA in Asn-69 (N69), emphasizing α-1,6-core fucosylation. [Figure 2] Glycopeptides used for immunization and screening of fucPSA-specific antibodies. Figure 2A shows the fucosylated subsequence of the PSA glycopeptide used for immunization and positive screening (PSA(67-79)-G0F, i.e., the glycopeptide of formula I having the sequence of SEQ ID NO: 18). Figure 2B shows the glycosylated subsequence of the α-1,6-core-fucose-deficient PSA glycopeptide used for negative selection (similarly, PSA(67-79)-G2, i.e., the glycopeptide of formula II having the sequence of SEQ ID NO: 18). Figure 2C shows the isolated core-fucosylated glycan, i.e., glycosylated asparagine of formula III, used for negative selection. The symbols used in Figures 2A to 2C are the same as those used in Figure 1, i.e., they represent the same molecules as defined in the legend of Figure 1. The amino acid sequences of the glycopeptides in Figures 2A and 2B are shown in SEQ ID NO: 18. [Figure 3A] SRP Biacore kinetic data for binding to α-1,6-core-fucosylated PSA glycopeptide (PSA(67-79)-G0F / glycopeptide of formula (I) shown as "G0F" in the figure) and non-fucosylated PSA glycopeptide (PSA(67-79)-G2 / glycopeptide of formula (II) shown as "G2" in the figure) for six selected antibodies and two unselected antibodies. The figure shows a Biacore sensorgram overlay plot. The solid black line is the raw data, and the dotted gray line is the Langmuir fitting curve: A~B (13C5), C~D (2C11), E~F (2H9), G~H (2E9), I~J (3H6), K~L (3B10), M~N (13E12), and O~P (15F10). Binding to PSA(67~79)-G0F, injection of 0~900 nM; fitting to Langmuir 1:1. Binding to PSA(67~79)-G2, injection of 900 nM; fitting to Langmuir 1:1. [Figure 3B] Refer to the explanation in Figure 3A. [Figure 3C] Refer to the explanation in Figure 3A. [Figure 3D]Refer to the explanation in Figure 3A. [Figure 3E] Refer to the explanation in Figure 3A. [Figure 3F] Refer to the explanation in Figure 3A. [Figure 3G] Refer to the explanation in Figure 3A. [Figure 3H] Refer to the explanation in Figure 3A. [Figure 3I] Refer to the explanation in Figure 3A. [Figure 3J] Refer to the explanation in Figure 3A. [Figure 3K] Refer to the explanation in Figure 3A. [Figure 3L] Refer to the explanation in Figure 3A. [Figure 3M] Refer to the explanation in Figure 3A. [Figure 3N] Refer to the explanation in Figure 3A. [Figure 3O] Refer to the explanation in Figure 3A. [Figure 3P] Refer to the explanation in Figure 3A. [Figure 4] Exemplary Western blot analysis of native PSA isolated from semen using the anti-1,6-fucPSA antibody 2H9. Figure 4A: Lanes 1-4 contain decreasing concentrations of native PSA: 5 μg, 2.5 μg, 1 μg, and 0.5 μg, respectively. Figure 4B: Reactivity of 2H9 to 5 μg of native PSA (sample containing approximately 80% fucosylated PSA; lane 1) or to 5 μg of deglycosylated native PSA (lane 2). [Figure 5] Representative images of immunohistochemical staining of total PSA in formalin-fixed paraffin-embedded (FFPE) biopsies of prostate adenocarcinoma, detected using a commercially available anti-PSA mouse monoclonal antibody (ER-PR8, Roche Tissue Diagnostics, catalog number: 760-4271), or core-fucosylated PSA, detected using the anti-1,6-fucPSA antibodies of the present invention (2E9, 3B10, 3H6, 13C5, 2H9, and 2C11). [Figure 6]The glycopeptides tested for inhibition of binding of the anti-1,6-fucPSA antibody of the present invention to antigens in FFPE prostate adenocarcinoma samples are: (A) Fucosylated PSA glycopeptide containing a disaccharide having α-1,6-core fucose, "DP"; (B) Non-fucosylated PSA glycopeptide containing a nonasaccharide lacking α-1,6-core fucose, the glycopeptide of PSA(67-79)-G2 / formula (II); (C) Aglycosylated PSA fragment (SEQ ID NO: 18); (D) Fucosylated non-PSA glycopeptide (alpha-fetoprotein referred to as "AFP") containing the same glycan structure as PSA (in particular, the same glycan as the glycopeptide of PSA(67-79)-G0F / formula (I)). The symbols used in Figures 6A to 6D are the same as those used in Figure 1, i.e., they represent the same molecules as defined in the legend of Figure 1. The amino acid sequences of the glycopeptides and / or peptides in Figures 6A to 6C are shown in Sequence ID No. 18. [Figure 7A] Inhibition of exemplary anti-1,6-fucPSA antibody binding by the PSA(67-79)-G0F / formula (I) glycopeptide (referred to as "G0F peptide" in the figure) in IHC analysis of FFPE samples of prostate adenocarcinoma. From top to bottom, exemplary anti-1,6-fucPSA antibodies: A: 2E9, 3B10, and 3H6, 2.5 μg / ml, 2.5 μg / ml, and 2.5 μg / ml, respectively; B: 13C5, 2H9, and 2C11, 3 μg / ml, 2.5 μg / ml, and 1 μg / ml, respectively. For A and B, the antibodies were pre-incubated with PBS buffer (control) or 5 × 10⁻⁹ M, 5 × 10⁻⁸ M, 5 × 10⁻⁷ M, 5 × 10⁻⁶ M, or 5 × 10⁻⁵ M PSA(67-79)-G0F, from left to right. [Figure 7B] See the explanation in Figure 7A. [Figure 8A]Inhibition of anti-1,6-fucPSA antibody binding by fucosylated PSA disaccharide peptide (DP) in IHC analysis of FFPE samples of prostate adenocarcinoma. From top to bottom, exemplary anti-1,6-fucPSA antibodies: A: 2E9, 3B10, and 3H6, 2.5 μg / ml, 2.5 μg / ml, and 2.5 μg / ml, respectively; B: 13C5, 2H9, and 2C11, 3 μg / ml, 2.5 μg / ml, and 1 μg / ml, respectively. For A and B, the antibodies were pre-incubated with PBS buffer (control) or with 5 × 10⁻⁹ M, 5 × 10⁻⁸ M, 5 × 10⁻⁷ M, 5 × 10⁻⁶ M, or 5 × 10⁻⁵ M DP, from left to right. [Figure 8B] See the explanation in Figure 8A. [Figure 9A] In IHC analysis of FFPE samples of prostate adenocarcinoma, exemplary anti-1,6-fucPSA antibody binding is not inhibited by non-fucosylated PSA glycopeptides (PSA(67-79)-G2 / formula (II) glycopeptides, referred to as "G2" peptides). From top to bottom, the columns are: A: 2E9, 3B10, and 3H6, 2.5 μg / ml, 2.5 μg / ml, and 2.5 μg / ml respectively; B: 13C5, 2H9, and 2C11, 3 μg / ml, 2.5 μg / ml, and 1 μg / ml respectively. For A and B, the antibodies were pre-incubated with PBS buffer (control) or with 5 × 10⁻⁹ M, 5 × 10⁻⁸ M, 5 × 10⁻⁷ M, 5 × 10⁻⁶ M, or 5 × 10⁻⁵ M G2, from left to right. [Figure 9B] See the explanation in Figure 9A. [Figure 10A]Aglycosylated PSA fragments (aa67-79); exemplary anti-1,6-fucPSA antibody binding is not inhibited by SEQ ID NO: 18. From top to bottom, exemplary anti-1,6-fucPSA antibodies: A: 2E9, 3B10, and 3H6, 2.5 μg / ml, 2.5 μg / ml, and 2.5 μg / ml, respectively; B: 13C5, 2H9, and 2C11, 3 μg / ml, 2.5 μg / ml, and 1 μg / ml, respectively. For A and B, the antibodies were pre-incubated with PBS buffer (control) or with 5 × 10⁻⁹ M, 5 × 10⁻⁸ M, 5 × 10⁻⁷ M, 5 × 10⁻⁶ M, or 5 × 10⁻⁵ M aglycosylated PSA fragments, from left to right. [Figure 10B] Refer to the explanation in Figure 10A. [Figure 11A] Exemplary anti-1,6-fucPSA antibody binding is not inhibited by core-fucosylation-independent peptides containing the same glycan structure as PSA(67-79)-G0F (referred to as the "AFP" peptide). Exemplary anti-1,6-fucPSA antibodies: A: 2E9, 3B10, and 3H6, 2.5 μg / ml, 2.5 μg / ml, and 2.5 μg / ml, respectively; B: 13C5, 2H9, and 2C11, 3 μg / ml, 2.5 μg / ml, and 1 μg / ml, respectively. For A and B, the antibodies were pre-incubated with PBS buffer (control) or with 5 × 10⁻⁹ M, 5 × 10⁻⁸ M, 5 × 10⁻⁷ M, 5 × 10⁻⁶ M, or 5 × 10⁻⁵ M AFP, from left to right column. [Figure 11B] Refer to the explanation in Figure 11A. [Figure 12A] Reactivity of sandwich ELISA after spiking in an artificial serum matrix with either the anti-total PSA antibody K-54794 (capture antibody) or one of six exemplary anti-1,6-fucPSA antibodies 2E9, 3B10, 3H6, 13C5, 2H9, and 2C11 (detection antibodies), together with natural (rhomboid) and deglycosylated (square) PSA antigens. [Figure 12B] Refer to the explanation in Figure 12A. [Figure 12C] Refer to the explanation in Figure 12A. [Figure 12D] Refer to the explanation in Figure 12A. [Figure 12E]Refer to the explanation in Figure 12A. [Figure 12F] Refer to the explanation in Figure 12A. [Figure 13A] Alignment of the consensus sequence of the heavy chain variable domain of the anti-1,6-fucPSA antibody (SEQ ID NO: 19) with the heavy chain variable domains of exemplary antibodies 2E9, 2C11, 2H9, 3B10, 3H6, and 13C5 (SEQ ID NOs: 55, 59, 65, 61, 63, and 57, respectively). [Figure 13B] Alignment of the consensus sequence of the light chain variable domain of the anti-1,6-fucPSA antibody (SEQ ID NO: 20) with the light chain variable domains of exemplary antibodies 2E9, 2C11, 2H9, 3B10, 3H6, and 13C5 (SEQ ID NOs: 56, 60, 66, 62, 64, and 58, respectively). [Modes for carrying out the invention]

[0073] 5. Detailed explanation 5.1 Antibodies and antibody-antigen binding fragments that selectively and discriminatorily bind to α-1,6 core fucosylated PSA The present invention provides antibodies and antigen-binding fragments (hereinafter interchangeably referred to as anti-1,6-fucPSA antibodies and anti-1,6-FucPSA antibody-antigen-binding fragments) that specifically bind to core-fucosylated PSA and subsequences thereof containing core-fucose residues, as well as polynucleotides encoding such antibodies and antigen-binding fragments. The antibodies and antibody-antigen-binding fragments of the present invention are particularly useful as reagents for the specific binding of core-fucosylated PSA or its core-fucosylated fragments, which also distinguish target antigens, i.e., core-fucosylated PSA or its core-fucosylated fragments, from PSA and subsequences thereof lacking core-fucose residues (including aglycosylated PSA and its aglycosylated subsequences). In certain embodiments, the antibodies and antibody-binding fragments of the present invention also distinguish core-fucosylated PSA / PSA subsequences from core-fucosylated glycans in other contexts of PSA (e.g., core-fucosylated glycans of formula IIIb).

[0074] As used herein, the term "PSA" refers to the glycopeptide prostate-specific antigen and includes variants, isoforms, and species congeners of PSA. Accordingly, the antibodies and antibody-antigen binding fragments disclosed herein can bind to human PSA and can cross-react with PSA from non-human species, provided that the PSA or PSA sequence contains core-fucosylation and the antibodies and antigen-binding fragments also specifically bind to the glycopeptide of formula Ib. An exemplary amino acid sequence of PSA is provided as SEQ ID NO: 21, which has a single N-glycosylation site at Asn-69.

[0075] The antibodies and antibody-antigen binding fragments disclosed herein specifically bind to epitopes of core-fucosylated PSA that include an α-1,6-core-fucose residue and at least a portion of the amino acid sequence of SEQ ID NO: 18, which includes a PSA N-glycosylation site at Asn-69. Therefore, the antibodies and antibody-antigen binding fragments of the present invention do not significantly bind to PSA and PSA subsequences lacking core-fucose residues (for example, if the glycan lacks α-1,6-core-fucosylation as in formula IIb, it will not bind to glycosylated PSA, nor to aglycosylated PSA or its aglycosylated fragment). Furthermore, in certain embodiments, the antibodies and antigen-binding fragments disclosed herein do not significantly bind to core-fucosylated glycans and antigens including peptides as defined herein in the context of non-target proteins, nor do they bind to core-fucosylated peptides such as AFP (alpha-fetoprotein), nor do they bind to isolated forms, such as core-fucosylated glycans as shown in formula IIIb. It is most preferable that the anti-1,6-fucPSA antibody and antibody-antigen-binding domain of the present invention specifically bind to the core-fucosylated PSA glycopeptide of formula Ib, i.e., PSA(67-79)-G0F, and distinguish from / against both (i.e., not specifically bind) glycosylated PSA fragments lacking core-fucose residues, such as formula IIb, and core-fucosylated glycans of formula IIIb.

[0076] In humans, endogenously expressed PSA contains a single N-glycosylation site in asparagine corresponding to Asn-69 of Uniprot ID P07288; therefore, the terms “glycan” and “glycosylation of PSA” and similar terms used herein refer to this single carbohydrate structure bound to PSA. The glycan of endogenously expressed PSA may or may not contain a core-fucose residue, i.e., an α-1,6 fucose residue linked to the core GlcNac bound to Asn69 of PSA. As demonstrated herein, the antibodies and antibody-antigen binding fragments of the present invention recognize the epitope partially determined by this core-fucose residue, with the contribution of any remaining carbohydrate structure, if any. Therefore, the anti-1,6-fucPSA antibody and antibody-antigen binding fragment provided herein may also bind to PSA and / or PSA subsequences containing carbohydrate structures / glycans that are not endogenously expressed in humans or other animals (only if the carbohydrate structure contains core-fucose residues), i.e., the antibody and antigen binding fragment of the present invention specifically bind to the glycopeptide of formula I or glycoproteins containing the glycopeptide of formula I and distinguish them from the glycopeptide of formula II and / or glycoproteins containing the glycopeptide of formula II. Preferably, the antibody and antigen binding fragment of the present invention specifically bind to the glycopeptide of formula Ib and distinguish them from the glycopeptide of formula IIb (i.e., do not specifically bind). In certain embodiments, the antibody and antibody-antigen binding fragment of the present invention specifically bind to the glycopeptide of formula IV as well as the glycopeptide of formula Ib. Therefore, in certain embodiments, the antibody and antigen binding fragment of the present invention specifically bind to the glycopeptide of formula IV and distinguish them from the glycopeptide of formula IIb (i.e., do not specifically bind).

[0077] Furthermore, as demonstrated herein, the epitopes recognized by the antibodies and antibody-antigen binding fragments of the present invention include at least a portion of SEQ ID NO: 18. Therefore, it is preferable that the antibodies and antigen binding fragments of the present invention specifically bind to the glycopeptide of formula Ib and are distinct from (i.e., do not specifically bind to) the core-fucosylated glycan of formula IIIb. Most preferably, the antibodies and antigen binding fragments of the present invention specifically bind to the glycopeptide of formula Ib and are distinct from (i.e., do not specifically bind to) both the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb. In these most preferred embodiments, preferred bindings of the glycopeptide of formula Ib, the glycopeptides of formula IIb and formula IIIb in the glycans are shown in formulas Ic, IIc, and IIIc, respectively.

[0078] The antibodies and antibody-binding domains of the present invention are exemplified by several different embodiments (i.e., antibodies) disclosed herein, which can provide specific binding to core-fucosylated PSA and its subsequences containing core-fucosylation, enabling the determination of a consensus CDR structure that can provide identification of core-fucosylated glycans in PSA / PSA subsequences lacking core-fucose residues and / or in isolation or in other contexts (e.g., in the context of core-fucosylated AFP). However, it is well known in the art that some deviation from the consensus CDR sequence is possible while still retaining the functionality of specific and discriminative binding demonstrated by the exemplary antibodies, for example, as is known from at least standard humanization protocols. That is, it is known that certain CDR / variable region residues can be replaced, and sequence variants that maintain desired functional properties can be readily identified using only common knowledge in the art. Therefore, the present invention provides an antibody and / or antibody-antigen-binding fragment (preferably a monoclonal antibody or monoclonal antibody-antigen-binding fragment) that specifically and discriminatorily binds to core-fucosylated PSA and a subsequence thereof containing core-fucosylation, wherein the antibody or antibody-antigen-binding fragment includes the following: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Antibody heavy chain variable domain (HV1) ; and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Antibody light chain variable domain (LV1) .

[0079] In certain embodiments, the above-mentioned anti-1,6-fucPSA antibody or antibody-antigen binding fragment containing HV1 and / or LV1 may include the following: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Antibody heavy chain variable domain (HV2); and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single conservative amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Antibody light chain variable domain (LV2) .

[0080] In certain embodiments, the above anti-1,6-fucPSA antibody or antibody-antigen binding fragment, comprising one of HV1 to HV2 and / or one of LV1 to LV2, may include the following: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Antibody heavy chain variable domain (HV3) ; and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Antibody light chain variable domain (LV3) .

[0081] In a particular embodiment, the above anti-1,6-fucPSA antibody or antibody-antigen binding fragment, comprising one of HV1 to HV3 and / or one of LV1 to LV3, may include the following: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Heavy chain variable domain (HV4) ; and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by a single conservative amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified by up to two conservative amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Light chain variable domain (LV4) .

[0082] In certain embodiments, the above anti-1,6-fucPSA antibody or antibody-antigen binding fragment, comprising one of HV1 to HV4 and / or one of LV1 to LV4, may include: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Heavy chain variable domain (HV5) ; and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Light chain variable domain (LV5) .

[0083] In certain embodiments, the above anti-1,6-fucPSA antibody or antibody-antigen binding fragment, comprising any one of HV1 to HV5 and / or any one of LV1 to LV5, may include the following: (i) CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15. Heavy chain variable domain (HV6) ; and / or (ii) CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified by a single highly conserved amino acid substitution at positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof modified by up to two highly conserved amino acid substitutions at positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. Light chain variable domain (LV6) .

[0084] As disclosed above, the CDR may include the enumerated reference sequences or may differ from them by one or more amino acid substitutions. It will be understood that the amino acid substitutions disclosed above may not be present in the CDRs of the antibodies of the present invention, or may be present in one, two or more, or all of the CDRs. As used herein, the term “substitution” refers to replacing one amino acid with another. Deletion of an amino acid at a particular position and introduction of one (or more) amino acids at different positions are not expressly included in the term “substitution.” As stated above, the present invention encompasses conserved or highly conserved amino acid substitutions as defined herein.

[0085] CDRs described above as containing one or more substitutions are referred to herein as “variant CDRs.” It is clear that a variant CDR may be a functional variant, i.e., different from the reference amino acid sequence, but the different sequence has an amino acid sequence that exhibits or maintains the same functional activity as the reference sequence in the context 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 containing one or more variant CDRs exhibits specific and discriminative binding to the core-fucosylated PSA or fucosylated subsequences described herein. The anti-1,6-fucPSA antibody or antibody-antigen-binding fragment of the present invention containing heavy chain and / or light chain variable domains having one or more variant CDRs disclosed herein is most preferably specific to the glycopeptide of formula Ib. In certain embodiments, the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention also discriminatively (i.e., does not specifically bind) to the glycopeptide of formula IIb. In certain embodiments, the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention is discriminatory (i.e., does not specifically bind) to the core-fucosylated glycan of formula IIIb. Most preferably, the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention is discriminatory (i.e., does not specifically bind) to the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb.

[0086] The anti-1,6-fucPSA antibody or its antigen-binding fragment is most preferably bound to the glycopeptide of formula Ib (in the most preferred embodiment, the glycan of formula Ib preferably contains the binding shown in formula Ic) with a KD of 30 nM or less, more preferably 20 nM or less, and most preferably 15 nM or less. The assay conditions used to determine the antibody binding affinity are further preferably standardized such that the determined KD of the antibody, preferably a rabbit antibody, which includes the VH and VL domains of exemplary antibody 3B10 for the glycopeptide of formula Ib (containing the glycan having the preferred binding shown in formula Ic) (i.e., including SEQ ID NOs. 61 and 62, respectively), is 11 nM + the standard error of the particular assay.

[0087] The anti-1,6-fucPSA antibody or antibody-antigen binding fragment also preferably has an affinity for the target antigen, e.g., a KD value, that is at least 10 times, at least 20 times, preferably at least 50 times, and more preferably at least 100 times better (e.g., lower KD value) than its affinity for the non-target antigen. In this embodiment, it is most preferable that the target antigen is a glycopeptide of formula Ib and the non-target antigen is a glycopeptide of formula IIb. Furthermore, as described above, it is most preferable that the affinity for the target antigen, e.g., KD, is at least 100 times better than its affinity for the non-target antigen. Therefore, in relation to the most preferred embodiment, i.e., when the target antigen is a glycopeptide of formula Ib and the non-target antigen is a glycopeptide of formula IIb, it is most preferable that the anti-1,6-fucPSA antibody or its antibody-antigen binding fragment has an affinity for the glycopeptide of formula Ib, e.g., a KD value, that is at least 100 times better than its affinity for the glycopeptide of formula IIb. In these most preferred embodiments, preferred bindings in the glycopeptide of formula Ib and the glycopeptide of formula IIb in the glycan are shown in formulas Ic and IIc, respectively. If the non-target antigen does not show detectable binding, identification indicates that the anti-1,6-fucPSA antibody or antibody-antigen binding fragment has a binding affinity at least 100 times greater than the lowest detectable binding of the assay.

[0088] Alternatively or additionally, the anti-1,6-fucPSA antibody or its antigen-binding fragment of the present invention may be 1 × 10⁻⁶ 5 M -1 s -1 The above is fua2×10 5 M -1 s -1 In summary, the most preferred is 5 × 10 5 M -1 s -1 The above meeting rate (k a ) specifically binds to its target antigen. The antibodies or antigen-binding fragments provided herein are 1 × 10 5 M -1 s -1 The above is fua2×10 5 M -1 s-1 In summary, the most preferred is 5 × 10 5 M -1 s -1 The above meeting rate (k a It is preferable to bind to the glycopeptide of formula Ib in this manner. In exemplary embodiments, the antibody or antibody-conjugated fragment of the present invention is specific to core-fucosylated PSA and / or its core-fucosylated subsequence, most preferably the glycopeptide of formula Ib, and comprises one of the antibody 2E9, 13C5, 2C11, 3B10, 3H6, and 2H9, and the CDR of the heavy chain and / or light chain variable domains disclosed herein. Thus, the antibody or antibody-antigen-conjugated fragment of the present invention comprises: (i) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 22, CDR-H2 having the amino acid sequence of SEQ ID NO: 23, and CDR-H3 having the amino acid sequence of SEQ ID NO: 24, Heavy chain variable domain (HV-2E9) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 34, CDR-L2 having the amino acid sequence of SEQ ID NO: 35, and CDR-L3 having the amino acid sequence of SEQ ID NO: 36. Light chain variable domain (LV-2E9) ; (ii) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27, Heavy chain variable domain (HV-13C5) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 34, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38. Light chain variable domain (LV-13C5) ; (iii) CDR-H1 having the amino acid sequence of SEQ ID NO: 28, CDR-H2 having the amino acid sequence of SEQ ID NO: 29, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27, Heavy chain variable domain (HV-2C11) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 39, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40. Light chain variable domain (LV-2C11) ; (iv) CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 30, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27, Heavy chain variable domain (HV-3B10) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 41, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 42. Light chain variable domain (LV-3B10) ; (v) CDR-H1 having the amino acid sequence of sequence number 25, CDR-H2 having the amino acid sequence of sequence number 31, and CDR-H3 having the amino acid sequence of sequence number 32, Heavy chain variable domain (HV-3H6) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 43, CDR-L2 having the amino acid sequence of SEQ ID NO: 44, and CDR-L3 having the amino acid sequence of SEQ ID NO: 45. Light chain variable domain (LV-3H6) ; or (vi) CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 31, and CDR-H3 having the amino acid sequence of SEQ ID NO: 33, Heavy chain variable domain (HV-2H9) ; and / or This includes CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40. Light chain variable domain (LV-2H9) .

[0089] It is known in the art that the heavy or light chain variable domain of an antibody includes four framework domains in addition to the three CDRs defined above. Specifically, it is known that framework region 1 (FW1) represents most of the N-terminal portion of the variable chain domain, and framework region 4 (FW4) represents most of the C-terminal portion, and the general formula (V) FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4 (Formula V) Accordingly, CDRs are scattered across the framework domains.

[0090] Whether a framework region (FW) or complementarity-determining region (CDR) of a heavy or light chain variable domain is being referred to is clear from the context, but FWs and CDRs are distinguished herein by the labels "H" or "L". For example, the components of a heavy chain variable domain, FWs and CDRs, are referred to herein by formula (VI) (FW-H1)-(CDR-H1)-(FW-H2)-(CDR-H2)-(FW-H3)-(CDR-H3)-(FW-H4) (Formula VI) It is mentioned as something that is roughly represented by [this].

[0091] Similarly, the components FW and CDR of the light chain variable domain are expressed herein by formula (VII) (FW-L1)-(CDR-L1)-(FW-L2)-(CDR-L2)-(FW-L3)-(CDR-L3)-(FW-L4) (Formula VII) It is mentioned as something that is roughly represented by [this].

[0092] The anti-1,6-fucPSA antibody and / or anti-1,6-fucPSA antibody antigen-binding fragment according to the present invention comprises at least one heavy chain or light chain variable domain HV1, HV2, HV3, HV4, HV5, HV6, LV1, LV2, LV3, LV4, LV5, or LV6 as defined above, and preferably comprises an antibody Fv domain comprising paired heavy chain and light chain variable domains HV1 and LV1, HV2 and LV2, HV3 and LV3, HV4 and LV4, HV5 and LV5, or HV6 and LV6. The heavy-chain variable domains of the present invention, e.g., HV1, HV2, HV3, HV4, HV5, and HV6 (including HV-2E9, HV-13C5, HV-2C11, HV-3B10, HV-3H6, and HV-2H9), and the light-chain variable domains of the present invention, LV1, LV2, LV3, LV4, LV5, and LV6 (including LV-2E9, LV-13C5, LV-2C11, LV-3B10, LV-3H6, and LV-2H9), are characterized by the sequences of their CDRs as defined herein, which determine the specific and definitive binding to the core-fucosylated PSAs and their core-fucosylated subsequences described herein, as is known in the art. The sequences of the surrounding FW domains can be selected by those skilled in the art using standard methods routinely practiced in the art. It is understood that those skilled in the art will select a suitable sequence for the FW domain such that the obtained antibody or antibody-antigen binding fragment is an anti-1,6-fucPSA antibody or antigen binding fragment as defined herein, that is, it exhibits specific binding to core-fucosylated PSA and / or core-fucosylated subsequences as defined herein, exhibits differential binding (i.e., does not specifically bind) to PSA / PSA subsequences lacking core-fucosylation, and is further differential to the coreglycan of formula IIIb. The obtained anti-1,6-fucPSA antibody and antibody-antigen binding fragment of the present invention is most preferably specific to the glycopeptide of formula Ib. The anti-1,6-fucPSA antibody or antigen binding fragment of the present invention is also most preferably discriminatory (i.e., does not specifically bind) to the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb.In these most preferred embodiments, the preferred linkages of the glycopeptide of formula Ib, and the glycopeptides of formulas IIb and IIIb in the glycan are shown in formulas Ic, IIc, and IIIc, respectively.

[0093] In certain embodiments, the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention comprises the following: (i) FW-H1 having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 47; FW-H2 having an amino acid sequence having at least 95%, at least 90% sequence identity with SEQ ID NO: 48; FW-H3 having an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 49; and FW-H4 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 50; and / or the heavy chain variable domains HV1, HV2, HV3, HV4, HV5, or HV6 as defined herein above; and / or (ii) Light chain variable domains LV1, LV2, LV3, LV4, LV5, or LV6 as defined herein, further comprising FW-L1 having an amino acid sequence having at least 90%, at least 93%, at least 95%, or at least 97% sequence identity with SEQ ID NO: 51; FW-L2 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 52; FW-H3 having an amino acid sequence having at least 90%, at least 93%, or at least 95% sequence identity with SEQ ID NO: 53; and FW-H4 having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 54.

[0094] In a particular embodiment, the total number of mutations in FW1-4 of the heavy chain variable domain is a maximum of 5 amino acid substitutions in total with respect to the reference heavy chain framework sequence, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, and the total number of mutations in FW1-4 of the light chain variable domain is a maximum of 3 amino acid substitutions in total with respect to the reference light chain framework sequence, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.

[0095] In certain embodiments, the total number of mutations in the heavy chain variable domains FW1-4 is a maximum of 5, 4, 3, 2, and 1 amino acid substitutions in total with respect to the reference heavy chain framework sequence, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, and / or the total number of mutations in the light chain variable domains FW1-4 is a maximum of 3, 2, and 1 amino acid substitutions in total with respect to the reference light chain framework sequence, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54. In further embodiments, no mutations, such as substitutions, are present in the heavy chain variable domains FW1-4 when compared together with the reference heavy chain framework sequence, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, and / or no mutations, such as substitutions, are present in the light chain variable domains FW1-4 when compared together with the reference light chain framework sequence, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.

[0096] For all of the above-defined FWs relative to a reference sequence, it will be understood that different degrees of sequence identity may be permitted for different FWs, depending on the length of each FW sequence and its position within each variable chain domain. Specific FW sequences can be selected according to general knowledge in the art to maintain specificity for core-fucosylated PSA and its core-fucosylated sequences. Anti-1,6-fucPSA antibodies and antibody-antigen binding fragments comprising one or more variant CDRs and one or more variant FWs as defined above herein are most preferably specifically bound to the glycopeptide of formula Ib and distinguishable from the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb.

[0097] In certain embodiments, the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention is as follows: (i) Heavy chain variable domains having an amino acid sequence having at least 80%, at least 86%, at least 90%, or preferably at least 93% sequence identity with respect to SEQ ID NO: 19; and / or (ii) A light chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 90%, or preferably at least 96% sequence identity with respect to SEQ ID NO: 20; Includes, The antibody or antigen-binding fragment is characterized by specific and discriminant binding to core-fucosylated PSA, a core-fucosylated subsequence of PSA, or most preferably a glycopeptide of formula Ib. The anti-1,6-fucPSA antibody or antibody-antigen-binding fragment disclosed in this paragraph preferably contains both the heavy chain and light chain variable domains of (i) and (ii) described immediately above. In certain embodiments, the monoclonal antibodies and antigen-binding fragments disclosed herein comprise a heavy chain variable domain having an amino acid sequence having at least 93%, at least 95%, at least 96%, at least 97%, or at least 98% sequence identity to SEQ ID NO: 19; and / or (preferably and) a light chain variable domain having an amino acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20, wherein the antibody or antigen-binding fragment is characterized by specific and distinctive binding as described herein to core-fucosylated PSA, a core-fucosylated subsequence of PSA, or most preferably, a glycopeptide of formula Ib.

[0098] In exemplary embodiments, the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention includes the following: (i) A heavy chain variable domain having the sequence of SEQ ID NO: 55 and a light chain variable domain having the sequence of SEQ ID NO: 56 (the variable domain of antibody 2E9); (ii) A heavy chain variable domain having the sequence of SEQ ID NO: 57 and a light chain variable domain having the sequence of SEQ ID NO: 58 (variable domain of antibody 13C5); (iii) A heavy chain variable domain having the sequence of SEQ ID NO: 59 and a light chain variable domain having the sequence of SEQ ID NO: 60 (variable domain of antibody 2C11); (iv) A heavy chain variable domain having the sequence of SEQ ID NO: 61 and a light chain variable domain having the sequence of SEQ ID NO: 62 (variable domain of antibody 3B10); (v) A heavy chain variable domain having the sequence of SEQ ID NO: 63 and a light chain variable domain having the sequence of SEQ ID NO: 64 (variable domain of antibody 3H6); or (vi) A heavy chain variable domain having the sequence of SEQ ID NO: 65 and a light chain variable domain having the sequence of SEQ ID NO: 66 (the variable domain of antibody 2H9).

[0099] As used herein, the term “sequence identity %” in relation to the amino acid sequence and / or nucleic acid sequence or nucleic acid molecule of a polypeptide / peptide represents the number of identical amino acid or nucleic acid residue matches of two or more aligned sequences compared to the number of residues that make up the full length of the sequence being compared (or the portion being compared as a whole). The percentage of identical residues using the alignment of two or more sequences or subsequences may be determined by comparing the (sub)sequences on a comparison window or on a designated region measured using a sequence comparison algorithm known in the art, and aligning for the greatest match, or by manual alignment and visual inspection. Non-exclusive examples of algorithms used 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, Nucleic Acids Res. 2 (1994), 4673-4680), or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 85 (1988), 2444). The FASTA algorithm typically does not consider internal mismatched deletions or additions, i.e., gaps, in its calculations, although this can be manually corrected to avoid overestimating % sequence identity. However, CLUSTALW does consider sequence gaps in its identity calculations. The BLAST and BLAST 2.0 algorithms (Altschul et al., Nucl Acids Res., 25(1977), 3389) are also available.

[0100] The present invention also provides an anti-1,6-fucPSA antibody or anti--1,6-fucPSA antibody antigen-binding fragment that binds to the same epitope of core-fucosylated PSA or its core-fucosylated subsequence, most preferably a glycopeptide of formula Ib, as an antibody or antigen-binding fragment comprising: (i) A heavy chain variable domain having the sequence of sequence number 55 and a light chain variable domain having the sequence of sequence number 56; (ii) A heavy chain variable domain having the sequence of Sequence ID No. 57 and a light chain variable domain having the sequence of Sequence ID No. 58; (iii) A heavy chain variable domain having the sequence of Sequence ID No. 59 and a light chain variable domain having the sequence of Sequence ID No. 60; (iv) A heavy chain variable domain having the sequence of Sequence ID No. 61 and a light chain variable domain having the sequence of Sequence ID No. 62; (v) A heavy chain variable domain having the sequence of SEQ ID NO: 63 and a light chain variable domain having the sequence of SEQ ID NO: 64; or (vi) A heavy chain variable domain having the sequence of sequence number 65 and a light chain variable domain having the sequence of sequence number 66.

[0101] The specific epitope of core-fucosylated PSA or its core-fucosylated subsequence, most preferably the glycopeptide of formula Ib, which is conjugated by either the antibody or antigen-binding fragment defined above, can be identified in combination with any suitable epitope mapping method known in the art by any suitable method. An example of such a method is to screen peptides of various lengths derived from core-fucosylated PSA, preferably the glycopeptide of formula Ib, which contain core-fucosylated glycan residues, for binding to the antibody of the present invention as defined above, to identify the smallest glycosylated fragment (i.e., the smallest glycopeptide) that can specifically bind to the antibody. The glycopeptide that binds to the antibody can be identified, for example, by mass spectrometry. In another example, the epitope to which the antibody of the present invention binds can be identified using NMR spectroscopy or X-ray crystallography. Once identified, the epitope fragment that binds to the antibody of the present invention can be used as an immunogen to obtain further antibodies that bind to the same epitope.

[0102] 5.2 Preparation and manipulation of antibody polypeptides and their antigen-binding fragments Unless otherwise specified, the terms “antibody,” “antibody(s),” and similar terms encompass, with respect to complete immunoglobulin molecules, naturally occurring forms of antibodies (including, but not limited to, IgG, IgA, IgM, and IgE), as well as recombinant antibody constructs, including, but not limited to, single-chain antibodies, chimeric antibodies, humanized antibodies, antibody fusion proteins, and multispecific antibodies; and all of the antigen-binding fragments and derivatives described above. As is known in the art, an antibody includes a variable region (known in the art as the “Fv region” and / or “Fv domain”) formed from paired variable domains from both the heavy and light chains, which interact with the antigen. The term “Fv region” does not include the constant regions of the heavy and / or light chains.

[0103] As used herein, the terms “antibody,” “antibody(s),” and similar terms also refer to their antigen-binding fragments, which may be referred herein as antibody-antigen-binding fragments and / or simply antigen-binding fragments. These terms refer to one or more fragments of an antibody that possess the ability to specifically bind to a target antigen, such as core-fucosylated PSA or its core-fucosylated subsequence, as known in the art, and include, but are not limited to, antigen-binding fragments containing paired heavy-chain and light-chain variable domains, such as Fv domains, i.e., Fab, Fab', F(ab')2 and Fv fragments, as well as recombinant constructs, such as single-chain Fv domains known in the art as scFv. The terms also include antibody-antigen-binding fragments containing a single unpaired heavy-chain or light-chain variable domain known in the art that possess the ability to specifically and selectively bind to an antigen as defined herein, and which include, but are not limited to, single-domain antibodies based on camel heavy chains (also referred to in the art as sdAb, dAb, and / or nanobodies) and V H It includes the H domain.

[0104] The antibodies and antigen-binding fragments of the present invention may be polyclonal or monoclonal, preferably monoclonal. Where used herein with respect to antibodies or their antigen-binding fragments, the terms “monoclonal,” “monoclonal composition,” and similar terms refer to a population of antibody polypeptides or fragments thereof produced from a single B cell clone, the population comprising only one species of antigen-binding site capable of immunoreacting with a specific epitope of an antigen. This is in contrast to the term “polyclonal” antibodies and compositions, which refers to a population of antibody polypeptides or antigen-binding fragments comprising multiple species of antigen-binding sites. The present invention also includes modified forms of monoclonal antibodies, e.g., humanized or chimeric versions thereof, as well as recombinant antibody constructs, e.g., antibody (or antigen-binding fragment) fusion proteins, where the antibody or antigen-binding fragment comprises, for example, additional domains for the isolation and / or preparation of recombinantly produced antibodies / fragments / constructs.

[0105] The antibodies and antigen-binding fragments of the present invention can be prepared by various techniques routinely used in the art. For example, antibodies can be prepared by immunizing non-human animals with an antigen of interest for isolation, and then isolating antigen-reactive antibody-producing B cells. It is most preferable to identify positive clones using the glycopeptide of formula Ib, i.e., clones that produce antibodies that specifically bind to this glycopeptide. In this most preferred embodiment, the binding of formula Ib in the glycan is preferably as shown in formula Ic. The positive clones are further subjected to negative selection to exclude clones that react specifically or significantly with the glycopeptide of formula IIb and / or the core-fucosylated glycan of formula IIIb, and more preferably clones that react with both the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb. In these preferred embodiments, preferred bindings of the glycopeptides of formula IIb and formula IIIb in the glycan are shown in formulas IIc and IIIc, respectively. Methods for isolating and / or selecting (positive or negative) clones that produce antibodies with desired characteristics are well known in the art. For example, non-limiting exemplary methods for the production and isolation of antigen-reactive antibody-producing B cells include immunizing a non-human animal, preferably a rabbit, e.g., a NZW rabbit, with a core-glycosylated peptide fragment of PSA, most preferably a glycopeptide of formula Ib, PSA(67-79)-G0F. As is known in the art, peptide immunogens may be conjugated to an adjuvant carrier, e.g., keyhole limpet hemocyanin (KLH), and / or administered with an adjuvant composition, e.g., Freund's complete or incomplete adjuvant, to improve immunogenicity. Animals may be immunized according to standard schedules such as weekly, monthly, or a combination of weekly and monthly, depending on the animal, antigen, and titer of the antibody produced. To determine the animal's response, antibody titers in serum may be tested according to standard procedures.Reactive B cells can be purified from serum by isolating the peripheral blood mononuclear cell (PBMC) fraction of positive animals and purifying antigen-reactive B cells using standard techniques such as ELISA or column-based techniques, as described, for example, Seeber et al., PLoS One.9(2014),e86184. As mentioned, such a screening method may or may not include a negative selection step to identify and eliminate clones that exhibit cross-reactivity with undesirable antigens, such as PSA or PSA fragments lacking core-fucose residues, such as the glycopeptide of formula IIb ("PSA(67-79)-G2") and core-fucosylated glycans of formula IIIb. The selected positive clones, i.e., clones that bind to the screening peptide, can then be selected for subsequent recombination.

[0106] Another suitable method for generating or isolating the antibodies and antibody-antigen binding fragments of the present invention is, but is not limited to, a method of selecting recombinant antibodies from a peptide or protein library (e.g., bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or yeast display library) using the desired binding activity. For example, the antibody or antigen binding fragment can be selected from such a library by positive selection for specific binding to the glycopeptide of formula Ib and negative selection for binding to the glycopeptide of formula IIb and / or the core-fucosylated glycan of formula IIIb. It is most preferable that the antibody or antigen binding fragment of the present invention be selected by positive selection for specific binding to the glycopeptide of formula Ib and negative selection for binding to both the glycopeptide of formula IIb and the core-fucosylated glycan of formula IIIb. Display libraries are well known in the art and are available from various commercial suppliers, including, but are not limited to, Cambridge Antibody Technologies (Cambridgeshire, UK), MorphoSys (Martinsried / Planegg, Del.), Biovation (Aberdeen, Scotland, UK), and Bioinvent (Lund, Sweden). Here again, selected clones can be processed according to conventional methods for subsequent recombination.

[0107] Thus, the present invention also provides a nucleic acid molecule encoding the anti-1,6fucPSA antibody or an antigen-binding fragment of the anti-1,6fucPSA antibody disclosed herein, particularly a nucleic acid molecule encoding an anti-1,6fucPSA heavy chain and / or light chain variable domain as defined hereinabove. As used herein, the terms "nucleic acid molecule", "nucleic acid sequence", "polynucleotide" and similar terms include both genomic DNA and cDNA, as well as RNA capable of driving the expression of the antibody or antigen-binding fragment of the present invention. The term "RNA" as used herein includes mRNA, tRNA and rRNA, and is understood to include all forms of RNA including genomic RNA, such as in the case of the RNA of an RNA virus. Preferably, embodiments described as "RNA" relate to mRNA. The nucleic acid molecule / nucleic acid sequence of the present invention can be of natural as well as synthetic or semi-synthetic origin. Thus, the nucleic acid molecule can be, for example, a nucleic acid molecule synthesized according to conventional protocols of organic chemistry, synthesized according to recombinant methods, or produced semi-synthetically, for example by combining chemical synthesis and recombinant methods. Those skilled in the art are proficient in the preparation and use of such nucleic acid molecules.

[0108] In certain embodiments, the present invention particularly provides a polynucleotide encoding the following, as defined hereinabove: (i) an antibody heavy chain variable domain (HV1) and / or an antibody light chain variable domain (LV1); (ii) an antibody heavy chain variable domain (HV2) and / or an antibody light chain variable domain (LV2); (iii) an antibody heavy chain variable domain (HV3) and / or an antibody light chain variable domain (LV3); (iv) an antibody heavy chain variable domain (HV4) and / or an antibody light chain variable domain (LV4); (v) an antibody heavy chain variable domain (HV5) and / or an antibody light chain variable domain (LV5); or (vi) an antibody heavy chain variable domain (HV6) and / or an antibody light chain variable domain (LV6).

[0109] Vectors comprising nucleic acid molecules encoding the antibody or antibody-antigen binding fragment of the present invention are also provided. As used herein, the term “vector” refers to a cyclic or linear nucleic acid molecule capable of self-replicating in the introduced host cell. Non-limiting examples of vectors suitable for use in the present invention include cosmids, plasmids (e.g., naked or liposome-encapsulated), viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses), and bacteriophages. However, the art provides many suitable vectors, and their selection 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). The vectors used in connection with the present invention include nucleic acid sequences that encode a full-length anti-1,6-fucPSA antibody or an anti-1,6-fucPSA antibody antigen-binding fragment, as disclosed herein. Therefore, the vectors used in connection with the present invention may encode the following, as defined herein above: (i) Antibody heavy chain variable domain (HV1) and / or antibody light chain variable domain (LV1); (ii) Antibody heavy chain variable domain (HV2) and / or antibody light chain variable domain (LV2); (iii) Antibody heavy chain variable domain (HV3) and / or antibody light chain variable domain (LV3); (iv) Antibody heavy chain variable domain (HV4) and / or antibody light chain variable domain (LV4); (v) antibody heavy chain variable domain (HV5) and / or antibody light chain variable domain (LV5); or (vi) Antibody heavy chain variable domain (HV6) and / or antibody light chain variable domain (LV6).

[0110] With respect to the term “vector containing ~” as used herein, it is understood in the Art that a vector contains further nucleic acid sequences necessary and / or sufficient for the desired vector activity in host cells, such as a nucleic acid sequence that drives the replication of the vector (and thus encodes it) and / or instructs 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 the expression of the desired sequence in a particular cell line. For example, a vector may contain nucleic acid molecules encoding the antibody or antibody-antigen-binding fragment of the present invention, which are operably linked and / or under the control of regulatory sequences. The term “regulatory sequence” refers to the DNA sequences necessary to bring about the expression of the encoding sequence to which they are operably linked. The term “control sequence” is intended to include at least all components whose presence may also be necessary for expression, and may further include additional advantageous components, for example, to enable replication. As understood in the Art, the nature of such regulatory and control sequences varies depending on the host organism. For example, in prokaryotes, control sequences generally include promoters, ribosome-binding sites, and terminators. In eukaryotes, regulatory sequences generally include promoters, terminators, and in some cases enhancers, transcription activators, or transcription factors.

[0111] The vector used in the present invention is preferably an expression vector. An expression vector can direct the replication and expression of the nucleic acid molecule of the present invention in host cells, and thus, for example, provides the expression of the heavy chain and / or light chain variable domains of the anti-1,6-fucPSA antibody disclosed herein. In some embodiments, the vector may include further sequences to ensure the expression of not only the heavy chain and light chain variable domains but also the remaining heavy chain and light chain constant regions, so that a full-length IgG antibody containing the heavy chain and light chain variable domains of the present invention is expressed. Suitable expression vectors are widely described in the literature, and the determination of a suitable expression vector for a particular cell line can be easily performed by those skilled in the art using conventional methods. Preferably, the vector disclosed herein comprises a recombinant polynucleotide (i.e., a nucleic acid sequence encoding the anti-1,6-fucPSA antibody or its antigen-binding fragment) and an expressibly linked regulatory sequence. The vectors provided herein preferably further include a promoter. The vectors described herein may also include a selection marker gene and an origin of replication to ensure replication in the host. Furthermore, the vectors provided herein may also include a termination signal for transcription. Expression vectors known in the art can drive transient or constitutive expression in host cells.

[0112] The nucleic acid molecules and / or vectors of the present invention can be designed for transfection into prokaryotic or eukaryotic host cells by any means known in the art or described herein. Non-limiting examples of suitable methods include chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nucleofection), non-chemical methods (electroporation, sonoporation, phototransfection, gene electrophoresis, hydrodynamic delivery, or innate transformation upon contact of cells with the nucleic acid molecules of the present invention), particle-based methods (gene gun, magnetofection, impulfection), phage vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, Semryki forest virus, or bovine papillomavirus can be used for transfection of nucleic acid molecules into target cell populations. Furthermore, baculovirus systems can also be used as vectors in eukaryotic expression systems for the nucleic acid molecules of the present invention.

[0113] The term "prokaryotes" includes all bacteria that can be transformed, transfected, or transfected with DNA or DNA or RNA molecules for the expression of the proteins of the present invention. Examples of prokaryotic hosts include Gram-negative and Gram-positive bacteria, such as Escherichia coli, Salmonella tiphimuria, Serratia marcescens, Corynebacterium (glutamicum), Pseudomonas (fluorescein), Lactobacillus, Streptomyces, Salmonella, and Bacillus subtilis. The term "eukaryotes" includes yeast, higher plants, insects, and mammalian cells. Non-limiting examples of host cells typically used in this art include HeLa, HEK293, H9, Per.C6, and Jurkat cells; mouse NIH3T3, NS / 0, SP2 / 0, and C127 cells; COS cells, such as COS1 or COS7, CV1; quail QC1-3 cells; mouse L cells; mouse sarcoma cells; Bose melanoma cells; and Chinese hamster ovary (CHO) cells.

[0114] When a recombinant expression vector encoding the antibody heavy chain and / or light chain variable domains disclosed herein is introduced into host cells, the antibody or antibody-antigen-binding fragment is produced by expressing the antibody or antigen-binding fragment in the host cells, or, preferably, culturing the host cells for a period of time sufficient to secrete the antibody or antigen-binding fragment into the growing medium. The antibody and / or antigen-binding fragment can be recovered from the medium using standard protein purification methods. Accordingly, the present invention also provides a method for producing the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody-antigen-binding fragment disclosed herein, comprising culturing the host cells of the present invention under appropriate conditions and isolating the produced antibody. The present invention further provides an antibody or antigen-binding fragment that can be obtained by any of the methods disclosed herein.

[0115] The host cells according to the present invention are preferably CHO cells. While antibodies and antigen-binding fragments as disclosed herein can be expressed in both prokaryotic and eukaryotic host cells, antibody expression in eukaryotic cells is preferred, and antibody expression in mammalian host cells is most preferred. This is because such eukaryotic cells (particularly mammalian cells, most preferably) are more likely to express properly folded antibodies / antibody fragments containing appropriate post-translational modifications that are immunologically active.

[0116] Transformed host cells can be grown in a bioreactor and cultured by techniques known in the art to achieve optimal cell proliferation. The antibodies and / or antibody-antigen binding fragments of the present invention can then be isolated from cell fractions or growth media by any conventional means, for example, but not limited to affinity chromatography (e.g., using fusion tags such as Strep-tagII or His6 tag), gel filtration (size exclusion chromatography), anion exchange chromatography, cation exchange chromatography, hydrophobic interaction chromatography, high-pressure liquid chromatography (HPLC), reverse-phase HPLC, or immunoprecipitation.

[0117] Variations of the above procedure will be understood to be within the scope of the present invention. For example, recombinant DNA technology can be used to isolate or modify DNA sequences encoding heavy chain and / or light chain variable domains, for example, as defined above herein, which encode the antibodies and / or antibody-antigen binding fragments disclosed herein. For example, recombinant DNA technology can be used to remove portions of the coding sequence that are not necessary to maintain specific and selective binding to the antigen of interest. Molecules expressed from such cleaved DNA molecules are also included in the antibodies of the present invention. Furthermore, bifunctional antibodies are also provided, comprising the heavy chain and / or light chain variable domains of the present invention (for example, forming an antibody Fv domain that specifically and selectively binds to core-fucosylated PSA or a core-fucosylated PSA sub-sequence) and the heavy chain and / or light chain variable domains of another antibody specific to an antigen other than core-fucosylated PSA.

[0118] Antibody derivatives can be produced, for example, by adding exogenous sequences to modify immunogenicity, or by reducing, enhancing, or modifying binding, affinity, on-rate, off-rate, binding activity, specificity, half-life, or any other suitable characteristics. Generally, some or all of a non-human or human CDR sequence is maintained while the non-human sequences in the variable and constant regions are substituted with human or other amino acids.

[0119] Humanized versions of the antibodies disclosed herein, i.e., those comprising heavy and / or light chain CDRs as disclosed above herein, are also provided. As is well known in the art, “humanization” (for producing a humanized version of a parent antibody) refers to the recombinant operation of an antibody using a CDR derived from a non-human donor immunoglobulin in the context of a human-derived framework and constant domain. During the operation, the framework and / or CDR residues may be modified to retain binding affinity and activity, for example, specificity to core-fucosylated PSA and / or its core-fucosylated subsequences, and (i) PSA and its subsequences lacking the α-1,6-core-fucose residue; (ii) discriminative activity to core-fucosylated glycans of formula IIIb. It is most preferable that the antibody and antigen-binding fragments be manipulated to retain specificity to glycopeptides of formula Ib, and discriminative binding activity to glycopeptides of formula IIb and core-fucosylated glycans of formula IIIb. Methods for humanizing antibodies are known in the art and are described, for example, in Queen et al., Proc. Natl. Acad Sci USA 86(1989), 10029-10032; and Hodgson et al., Bio / Technology 9(1991) 421.

[0120] 5.3 Characterization of Binding Activity The anti-1,6-fucPSA antibody and anti-1,6-fucPSA antibody-binding fragment of the present invention exhibit specific binding to core-fucosylated PSA and / or its core-fucosylated subsequence, the subsequence comprising and including SEQ ID NO: 18. The antibody and antigen-binding fragment also recognize (i) PSA lacking the α-1,6-core-fucose residue and its subsequence (including aglycosylated PSA and its aglycosylated subsequence), preferably from glycoproteins of formula Ib. In certain embodiments, the antibody and antigen-binding fragment also recognize core-fucosylated glycans of PSA, e.g., alone or in combination (e.g., core-fucosylated AFP) of formula IIIb. As used herein, the phrase "specifically binds" in the context of an antibody or antibody-antigen binding fragment that reacts with core-fucosylated PSA and / or its core-fucosylated subsequence (glycopeptide antigen) indicates that the glycopeptide binds to the antibody or antibody-antigen binding fragment via an antigen-antibody reaction. As also described herein, the term "identifies from / against" indicates that the antibody and antigen binding fragment of the present invention specifically binds to the target antigen (i.e., core-fucosylated PSA and / or its core-fucosylated subsequence, most preferably the glycopeptide of formula IB), but does not specifically bind to PSA / PSA subsequences lacking core-fucose residues, and / or PSA core-fucosylated glycans in other circumstances, such as a single core-fucosylated asparagine as shown in formula III, most preferably formula IIIb.

[0121] When used herein, the anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention binds specifically to the antigen, and its key-dissociation (KD) to the antigen is 30 nM or less, preferably 20 nM or less, and most preferably 15 nM or less. In the most preferred embodiment, the anti-1,6-fucPSA antibody or its antigen-binding fragment provided herein binds to the glycopeptide of formula Ib with a KD of 30 nM or less, preferably 20 nM or less, and most preferably 15 nM or less. In this most preferred embodiment, the binding of the glycopeptide of formula Ib in the glycan is even more preferably as shown in formula Ic. The anti-1,6-fucPSA antibody or antibody-antigen binding fragment of the present invention has a capacity of 1 × 10⁻¹⁶ 5 M -1 s -1 The above is fua2×10 5 M -1 s -1 In summary, the most preferred is 5 × 10 5 M -1 s -1 The above meeting rate (k a It is even more preferable that the antibody or antigen-binding fragment provided herein specifically binds to the target antigen. 5 M -1 s -1 The above is fua2×10 5 M -1 s -1 In summary, the most preferred is 5 × 10 5 M -1 s -1 The above meeting rate (k a It is most preferable that the glycopeptide of formula Ib is bound in the form shown in formula Ic. In these embodiments, the preferred binding of the glycopeptide of formula Ib in the glycan is shown in formula Ic.

[0122] As understood in the art, "KD" refers to the dissociation constant of an antibody-antigen interaction, which can be determined by any conventional means known to those skilled in the art, or as described herein. In a preferred embodiment, the KD of the binding interaction is determined by surface plasmon resonance (SPR) spectroscopy.

[0123] Using SPR, KD and / or ka An exemplary protocol for determining the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention is immobilized directly or indirectly on a solid phase (known as a "tip") using a capture reagent (i.e., a reagent capable of binding the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention to a solid phase) according to the manufacturer's instructions. The capture reagent can be immobilized to, for example, a level of 1,000 response units (RU). The appropriate capture reagent depends on the binding molecule being tested, e.g., full-length immunoglobulin or its antigen-binding fragment, and can be selected according to standard methods known in the art. Suitable capture reagents include polyclonal antibodies specific to the immunoglobulin constant domain common to all full-length immunoglobulins and / or their antigen-binding fragments being tested. The antibody or antibody-antigen-binding fragment to be tested can be diluted, for example, in a suitable buffer which may contain a blocking agent, and exposed to the capture reagent on the solid phase, i.e., captured on the solid phase. The captured antibody or antibody-antigen-binding fragment is then exposed to a target antigen in a suitable buffer and the signal response is recorded. Once saturated, exposure to the target antigen can be stopped, and signals for dissociation can be monitored. The signals can be analyzed using any software known in the art, but preferably using software that includes or supports a specific SPR instrument.

[0124] In non-limiting examples, SPR analysis can be performed by capturing the monoclonal antibody or antigen-binding fragment of the present invention onto any suitable surface (sensor) according to the manufacturer's instructions and recommendations. As is known in the art, exemplary sensors typically have a metal layer coated with a material that allows the molecule of interest to be covalently bonded to its surface. A non-limiting example is a CM5 sensor chip having a matrix of carboxymethylated dextran covalently bonded to a gold surface. The molecule can be covalently bonded to the sensor surface by utilizing available amine, thiol, aldehyde, or carboxyl functional groups on the ligand. The analysis may include using HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w / v Tween 20®) supplemented with 1 mg / ml carboxymethylated dextran as the equilibrium and / or running buffer. The analysis temperature may be room temperature or 37°C, preferably 37°C. D and / or k a Analysis of the data collected to determine antibody / fragment-antigen binding parameters such as those mentioned above is performed, for example, using a Langmuir fitting model, preferably R MAX This can be determined by any suitable method known in the art, including fitting local surface plasmon resonance data.

[0125] In non-limiting embodiments, SPR spectroscopy is performed using a Biacore® 8k instrument. In the exemplary protocol, the Biacore instrument is preferably operated at 37°C, and the mounted CM5 research-grade sensor is normalized with system buffer according to the manufacturer's instructions. The capture reagent (e.g., a polyclonal antibody specific to Fcγ of the antibody, or specific to a portion of the antibody fragment to be tested) in the sample buffer, e.g., system buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran), is pre-concentrated in a flow cell to a maximum of 10,000 RU. The anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment is diluted in the sample buffer and captured in an 8 sample flow cell at 5 μl / min for 2 minutes. For example, the target antigen, diluted in the sample buffer at a series of concentrations of 0 nM (buffer), 11 nM, 33 nM, 100 nM, 300 nM, and 900 nM, is injected at 30 μl / min for an association time of 3 minutes. A control can be established, for example, by injecting one concentration level twice. After the association phase, the maximum concentration level tested may be set to quantify the signal response at antibody-ligand saturation. Antibody-antigen dissociation can then be monitored for 5 minutes. A superposition plot of concentration-dependent antigen binding can be created, and the kinetic rates can be measured using software provided by the manufacturer, e.g., R MAX The SPR parameter is determined using Biacore 8k evaluation software with a local Langmuir fitting model. In a preferred embodiment, the SPR parameter is standardized such that the determined KD of an antibody, preferably a rabbit antibody, containing VH and VL of exemplary antibody 3B10 for the glycopeptide of formula Ib (having a glycan containing the preferred binding shown in formula Ic) (i.e., containing SEQ ID NOs. 61 and 62, respectively), is 11 nM + standard error of the particular assay.

[0126] The binding analysis to establish KD need not be performed on a native protein or a fragment thereof that maintains the native three-dimensional conformation. In a preferred embodiment, the polypeptide to be tested is reduced prior to analysis. Reducing conditions suitable for the proteins and polypeptides to be assayed subsequently using antibody / antigen binding fragments are well known in the art and further described herein and, by way of non-limiting example, include incubation in a buffer containing β-mercaptoethanol (2-ME), dithiothreitol (DTT), and Tris(2-carboxyethyl)phosphine (TCEP). In these preferred embodiments, the analysis of antigen binding involves assaying an antibody or antigen binding domain for binding to the glycopeptide of formula Ib, thereby establishing the criteria for specific binding.

[0127] 5.4 Diagnostic and pharmaceutical compositions As described above herein, the anti-1,6fucPSA antibodies and antibody antigen binding fragments specifically bind to core-fucosylated PSA and its core-fucosylated partial sequences, and in particular, discriminate against PSA or PSA partial sequences lacking core-fucosylated residues. Thus, the antibodies and antibody antigen binding fragments of the present invention are suitable for specific and discriminatory binding of core-fucosylated PSA to other variants of PSA, and in particular, enable specific detection (and thus discrimination) of core-fucosylated PSA / PSA partial sequences against other variants of PSA. In the most preferred embodiment, the antibodies and antibody antigen binding fragments specifically bind to and detect the glycopeptide of formula Ib.

[0128] Thus, the present invention further relates to a composition, for example, a diagnostic or pharmaceutical composition, comprising at least one of (i) an antibody or antibody antigen binding fragment of the present invention, (ii) a nucleic acid molecule of the present invention, (iii) a vector of the present invention, (iv) a host cell of the present invention, and / or (v) an antibody produced or obtained by the method of the present invention.

[0129] 5.4.1 Diagnostic compositions As described herein, the anti-1,6-fucPSA antibodies and antigen-binding fragments provided herein are useful for the detection of core-fucosylated PSA and its core-fucosylated subsequences, particularly for comparison with and thus identification of other variants of the PSA / PSA subsequence that may be present in a sample. In the most preferred embodiment, the anti-1,6-fucPSA antibodies and antigen-binding fragments of the present invention are useful for the detection of glycopeptides of formula Ib or glycoproteins containing glycopeptides of formula Ib. Those skilled in the art will be well aware of methods for determining whether a sample contains core-fucosylated PSA / PSA subsequences using the antibodies or antibody-antigen-binding fragments of the present invention. Non-limiting examples of suitable methods include in vivo assays in which the antibodies or antigen-binding fragments of the present invention are conjugated to suitable detectable reagents or portions such as radionuclides or contrast agents (e.g., for MRI or CT), as well as in vitro assays such as immunohistochemical and immunocytochemical methods, Western blotting, ELISA, and immunoassays based on the detection of luminescence, fluorescence, chemiluminescence, or electrochemiluminescence. In one embodiment, the determination of core-fucosylated PSA or a subsequence thereof is by immunohistochemistry, i.e., by detecting the binding of antibodies or antigen-binding fragments to / from the sample. The diagnostic method may include the use of appropriate controls to ensure the reliability of any positive or negative result. Appropriate positive and negative controls can be designed and included in the experimental setup by those skilled in the art using conventional methods and the teachings of this disclosure, for example, including a glycopeptide of formula I as a positive control and / or an aglycosylated PSA or PSA subsequence and / or a glycopeptide of formula II as a negative control.

[0130] Biological specimens in which core-fucosylated PSA / PSA subsequences are detected according to the methods disclosed herein include specimens or preparations derived from the subject. Specimens derived from the subject may be any specimen known, determined, or suspected to contain core-fucosylated PSA or core-fucosylated PSA subsequences, and may include, but are not limited to, blood specimens and body fluid specimens of the subject. Blood specimens may be whole blood, serum, or plasma. Body fluid specimens may be urine, semen, or ejaculated semen. The methods also encompass the analysis of specimens derived from the subject in which the presence of core-fucosylated PSA or core-fucosylated PSA subsequences is unknown and / or has been ruled out.

[0131] As used herein, preparations derived from a subject also include tissue preparations. In particular, the present invention provides methods and compositions for immunohistochemical analysis of such tissue preparations for tissue slides prepared according to standard methods known in the art. The sample for immunohistochemical analysis by the method of the present invention is preferably a formalin-fixed paraffin-embedded (FFPE) sample.

[0132] Samples for immunohistochemical analysis (e.g., FFPE samples) can be pre-treated before exposure to the antibody or antigen-binding fragment of the present invention. Such pre-treatments include epitope retrieval methods known in the art. Suitable methods of antigen retrieval include protease-induced epitope retrieval (PIER) and heat-induced epitope retrieval (HIER), which can be applied to retrieval. In the method of the present invention, it is preferable that the sample be subjected to antigen retrieval, including heat-induced epitope retrieval in the presence of a basic epitope retrieval solution suitable for HIER, having a pH in the range of approximately pH 8 to approximately pH 10. Exemplary types of basic epitope retrieval solutions include ethylenediaminetetraacetic acid ("EDTA")-based solutions, tris(hydroxymethyl)aminomethane ("Tris")-based solutions, EDTA / Tris-based solutions, and Tris-buffered saline-based solutions. Examples of commercially available basic epitope retrieval solutions include VENTANA cell conditioning solution 1 (CC1), a Tris-based solution at pH 8.5 (Roche); EnVision FLEX Target Retrieval, High pH (Agilent), a Tris / EDTA-based solution at pH 9; eBioscience® IHC Antigen Retrieval Solution-High pH (ThermoFisher), a Tris / EDTA-based solution at pH 9; BOND Epitope Retrieval Solution 2 (Leica Biosystems), an EDTA-based solution at pH 8.9-9.1; BOND Novocastra® Epitope Retrieval Solution pH 8, an EDTA-based solution at pH 8; and BOND Novocastra® Epitope Retrieval Solution pH 9, a Tris / EDTA-based solution at pH 9. In preferred embodiments, the epitope retrieval solution is a Tris-based solution. Following any (but preferred) pretreatment, the tissue sample is reacted with the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention. The reaction is carried out under conditions suitable for the recognition of epitopes in the antigen and the subsequent formation of antigen-antibody complexes.The reaction conditions can be appropriately modified within a range suitable for the recognition of the epitope in the antigen by the antibody and the subsequent formation of an antigen-antibody complex, but these are conventional modifications well known in the art. The immunohistochemical method may include the use of the antibody or antigen-binding fragment of the present invention directly conjugated to a labeled material that enables visualization by standard methods known in the art, or it may include the use of a secondary antibody that recognizes the antibody or antigen-binding fragment of the present invention, and this secondary antibody can be visualized in that manner.

[0133] The present invention also provides a method for detecting and identifying core-fucosylated PSA and its core-fucosylated subsequences in a sample, including the use of ELISA-based methods known in the art. Samples for ELISA analysis can be pre-treated before exposure to the antibody or antigen-binding fragment of the present invention. It is preferable that the sample be subjected to reducing conditions prior to ELISA analysis so that the core-fucosylated PSA / PSA subsequences (if present) in the sample are reduced and / or linearized. Suitable reducing conditions for proteins and polypeptides subsequently assayed with antibody / antigen-binding fragments are well known in the art and, in non-limiting examples, include incubation in a buffer containing β-mercaptoethanol (2-ME), dithiothreitol (DTT), and Tris(2-carboxyethyl)phosphine (TCEP). The pre-treatment buffer for reducing the sample may also contain other agents conventionally present in such buffers, such as chelating agents to reduce or inhibit potential protease activity in the sample. In the ELISA method of the present invention, it is preferable to pre-treat the sample with Tris buffer containing TCEP and EDTA before exposure to the antibody or antigen-binding fragment of the present invention. Non-limiting examples of such pre-treatment buffers include 100 mM Tris (pH 12.9), 30.6 mM TCEP, and 2 mM EDTA.

[0134] In some embodiments, the antibodies and antigen-binding fragments of the present invention are used as primary antibodies in the immunohistochemical (IHC) or immunocytochemical (ICC) methods described above. IHC and ICC methods typically involve staining an antibody-reactive biomarker (i.e., a target antigen) in a tissue section (e.g., from fresh, frozen, or formalin-fixed paraffin-embedded (FFPE) sample) or cell specimen (e.g., a smear, liquid-based cytology (LBC) sample, or fine-needle aspiration cytology (FNA)) described herein by applying one or more antibodies or antigen-binding fragments of the present invention in combination with a suitable set of detection reagents to produce biomarker-stained sections. The sample is brought into contact with the primary antibody (i.e., the antibody or antigen-binding fragment of the present invention) under conditions that promote specific binding of the primary antibody to the biomarker / target antigen (i.e., core-fucosylated PSA or core-fucosylated PSA subsequence). The primary antibody bound to the sample promotes the deposition of a detectable portion adjacent to the biomarker, thereby generating a detectable signal localized to the biomarker.

[0135] The terms “detectable portion,” “detectable reagent,” and similar terms include any type of molecule that can be used to identify a region of a sample to which the antibody or antigen-binding fragment of the present invention has bound when used as a primary antibody. Exemplary detectable portions include chromogenic, fluorescent, phosphorescent, and luminescent molecules and materials, as well as mass tags (disclosed, e.g., in Levenson et al., Lab Invest 95 (2015), 397-405). In some examples, the detectable portion is a fluorophore belonging to several common chemical classes, including coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorphine, luminophores, and cyanines. Further examples of fluorescent molecules can be found, for example, in Molecular Probes Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher Scientific, 11th edition. In other embodiments, the detectable portion is a pigment or colored precipitate containing diaminobenzidine (DAB), 4-(dimethylamino)azobenzene-4'-sulfonamide (DABSYL), tetramethylrhodamine (DISCOVERY Purple), N,N'-biscarboxypentyl-5,5'-disulfonato-indodicarbocyanine (Cy5), and rhodamine 110 (Rhodamine). In other embodiments, the detectable portion is the result of a metallographic detection scheme. The metallographic detection method involves using an enzyme such as alkaline phosphatase in combination with a water-soluble metal ion and a substrate inert to the redox reaction of the enzyme. In some embodiments, the substrate is converted into a redox activator by an enzyme, which reduces the metal ions and forms a detectable precipitate (disclosed, for example, in U.S. Patent Application No. 2005 / 0100976, PCT Publication No. 2005 / 003777, and U.S. Patent Application Publication No. 2004 / 0265922).Metallographic detection methods include using an oxidoreductase (such as horseradish peroxidase) together with a water-soluble metal ion, an oxidizing agent, and a reducing agent to form a detectable precipitate (for example, disclosed in U.S. Patent No. 6,670,113).

[0136] IHC and ICC staining methods can generally be divided into “direct” and “indirect” methods. In direct methods, the primary antibody (i.e., the anti-1,6-fucPSA antibody or antigen-binding fragment of the present invention) is labeled to be detectable. In indirect methods, the detectable portion is localized to the primary antibody by another agent that binds to the primary antibody. An exemplary indirect method includes a method that uses an antibody that is at least specific to the primary antibody (called a secondary antibody) to localize the detectable portion to the primary antibody. In certain embodiments, indirect methods are used to localize a detectable portion to the primary antibody by a method selected from the following: (a) a secondary antibody is bound to the primary antibody, and the secondary antibody is detectably labeled; (b) a secondary antibody is bound to the primary antibody and to an antibody specific to the secondary antibody (referred to as a tertiary antibody), and the tertiary antibody, or both the secondary and tertiary antibodies, are detectably labeled; (c) an epitope-tagged secondary antibody (such as a hapten-tagged secondary antibody) is bound to the primary antibody, and a tertiary antibody that is detectably labeled to the epitope tag is bound to the secondary antibody; (d) an enzyme-conjugated secondary antibody is bound to the primary antibody, and the signal transduction conjugate reacts with the enzyme, and the signal transduction conjugate reacts with the epitope tag (hapten tag). (e) a hapten and a potentially reactive moiety, wherein the enzyme catalyzes the transformation of the potentially reactive moiety into a reactive species that binds to the sample, and the tertiary antibody binds to the epitope tag of the signaling conjugate that binds to the sample, and the enzymes for the secondary and tertiary antibodies are the same; and (e) a secondary antibody is conjugated to a primary antibody, the secondary antibody is conjugated to an epitope tag (hapten, etc.), the enzyme conjugates the tertiary antibody to the epitope tag, the sample is brought into contact with a signaling conjugate comprising an epitope tag (hapten, etc.) and a potentially reactive moiety, under conditions in which the enzyme catalyzes the transformation of the potentially reactive moiety into a form that binds to the sample, and an additional tertiary antibody binds to the epitope tag of the signaling conjugate that binds to the sample. Exemplary “potentially reactive moieties” discussed herein include quinone methide (QM) analogs, e.g., those described in International Publication No. 2015124703A1, and tyramide conjugates, e.g., those described in International Publication No. 2012003476A2.When used in this context, the term “detectably labeled antibody” refers to an antibody that has been directly conjugated to a detectable portion or an enzyme capable of producing a detectable portion (e.g., in a metallurgical or chromogenic detection scheme). In this context, it is understood that once an antibody is conjugated to an enzyme, the enzyme can then react with additional reagents to result in the deposition of a detectable portion on a sample (e.g., by a chromogenic or metallurgical detection scheme).

[0137] In some embodiments, the IHC or ICC method is performed in an automated staining system. An automated IHC / ISH slide staining system typically includes at least: reservoirs for the various reagents used in the staining protocol; a reagent dispensing unit for dispensing reagents onto the slides, which is in fluid communication with the reservoirs; a waste removal system for removing used reagents and other waste from the slides; and a control system that coordinates the operation of the reagent dispensing unit and the waste removal system. In addition to performing the staining step, many automated slide staining systems can also perform (or are compatible with) other processes associated with staining, such as slide baking (to adhere the sample to the slide), degreasing (also known as deparaffinization), antigen recovery, counterstaining, dehydration and clearing, and coverslip coating. Prichard, Arch Pathol Lab Med., 138(2014), 1578-1582, describes several specific examples and various functions of automated IHC / ISH slide staining systems, including intelliPATH (Biocare Medical), WAVE (Celerus Diagnostics), DAKO OMNIS and DAKO AUTOSTAINER LINK 48 (Agilent Technologies), BENCHMARK (Ventana Medical Systems, Inc.), Leica BOND, and Lab Vision Autostainer (Thermo Scientific). This list of staining platforms is not intended to be exhaustive, and fully or semi-automated systems can be used to perform biomarker staining.Non-limiting examples of commercially available detection reagents or kits suitable for use in automated IHC and ISH methods include: VENTANA ultraView detection system (secondary antibody conjugated to an enzyme, including HRP and AP), VENTANA iVIEW detection system (biotinylated anti-isotype secondary antibody and streptavidin-conjugating enzyme), VENTANA OptiView detection system (anti-isotype secondary antibody conjugated to a hapten and anti-hapten tertiary antibody conjugated to an enzyme polymer), VENTANA Amplification kit (unconjugated secondary antibody that can be used with any of the above VENTANA detection systems to amplify the number of deposited enzymes at the primary antibody binding site), VENTANA OptiView Amplification systems (including hapten-conjugated anti-isotype secondary antibodies, enzyme-multimer-conjugated anti-hapten tertiary antibodies, and tyramide conjugated to the same hapten. During use, the secondary antibody is brought into contact with the sample to induce binding to the primary antibody. The sample is then incubated with the anti-hapten antibody to induce association between the enzyme and the secondary antibody. Next, the sample is incubated with tyramide to induce attachment of additional hapten molecules. Finally, the sample is incubated again with the anti-hapten antibody to induce attachment of additional enzyme molecules. Finally, the sample is incubated with the detectable portion to induce dye attachment); VENTANA DISCOVERY, DISCOVERY OmniMap, DISCOVERY UltraMap anti-hapten antibodies, secondary antibodies, dyes, fluorophores, and dye kits (these are available from Ventana Medical Systems, Inc. (Tucson, Arizona)); PowerVision and PowerVision+IHC Detection Systems (a compact polymer obtained by directly polymerizing a secondary antibody with HRP or AP, resulting in a high enzyme-to-antibody ratio); and DAKO EnVision™ system (an enzyme-labeled polymer conjugated to a secondary antibody).

[0138] If desired, IHC or ICC stained slides may be counterstained. Examples of counterstains include colorimetric nuclear counterstains such as hematoxylin (stains blue to purple), methylene blue (stains blue), toluidine blue (stains the nucleus dark blue and polysaccharides pink to red), nuclea first red (also known as Kernectrot, stains red), and methyl green (stains green); eosin (4',6-diamino-2-phenylindole (DAPI, stains blue), propidium iodide (stains red), Hoechst stain (stains blue), Nuclear Green DCS1 (stains green), Nuclear Yellow (Hoechst This includes chromogenic nonnuclear stains such as S769121 (stains yellow at neutral pH and blue at acidic pH), DRAQ5 (stains red), and DRAQ7 (stains red); and fluorescent nonnuclear stains such as fluorophore-labeled phalloidin (stains filamentous actin, with color dependent on the conjugated fluorophore).

[0139] Using the methods described herein, it is possible to determine whether a subject has or is at risk of developing prostate cancer by, for example, determining the level of core-fucosylated PSA or a core-fucosylated PSA sub-sequence relative to the total amount of PSA in a subject using the antibody and antigen-binding fragment of the present invention. Thus, in certain embodiments, the disclosure also provides a method for determining whether a subject who has or is at risk of developing prostate cancer, as discussed herein and known in the art, is a candidate for anticancer therapy or treatment. Specifically, such a method may include (a) determining the concentration or amount of core-fucosylated PSA or a core-fucosylated PSA sub-sequence in a test sample from a subject, in particular, relative to the total amount of other types of PSA, using the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention by the method described herein or by a method known in the art; and (b) comparing the concentration or amount determined in step (a) to a predetermined level (or range of levels) determined in a subject known not to have cancer. If the concentration or amount determined in step (a) is within a predetermined range, the subject is determined to be cancer-free or at risk thereof, as discussed herein and known in the art. However, if the concentration or amount determined in step (a) is outside the range, in particular below a predetermined level, the subject is determined to be prostate cancer-free or at risk thereof, as discussed herein and known in the art.

[0140] The methods described herein can also be used to monitor the progression of prostate cancer, for example, in response to therapy, by monitoring the concentration or amount of core-fucosylated PSA and its core-fucosylated subsequences in test samples from a subject over time. Such a method may include (a) determining the concentration or amount of core-fucosylated PSA or core-fucosylated PSA subsequences in a test sample from a subject, particularly compared to the total amount of other types of PSA, using the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention by the method described herein or by a method known in the art; (b) determining the concentration or amount of core-fucosylated PSA or core-fucosylated PSA subsequences (particularly compared to the total amount of other types of PSA) in subsequent samples by the same method as in (a); and (c) comparing the concentration or amount determined in step (b) with the concentration or amount determined in step (a). If the relative concentration or amount determined in step (b) has not changed or has not changed significantly compared to the relative concentration or amount determined in step (a), the disease in the subject is determined to be persistent, progressing, or worsening, and / or the therapy is determined to be ineffective. If the relative concentration or amount determined in step (b) has increased compared to the relative concentration or amount determined in step (a), the disease in the subject is determined to be regressing or improving, and / or the therapy is determined to be effective. Accordingly, a method is also provided for monitoring or evaluating the effectiveness of prostate cancer therapy, which includes determining the change over time (e.g., before, during, and / or after therapy) of the level of core-fucosylated PSA or core-fucosylated PSA subsequence relative to the total level of all other PSA species.

[0141] 5.4.2 Pharmaceutical composition The anti-1,6-fucPSA antibody and antibody-antigen binding domain of the present invention, as well as methods for producing and using them, are intended not only to be provided as diagnostic tools, but also to have applicability in the treatment and improvement of diseases and disease symptoms, and applicability in model systems for investigating disease therapies. Accordingly, the present invention provides one or more pharmaceutically acceptable carriers, and a pharmaceutical composition comprising (i) an anti-1,6-fucPSA antibody and / or its antigen-binding fragment; (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).

[0142] The pharmaceutical compositions disclosed herein are formulated for administration to human or animal subjects. In the manufacture of the pharmaceutical formulation, the antibody or antigen-binding fragment of the present invention is mixed with a pharmaceutically acceptable carrier, excipient and / or diluent. The carrier, excipient and / or diluent must, of course, be acceptable in the sense that it is compatible with any other components in the formulation and must not be harmful to the subject. Examples of pharmaceutical carriers suitable for use with antibody-based compositions are well known in the art and can be formulated by conventional methods.

[0143] 5.5 Kit The present invention also provides a kit comprising any manufactured product (e.g., a package or container) comprising at least one reagent of the present invention, namely (i) an antibody or antibody-antigen binding fragment of the present invention, (ii) a nucleic acid molecule of the present invention, (iii) a vector of the present invention, (iv) a host cell of the present invention, and / or (v) an antibody or antibody-antigen binding fragment produced or obtained by the method of the present invention. The kit may be encouraged, distributed or sold as a unit for carrying out the method of the present invention.

[0144] The anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment disclosed herein can be used in core-fucosylated PSA detection kits. Such detection kits may include a capture reagent, a detection reagent, and / or a solid phase. The anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention may or may not be conjugated to a conjugate, or otherwise to a solid phase (e.g., magnetic microbeads). Furthermore, the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention may or may not be detectably labeled. The kit may optionally further include a second PSA-specific antibody that does not compete for binding to core-fucosylated PSA / core-fucosylated PSA subsequences having the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention. Preferably, the second antibody binds PSA in a glycosylation-independent manner. The anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody antigen-binding fragment of the present invention can be used as a capture reagent or a detection reagent. The detection reagent is preferably labeled. In such cases, the kit may further include a substrate and / or reagent that enables the detection of the label.

[0145] The detection kit may also include a pretreatment reagent suitable for the reduction and / or linearization of PSA / PSA fragments in the sample being tested. Suitable reduction pretreatment reagents include buffers containing β-mercaptoethanol (2-ME), dithiothreitol (DTT), Tris(2-carboxyethyl)phosphine (TCEP), and a chelating agent, such as EDTA. Non-limiting examples of such pretreatment buffers that may be included in the kit are 100 mM Tris (pH 12.9), 30.6 mM TCEP, and 2 mM EDTA.

[0146] The detection kit may further contain a pretreatment solution of a biological sample as a positive or negative control (e.g., a solution containing the glycopeptide of formula (I) and / or the glycopeptide of formula (II), respectively), a washing solution, and / or a calibration standard.

[0147] In non-limiting embodiments, the kit may include (a) the anti-1,6-fucPSA antibody or anti-1,6-fucPSA antibody-antigen conjugate fragment of the present invention in a ready-to-use format (optionally in a reagent container or dispenser for use in an automated immunohistochemistry / in-situ hybridization platform); or (b) optionally in a concentrated or solid format (e.g., powder, lyophilized, or crystalline form) combined with a diluent for reconstituting and / or diluting the antibody to a test concentration. The kit may further include any antibody / antibody-antigen conjugate fragment pre-packaged in a ready-to-use dispenser, such as those disclosed in U.S. Patents No. 7,378,058, 6,192,945, 6,416,713, 6, 45,759, 8,147,773, 9,341,641, 10,330,693 and 8,932,543. Other systems for dispensing readily usable antibodies are described, for example, in U.S. Patent Nos. 8,758,707 and 10,228,382.

[0148] The above detailed description of the present invention discloses several individual elements, features, techniques, and / or processes. It is readily apparent that each of these has benefits not only when considered or used individually, but also when considered and used in combination with one another. Therefore, in order to avoid excessively repetitive and redundant sections, this description avoids repeating all possible combinations and substitutions. Nevertheless, it is understood that such combinations, whether explicitly enumerated or not, are entirely within the scope of the subject matter of this disclosure.

[0149] Unless otherwise defined, all technical and scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art. References to technologies used herein are intended to refer to technologies commonly understood in the art, including variations or substitutions of equivalent technologies that would be obvious to those skilled in the art.

[0150] All amino acid sequences provided herein begin with the most N-terminal residue and end with the most C-terminal residue (N→C), as is customary in the art, and the one- or three-letter code abbreviations used to identify amino acids throughout the invention correspond to those commonly used for amino acids.

[0151] This specification references numerous documents, including patent applications and manufacturers' manuals. While the disclosures of these documents are not considered relevant to the patentability of the present invention, their entirety is incorporated by reference with this specification. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference. [Examples]

[0152] 6.1 Example 1: Generation of antibodies specific to fucosylated PSA Prostate-specific antigen (PSA) is a 28-32 kDa glycoprotein with 237 amino acids (SEQ ID NO: 21) and has a single N-glycosylation site in asparagine corresponding to Asn-69 Uniprot ID P07288 (SEQ ID NO: 21). The N-glycosylation includes α-1,6 core fucose residues, as schematically shown in Figure 1. Antibodies specific to core-fucosylated PSA ("fucPSA") have an octase containing α-1,6 core fucose, and are Asn-69 The compounds were produced by rabbit immunization with a glycopeptide consisting of amino acids 67-79 of PSA (SEQ ID NO: 18) including N-glycosylation (glycopeptide of formula Ib, specifically having a glycan with the linkage shown in formula Ic, and referred to herein as "PSA(67-79)-G0F"), followed by (i) positive selection with the same glycopeptide, i.e., PSA(67-79)-G0F; and (ii) negative selection with the same peptide sequence (SEQ ID NO: 18) containing a nonasaccharide but lacking α-1,6-core-fucose (glycopeptide of formula IIb, specifically having a glycan with the linkage shown in formula IIc, and referred to herein as "PSA(67-79)-G2"), and an octasaccharide with α-1,6-core-fucose (glycan without the PSA peptide, representing fucosylated asparagine of formula IIIb, specifically having a glycan with the linkage shown in formula IIIc). PSA(67-79)-G0F, PSA(67-79)-G2, and fucosylated asparagine of formula III are schematically shown in Figures 2A, 2B, and 2C, respectively. The three drugs used for screening (one positive and two negative) are hereafter referred to as screening agents.

[0153] 6.1.1 Synthesis of peptide immunogens and screening reagents 6.1.1.1 Peptides (PSA and AFP) containing complex glycans (G0F, G2) The peptides were synthesized following conventional procedures as disclosed in Seifert and Unverzagt, Tetrahedron Lett 38 (1997), 7857-7860. In particular, the peptides were synthesized by fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis using a peptide synthesizer (e.g., Protein Technologies, Inc.). Five equivalents of each amino acid derivative were used for amino acid coupling. The amino acid derivatives were dissolved in dimethylformamide containing one equivalent of 1-hydroxy-7-azabenzotriazole (HOAt). The peptides were synthesized on Sieber Amide resin. The coupling reaction was carried out for 5 minutes in dimethylformamide containing 5 equivalents of HATU and 10 equivalents of N,N-diisopropylethylamine relative to the filling resin. The Fmoc group was cleaved for 8 minutes after each synthesis step using 20% ​​piperidine in dimethylformamide. Lysine-serine dipeptide was used as the Fmoc-protected pseudoproline derivative. For the glycopeptides (immunogens) used in the KLH conjugate, an 8-amino-3,6-dioxaoctanoic acid linker and a cysteine ​​residue were incorporated into the C-terminus during solid-phase peptide synthesis. For the biotinylated peptides used in screening, a glutamic acid derivative with a PEG3-biotin side chain was attached to the N-terminus during solid-phase peptide synthesis. Peptide assembly by resin cleavage of Asp(ODmab) was achieved by washing the resin with 2% hydrazine in N,N-dimethylformamide (DMF) for 5 × 5 minutes, followed by treatment with 5 mM NaOH in water / MeOH (1:1) for 1 hour. Peptide release from the synthetic resin was achieved by incubation with 1% TFA in DCM for 10 × 3 minutes. Subsequently, the reaction solution was extracted with water and evaporated to dryness. The crude material was purified by flash chromatography. The identity of the purified material was analyzed by ion spray mass spectrometry.

[0154] Glycosyl azides (PSA(67-79)-G0F-azide and PSA(67-79)-G2-azide) were synthesized as described in J. Seifert, C. Unverzagt, Tetrahedron Lett. 38 (1997), 7857-7860. Each glycosyl azide was reduced to the corresponding amine by adding 1,3-propanedithiol (40 equivalents) and N,N-diisopropylethylamine (DIPEA) (30 equivalents) in MeOH. After stirring for 4 hours, the glycanamines were precipitated by adding cold diisopropyl ether. Glycan coupling to the peptide was achieved overnight using 2 equivalents of each glycanamine (PSA(67-79)-G0F amine or PSA(67-79)-G2 amine), 2 equivalents of HATU, 2 equivalents of HOAt, and 8 equivalents of DIPEA in DMF / DMSO (1:1). Subsequently, cleavage of the acid-instability protecting group was achieved at room temperature over 2 hours using 9.5 ml of trifluoroacetic acid, 0.25 ml of triisopropylsilane, and 0.25 ml of water. The reaction solution was then mixed with cold diisopropyl ether to precipitate the peptide. The precipitate was filtered, washed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid solution, and lyophilized. The crude material was purified by preparative RP-HPLC using an acetonitrile / 0.1% trifluoroacetic acid-containing water gradient. The identity of the purified material was analyzed by ion spray mass spectrometry.

[0155] 6.1.1.2 Complex glycoamino acids (biotinylated Asn with GF, also called Asn-GF) Asp-OBzl, biotin-PEG12-NHS ester (1 equivalent), and trimethylamine (8 equivalents) were dissolved in DMF and stirred for 2.5 hours. The crude product was purified by preparative RP-HPLC using an acetonitrile / 0.1% trifluoroacetic acid-containing water gradient.

[0156] Asn-G0F-azide was reduced to the corresponding amine by adding 1,3-propanedithiol (40 equivalents) and DIPEA (30 equivalents) in MeOH. After stirring for 4 hours, the glycan was precipitated by adding cold diisopropyl ether. Glycan coupling to biotin-PEG12-Asp-OBzl was achieved overnight using 0.5 equivalents of sugar amine, 1 equivalent of HATU, 1 equivalent of HOAt, and 4 equivalents of DIPEA in DMF / DMSO (1:1). The reaction solution was then mixed with cold diisopropyl ether. The precipitate was filtered, washed again with cold diisopropyl ether, dissolved in a small amount of aqueous acetic acid, and lyophilized. The crude material was purified by preparative RP-HPLC using an acetonitrile / 0.1% trifluoroacetic acid-containing water gradient. The identity of the purified material was analyzed by ion spray mass spectrometry.

[0157] 6.1.1.3 Peptides containing monosaccharides and disaccharides Peptides containing monosaccharides and disaccharides (GlcNAc and Fuc-GlcNAc) were synthesized on Tentagel resin according to the protocol described above. Fmoc-protected sugar amino acids (1.2 equivalents) were coupled for 1 hour in dimethylformamide containing 1.2 equivalents of HATU, 1.2 equivalents of HOAt, and 10 equivalents of N,N-diisopropylethylamine relative to the filling resin. After Tfa cleavage, the peptides were dissolved in methanol, and sodium methanolate was added dropwise until the pH reached 10. The solution was stirred for 4 hours, followed by neutralization with acetic acid. After removing the solvent, the peptides were purified by preparative RP-HPLC using an acetonitrile / 0.1% trifluoroacetic acid-containing water gradient. The identity of the purified material was analyzed by ion spray mass spectrometry.

[0158] 6.1.1.4 Synthesis of immunogens N-hydroxysuccinimide 3-(maleimide)propionic acid was added to a solution of KLH in phosphate buffer (20 mM, pH 7.2). The reaction was incubated at room temperature for 5 hours and then dialyzed against phosphate buffer (0.1 M, pH 7.0). A cysteine-containing glycopeptide was dissolved in DMSO and added to a solution of maleimide-activated KLH containing 0.1 M EDTA. The solution was incubated at room temperature for 5 hours and then dialyzed against phosphate buffer (0.1 M, pH 7.0) to obtain a KLH-peptide conjugate.

[0159] 6.1.2 immunization 12-16 week old New Zealand White (NZW) rabbits were immunized with PSA(67-79)-G0F (Figure 2). To enhance the immunogenicity of the peptide, it was conjugated to keyhole limpet hemocyanin (KLH) as a carrier protein. The immunogen used was PSA(67-79)-G0F-KLH. During the first month, animals were immunized weekly. From the second month onward, the immunization schedule was reduced to once a month. For the initial immunization, 500 μg of KLH-conjugated peptide was dissolved in 0.9% NaCl and emulsified in 2 ml of complete Freund's adjuvant (CFA). For all subsequent immunizations, CFA was replaced with 1 ml of incomplete Freund's adjuvant (IFA) emulsion.

[0160] 6.1.3 Potency analysis Titer analysis was performed using the ELISA protocol. Serum titration was performed using biotinylated PSA(67-79)-G0F as the positive control and biotinylated PSA(67-79)-G2 as the negative control.

[0161] Biotinylated screening peptides were immobilized on the surface of 96-well streptavidin-coated microtiter plates by incubation of 100 μl of 16 ng / ml solution per well at room temperature for 60 minutes. Subsequent washing was performed using an automated system (Biotek) according to the manufacturer's instructions. Small amounts of serum (2–3 ml per animal) were collected from each rabbit at days 35 and 165 after the start of the immunization program. Serum from each rabbit was diluted in PBS containing 1% BSA at ratios of 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 plates pre-prepared with the screening peptides and incubated at room temperature for 60 minutes. The bound antibodies were detected using HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche). The titer of the analyzed animals was set to the 50% signal decrease on the dilution curve.

[0162] (Table 1) Exemplary titers after immunization with PSA(67-79)-G0F TIFF0007901069000019.tif31128

[0163] As demonstrated by the results in Table 1, polyclonal serum from immunized animals bound to the PSA(67-79)-G0F screening peptide. Therefore, all animals were suitable for subsequent antibody development.

[0164] 6.1.4 B cell cloning To enrich antigen-reactive B cells, 100 ng / ml of biotinylated PSA(67-79)-G0F was pre-incubated with a pool of peripheral blood mononuclear cells (PBMCs) derived from immunized animals at 4°C for 15 minutes. After washing, antigen-reactive B cells bound to biotinylated PSA(67-79)-G0F were incubated with streptavidin-coated beads (Miltenyi) at 4°C for 15 minutes. Selection of positive B cells using a MACS column (Miltenyi) and subsequent incubation were performed as described in Seeber et al., PLoS One 9 (2014), issue 2, e86184, except that the MACS column (Miltenyi) was involved in the selection of positive B cells instead of plate binding.

[0165] Next, B cells expressing antibodies with the desired binding properties, i.e., antibodies that bind to the PSA(67-79)-G0F peptide and can distinguish between PSA(67-79)-G2 and Asn-G0F, were identified using Hit-ELISA (i.e., an ELISA that tests binding to the screening agent). The biotinylated screening agents PSA(67-79)-G0F, PSA(67-79)-G2, and Asn-G0F were immobilized on the surface of streptavidin-coated 96-well plates (Nunc) by incubating 100 μl of 100 ng / ml solution per well at room temperature for 60 minutes. The plates were washed, and 30 μl of rabbit B cell culture supernatant was transferred to each well and incubated at room temperature for 1 hour. For detection of antibodies bound to the screening agent, HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS substrate solution (Roche) were used according to the manufacturer's instructions. Sixteen clones were identified from the negative screening agent after binding to PSA(67-79)-G0F, according to selected cutoffs (OD greater than 0.6 for positive screening and less than 0.6 for negative screening). These 16 clones were selected for subsequent molecular cloning and recombinant expression, as described in Seeber et al., PLoS One 9 (2014), issue 2, e86184. For 15 of the 16 clones, the sequences were successfully cloned, and the sequences encoding VH and VL could be clearly determined. Subsequently, Hit-ELISA using the screening agent was repeated with the supernatant of recombinant expression, and stricter cutoff criteria (OD greater than 1 for positive screening agents and OD lower than the mean background signal for negative screening agents) were met for all 15 previously selected clones from which explicit VH and VL sequences were recovered.

[0166] 6.2 Example 2: Production of an antibody that is more selective to fucosylated PSA than to non-fucosylated PSA The VH and VL coding sequences were successfully determined, and recombinant monoclonal antibodies from 15 clones that met the screening criteria identified in Example 1 were further investigated for their kinetic kinetics and antigen-binding specificity to fucosylated and non-fucosylated PSA-derived peptides.

[0167] A Series S CM5 research-grade sensor was mounted on a Biacore 8k instrument (GE Healthcare) and normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w / v Tween 20) according to the manufacturer's instructions. The sample dilution buffer was the system buffer, i.e., HBS-ET buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, SIGMA). The system was operated at 37°C.

[0168] Polyclonal goat anti-rabbit IgG (GARb-Fcγ, Jackson Laboratories) conjugated to Fcγ fragments (30 μg / ml) in 10 mM sodium acetate buffer (pH 4.5) was pre-concentrated in all 16 flow cells, and up to 10,000 RU of GARb-Fcγ were immobilized using EDC / NHS chemistry according to the manufacturer's instructions. Rabbit IgG antibody (150 kDa) containing primary rabbit B cell culture supernatant was diluted 3-fold with sample buffer and captured on 8 sample flow cells at a flow rate of 5 μl / min for 2 minutes. The antibody capture level (CL) was quantified in response units (RU) for each antibody.

[0169] The non-fucosylated peptide analytes PSA(67-79)-G2 (3.7 kDa) and the fucosylated peptide analytes PSA(67-79)-G0F (3.9 kDa) were diluted in sample buffer at a series of concentrations: 0 nM (buffer), 11 nM, 33 nM, 100 nM, 300 nM, and 900 nM. Two injections of the 100 nM analyte sample were used as a control. The analytes were injected at a rate of 30 μl / min for a 3-minute association time. After the association phase of the 900 nM analyte injection, a report point (binding rate) was set, and the signal response at antibody-ligand saturation was quantified. Antibody-antigen dissociation was monitored for 5 minutes. The GARb-Fcγ antibody capture system was regenerated by injecting 10 mM glycine pH 2 at 30 μl / min for 1 minute, followed by two consecutive injections of 10 mM glycine pH 2.25 at 30 μl / min for 1 minute each. A concentration-dependent antigen binding superposition plot was created, and the kinetic rate was measured using R. MAX The half-life of the antibody / antigen complex was determined using Biacore 8k evaluation software with a local Langmuir fitting model. The half-life was calculated using formula t 1 / 2 diss =ln(2) / 60*k d The calculation was performed in minutes according to the instructions.

[0170] The molar ratio (MR) representing the antibody-antigen binding stoichiometry was also determined and calculated as MR = (Analyte Binding Rate (RU) / Antibody Capture Level (RU)) × (MW (Antibody) / MW (Analyte)).

[0171] The results of Biacore analysis of 15 antibodies are reported in Table 2 below. From the 15 antibodies tested, six sets (shown in bold and non-italicized text) were selected based on the following preferred characteristics: - Specific kinetic interactions with fucosylated antigen PSA(67-79)-G0F; - No detectable cross-reactivity with non-fucosylated antigen PSA(67-79)-G2 (see Figure 3); - Functional 1:1 or 1:2 antigen-binding stoichiometric ratios; and - Faster meeting rate k a (1 / Ms), and slow dissociation rate kd (1 / s).

[0172] (Table 2) Dynamical data of antibody binding activity of 15 clones identified in Example 1 against PSA(67-79)-G0F. Selected antibodies (13C5, 2C11, 2H9, 2E9, 3H6, 3B10) are highlighted in bold, and non-selected antibodies are highlighted in bold and italics (13E12, 15F10). TIFF0007901069000020.tif168170

[0173] Figure 3 shows sensorgram data for the binding of the above selected and unselected antibodies to PSA(67-79)-G0F; AB(13C5), CD(2C11), EF(2H9), GH(2E9), IJ(3H6), KL(3B10), MN(13E12), OP(15F10). Figure 3 also shows the binding of antibodies to PSA(67-79)-G2, the same PSA fragment but with glycosylation lacking core-fucose residues. Panels B, D, F, H, J, L, and N exemplify that antibodies 13C5, 2C11, 2H9, 2E9, 3H6, and 3B10, respectively, did not show detectable interaction / cross-reactivity with the non-core-fucosylated PSA(67-79)-G2 peptide, or that binding was below the detection limit, i.e., in the micromolar range.

[0174] As demonstrated, the immunization procedure yielded at least six individual IgG clones that specifically reacted with the α-1,6-core-fucosylated PSA-specific glycopeptide (PSA(67-79)-G0F) but not with the core-fucose residue-free glycopeptide (PSA(67-79)-G2). In particular, non-binding to Asn-G0F was also screened in the HIT-ELISA of Example 2 above.

[0175] Figure 13 shows the amino acid sequence alignments of the VH and VL regions of the six selected antibodies. Surprisingly, these alignments revealed that all six antibodies share remarkably high sequence similarity in the VH and VL regions, specifically in the CDR sequence.

[0176] In parallel, SPR analysis was performed using native PSA isolated from semen (approximately 80% 1,6-fucPSA fraction by MS-analytics; Scripps Laboratories). Interestingly, none of the selected antibodies reacted with the native protein, suggesting affinity for the linear epitopes of PSA and / or glycopeptides used for immunization. This suggests that denaturation and / or reduction of native PSA may be necessary for good recognition of the glycostructure epitopes in the context of native PSA using the selected antibodies. Indeed, in this regard, the selected antibodies were demonstrated to be reactive to and therefore able to identify native PSA when used in SDS-PAGE Western blot analysis. See Figure 4A. In contrast, no reactivity was observed for deglycosylated native PSA (Figure 4B), further demonstrating not only the specificity of the identified antibodies against 1,6-fucPSA but also their selectivity for 1,6-fucPSA compared to non-1,6-fucPSA, i.e., non-corefucosylated PSA. Furthermore, this confirms that natural 1,6-fuc PSA can be detected by Western blot analysis.

[0177] 6.3 Example 3: Use of an antibody selective for fucosylated PSA in an immunohistochemistry (IHC) assay Anti-1,6-fucPSA antibodies selected according to Example 2 were evaluated for their ability to detect 1,6-fucPSA in formalin-fixed paraffin-embedded (FFPE) samples of prostate adenocarcinoma using chromogenic immunohistochemistry assays. The VENTANA OptiView DAB IHC detection kit and the automated VENTANA BenchMark ULTRA platform were used with anti-1,6-fucPSA rabbit monoclonal antibodies 2E9, 3B10, 3H6, 13C5, 2H9, and 2C11. Assay development and optimization included identifying optimal antibody titers, selecting diluents, assay conditions (e.g., antigen retrieval, primary antibody incubation time, signal amplification), and specificity testing, as outlined in Table 4 below. Samples for assay development and optimization included FFPE human prostate carcinoma cell lines (PC3, LNCaP), benign human tissues (tonsils, kidneys, and colon), and prostate carcinoma tissue.

[0178] (Table 3) IHC conditions tested for optimization TIFF0007901069000021.tif100170

[0179] Samples stained according to the above combinations of preparation parameters were evaluated by certified pathologists. Optimal staining conditions were determined based on acceptable morphology, specific staining pattern and intensity, and non-specific (off-target) staining, as shown in Table 5 below.

[0180] (Table 4) Optimal assay conditions for IHC of FFPE samples containing anti-1,6-fucPSA antibody TIFF0007901069000022.tif86170

[0181] In IHC assays, the 1,6-fucPSA assay showed a similar staining pattern for FFPE prostate adenocarcinoma specimens to that of whole PSA (evaluated using the commercially available antibody ER-PR8, Roche Tissue Diagnostics), with slightly weaker staining intensity and lower coverage. See Figure 5. As can be seen, prostatic secretory epithelial cells showed moderate to strong cytoplasmic staining with 1,6-fucPSA, with increased staining intensity at the apical end of the prostatic epithelium. As with whole PSA staining, weak 1,6-fucPSA staining was occasionally detected in stromal cells. Although stromal cells do not express PSA, it is thought that PSA may diffuse into the vicinity of the prostate during sample preparation, potentially resulting in weaker staining, and therefore acceptable.

[0182] To verify that the anti-1,6-fucPSA antibody specifically and selectively recognizes core-fucosylated PSA epitopes, staining specificity was tested by peptide inhibition analysis. Specifically, the optimal IHC staining protocols described above were repeated, but before their use, anti-1,6-fucPSA antibodies were pre-incubated with various concentrations of PSA(67-79)-G0F and their variants, also schematically shown in Figure 6: (A) a glycopeptide of PSA fragment (SEQ ID NO: 18) containing a disaccharide with α-1,6-core fucose ("DP"); (B) a glycopeptide of PSA fragment (SEQ ID NO: 18) containing a nonasaccharide lacking α-1,6-core fucose ("PSA(67-79)-G2"); (C) an aglycosylated PSA fragment SEQ ID NO: 18, i.e., "PSA skeleton"; and (D) a fucosylation-unrelated (non-targeted) octaglycopeptide having the same glycan structure as PSA(67-79)-G0F, i.e., an octasaccharide with α-1,6-cofucose ("AFP").

[0183] As shown in Figure 7, PSA(67-79)-G0F specifically bound to anti-1,6-fucPSA antibodies in the IHC assay and inhibited their binding to target epitopes. Similarly, complete inhibition of anti-1,6-fucPSA antibody binding was also achieved by 5 × 10⁻⁶ -6 This was observed by pre-incubation with DP at M concentration. See Figure 8.

[0184] In contrast, anti-1,6-fucPSA antibodies showed at least 100 times higher levels (5 × 10⁻¹⁰). -5 No inhibition was observed when pre-incubated with PSA(67-79)-G2 at concentration M. See Figure 9. Furthermore, neither the aglycosylated PSA peptide backbone (aa67-79; SEQ ID NO: 18) nor AFP inhibited the binding of anti-1,6-fucPSA antibodies to 1,6-fucPSA in FFPE prostate tissue specimens (see Figures 10 and 11).

[0185] The summary of the above results, provided in Table 5 below, demonstrates that the anti-1,6-fucPSA antibody possesses not only high specificity but also high selectivity for 1,6-fucPSA in FFPE prostate tissue specimens.

[0186] (Table 5) TIFF0007901069000023.tif61170

[0187] The specificity of the anti-1,6-fucPSA antibody clone was further tested in FFPE tissue arrays of normal (tour of body, TOB) and diseased (tour of tumor, TOT) specimens. Staining of 1,6-fucPSA using the selected antibody from Example 2 was compared to staining of total PSA using the commercially available antibody ER-PR8. The anti-1,6-fucPSA antibody showed strong specific staining (intensity 2.5–3.5) in prostate tissue samples. However, nonspecific staining (0.25–1.25) was detected in selected normal (Table 6) and neoplasm (Table 7) tissue specimens, yet no PSA expression was observed in these histological types. Efforts to eliminate nonspecific staining also led to a decrease in specific staining intensity in prostate adenocarcinoma. Therefore, the 1,6-fucPSA assay conditions were not changed to avoid false negatives.

[0188] (Table 6) IHC staining of normal tissue specimens using anti-1,6-fucPSA antibody from Example 2 TIFF0007901069000024.tif101170

[0189] (Table 7) IHC staining of tumor tissue specimens using anti-1,6-fucPSA antibody from Example 2 TIFF0007901069000025.tif220170

[0190] The assay performance of the 1,6-fucPSA IHC assay has also been evaluated in FFPE samples derived from prostate hyperplasia and adenocarcinoma, including a wide range of Gliason scores (n=50). The data demonstrate that the 1,6-fucPSA antibody of the present invention can specifically detect the 1,6-fucPSA protein in FFPE prostate samples in the IHC assay system.

[0191] 6.4 Example 4: Use of an antibody selective for fucosylated PSA in a sandwich ELISA The applicability of anti-1,6-fucPSA antibodies in sandwich ELISA was tested by combining the selected antibodies from Example 2 with monoclonal and polyclonal antibodies directed against total PSA on the Roche IMPACT (Immunological Multi-Parameter Chip Technology) platform; Claudon et al., Clinical Chemistry, 54 (2008), 1554-1563. The most reactive immunological sandwich was formed by combining the capture rabbit polyclonal anti-total PSA antibody K-54794 (Novus) with one of the six anti-1,6-fucPSA monoclonal antibodies for detection. Sample pretreatment with 100 mM Tris (pH 12.9), 30.6 mM TCEP, and 2 mM EDTA, resulting in protein reduction, was necessary to linearize the natural glycopeptide epitopes to enable reactivity with the antibodies. Under these conditions, all sandwich assays reacted with purified semen PSA antigen spiked into an artificial serum matrix, but not with corresponding concentrations of deglycosylated PSA (Figure 12) or the unrelated glycoprotein CD59 (not shown). This assay format is applicable to serum, plasma, and other biological fluid samples.

[0192] The present invention further includes the following items.

[0193] 1. A monoclonal antibody or antigen-binding fragment thereof that is specific to α-1,6-core-fucosylated prostate-specific antigen (PSA) or a partial sequence thereof containing the α-1,6-core-fucosylated portion.

[0194] 2. The monoclonal antibody or antigen-binding fragment described in item 1, wherein the subsequence includes or consists of SEQ ID NO: 18.

[0195] 3. The α-1,6-core-fucosylated prostate-specific antigen (PSA) or the partial sequence containing the α-1,6-core-fucosylated portion thereof is a glycopeptide of formula Ib. A monoclonal antibody or antigen-binding fragment thereof as described in item 1 or 2, comprising or consisting of TIFF0007901069000026.tif58128.

[0196] 4. The α-1,6-core-fucosylated prostate-specific antigen (PSA) or the glycopeptide of formula IV having a partial sequence containing the α-1,6-core-fucosylated form. A monoclonal antibody or antigen-binding fragment thereof as described in item 1 or 2, comprising or consisting of TIFF0007901069000027.tif22128.

[0197] 5. A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 4, wherein the antibody or fragment distinguishes between (i) a partial sequence of α-1,6-core-fucosylated PSA or PSA containing α-1,6-core-fucosylation and (ii) PSA lacking α-1,6-core-fucose residues or a partial sequence thereof.

[0198] 6. The monoclonal antibody or antigen-binding fragment described in item 5, wherein the partial sequence of PSA lacking the α-1,6-core-fucose residue includes or consists of SEQ ID NO: 18.

[0199] 7. The PSA lacking the α-1,6-core-fucose residue or a partial sequence thereof is a glycopeptide of formula IIb. A monoclonal antibody or antigen-binding fragment as described in item 5 or 6, comprising or consisting of TIFF0007901069000028.tif71128.

[0200] 8. The antibody or fragment comprises (i) a partial sequence of α-1,6-core-fucosylated PSA or PSA containing α-1,6-core-fucosylated material, and (iii) an α-1,6-core-fucosylated glycan of formula (IIIb). A monoclonal antibody or antigen-binding fragment described in any one of items 1-7, which identifies TIFF0007901069000029.tif58128.

[0201] 9. A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 8, wherein the antibody or fragment distinguishes between a glycopeptide according to formula Ib and both a glycopeptide according to formula IIb and a core-fucosylated glycan according to formula IIIb.

[0202] 10. A monoclonal antibody or antigen-binding fragment according to any one of items 5 to 7, wherein (i) the binding affinity to the α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion is at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than (ii) the binding affinity to the PSA lacking the α-1,6-core-fucose residue or a subsequence thereof of the PSA lacking the α-1,6-core-fucose residue, and the binding to (i) and (ii) is performed under the same conditions.

[0203] 11. A monoclonal antibody or antigen-binding fragment as described in item 8, wherein (i) the binding affinity to the α-1,6-core-fucosylated PSA or the subsequence thereof containing the α-1,6-core-fucosylated portion is at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than (ii) the binding affinity to the core-fucosylated glycan of formula IIIb, and the binding to (i) and (ii) is performed under the same conditions.

[0204] 12. The monoclonal antibody or antigen-binding fragment thereof according to item 10 or 11, wherein the binding affinity of the antibody or fragment to the glycopeptide of formula Ib is at least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than its binding affinity to the glycopeptide of formula IIb.

[0205] 13. A monoclonal antibody or antigen-binding fragment according to any one of items 10 to 12, wherein the binding affinity is determined as KD.

[0206] A monoclonal antibody or antigen-binding fragment according to any one of items 1 to 13, which binds to the glycopeptide of formula Ib with a KD of 14.30 nM or less, preferably 20 nM or less, and more preferably 11 nM or less.

[0207] 15. Association rate of the glycopeptide of Equation 1B k a at least 10 5 M -1 s -1 The monoclonal antibody or antigen-binding fragment antibody described in item 14.

[0208] 16. The KD and / or the k a However, a monoclonal antibody or antigen-binding fragment as described in any one of items 13-15, as determined by surface plasmon resonance spectroscopy.

[0209] 17. The monoclonal antibody or antigen-binding fragment according to item 16, wherein the surface plasmon resonance spectroscopy comprises binding or capturing the monoclonal antibody or antigen-binding fragment to a CM5 sensor chip and injecting a glycopeptide of formula Ib as an analyte, and the determination is performed at 37°C using HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w / v Tween 20®) supplemented with 1 mg / ml carboxymethyl dextran.

[0210] 18. The monoclonal antibody or antigen-binding fragment described in item 17, wherein the binding or capture level of the monoclonal antibody or antigen-binding fragment on the CM5 sensor chip is selected to be a molar ratio of 1 or 2.

[0211] 19. The settings for surface plasmon resonance spectroscopy are as follows: (1) A rabbit antibody comprising a heavy chain variable domain having the sequence of SEQ ID NO: 61 and a light chain variable domain having the sequence of SEQ ID NO: 62, and (2) The glycopeptide of formula Ib as defined in item 3. A monoclonal antibody or antigen-binding fragment as described in any one of items 16-18, selected such that the KD of the bond between them is determined to be 11 nm within the standard error range of surface plasmon resonance spectroscopy.

[0212] 20. A monoclonal antibody or antigen-binding fragment as described in any one of items 16-19, wherein surface plasmon resonance spectroscopy is performed using a Biacore 8k instrument.

[0213] 21. The above KD and / or k a The determination is made using the Langmuir fitting model, preferably R MAX A monoclonal antibody or antigen-binding fragment as described in any one of items 16-20, including fitting surface plasmon resonance data locally.

[0214] 22. (i) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 2, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 3, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and (ii) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 4, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 5, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 6, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. A monoclonal antibody or antigen-binding fragment antibody as described in any one of items 1 to 21, including the above.

[0215] 23. (i) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 1, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 7, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 8, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and (ii) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 9, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 10, or a variant thereof modified by a single conservative amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 11, or a variant thereof modified by up to two conservative amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. A monoclonal antibody or antigen-binding fragment as described in any one of items 1 to 22, including the above.

[0216] 24. (i) A heavy chain variable domain (VH) comprising: CDR-H1 having the amino acid sequence of SEQ ID NO: 12, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 2, 3, 4, 5, or 6; CDR-H2 having the amino acid sequence of SEQ ID NO: 13, or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13, 15, 16, and 18; and CDR-H3 having the amino acid sequence of SEQ ID NO: 14, or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 3, 4, 6, 8, 9, 10, 11, 12, and 15; and (ii) A light chain variable domain (VL) comprising: CDR-L1 having the amino acid sequence of SEQ ID NO: 15, or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 6, 7, and 9; CDR-L2 having the amino acid sequence of SEQ ID NO: 16, or a variant thereof modified by a single highly conserved amino acid substitution at amino acid positions 1, 3, 5, 6, or 7; and CDR-L3 having the amino acid sequence of SEQ ID NO: 17, or a variant thereof modified by up to two highly conserved amino acid substitutions at amino acid positions selected from 1, 2, 5, 6, 9, 10, 12, 13, 14, and 15. A monoclonal antibody or antigen-binding fragment as described in any one of items 1 to 23, including the above.

[0217] 25. The aforementioned conservative antibody amino acid substitution is an amino acid substitution by another amino acid selected from the same group, and the group of amino acids is a) Nonpolar hydrophobic amino acids consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, and Met; b) Polar neutral amino acids consisting of Ser, Thr, Asn, and Gln; c) Basic amino acids having a positive charge, consisting of Arg, Lys and His, and d) An acidic amino acid having a negative charge, consisting of Asp and Glu, A monoclonal antibody or antigen-binding fragment as described in item 22 or 23, wherein Cys is substituted with Ser or Ala when conservatively substituted, and Pro is substituted with Ala when conservatively substituted.

[0218] 26. The above-mentioned highly conserved amino acid substitutions are a) Substitution of Ala by Val, Leu, Ile, or Gly; b) Substitution of Arg by Lys; c) Replacement of Asn with Gln; d) Replacement of Asp with Glu; e) Substitution of Cys by Ser; f) Substitution of Gln by Asn; g) Substitution of Glu by Asp; h) Substitution of Gly by Ala; i) Substitution of His by Arg; j) Substitution of Ile with Leu, Val, or Ala; k) Substitution of Leu with Ile, Val, or Ala; l) Substitution of Lys with Arg; m) Substitution of Met with Leu, Ile, or Val; n) Substitution of Phe with Tyr or Trp; o) Replacement of Pro with Ala; p) Substitution of Ser by Thr; q) Substitution of Thr by Ser; r) Substitution of Trp with Phe or Tyr; s) Substitution of Tyr with Phe or Trp; t) Substitution of Val with Leu, Ile, or Ala A monoclonal antibody or antigen-binding fragment antibody as described in item 24, selected from the following.

[0219] 27. (i) Heavy chain variable domains having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or preferably at least 93% sequence identity with respect to SEQ ID NO: 19; and (ii) Light chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or preferably at least 96% sequence identity with SEQ ID NO: 20 A monoclonal antibody or antigen-binding fragment as described in any one of items 1 to 26, including the above.

[0220] 28. A monoclonal antibody or antigen-binding fragment according to item 14, wherein CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined in any one of claims 22 to 26.

[0221] 29. (i) The heavy chain or heavy chain variable domain of a monoclonal antibody or antigen-binding fragment described in any one of items 1 to 28, and / or (ii) The light chain or light chain variable domain of the monoclonal antibody or antigen-binding fragment described in any one of items 1 to 28. A polynucleotide that codes for something.

[0222] 30. A vector containing the polynucleotides described in item 29.

[0223] 31. Host cells containing the polynucleotides described in item 29, or the vectors described in item 30.

[0224] 32. A host cell, as described in item 31, which is a prokaryotic or eukaryotic cell.

[0225] 33. A host cell as described in item 32, which is a eukaryotic cell and is a CHO cell.

[0226] 34. A method for producing a monoclonal antibody or antigen-binding fragment as described in any one of items 1 to 28, comprising culturing a host cell as described in any one of items 31 to 33, and isolating the antibody or antigen-binding fragment.

[0227] 35. An antibody described in any one of items 1-28, which can be obtained by the method of item 34.

[0228] 36. A composition comprising an antibody described in any one of items 1-28 and 35, a polynucleotide described in item 29, a vector described in item 30, or a host cell described in any one of items 31-33.

[0229] 37. A diagnostic composition comprising an antibody described in any one of items 1 to 28 and 35.

[0230] 38. Use of an antibody described in any one of items 1-28 and 35 or a composition described in item 37 for in vitro chromoly no assay.

[0231] 39. Use as described in item 38, where the immunoassay is a heterologous immunoassay.

[0232] 40. Use as described in item 27 or 28, where the immunoassay is an immunohistochemistry (IHC) assay.

[0233] 41. The use described in any one of items 38-40, wherein the sample for the immunoassay is a sample prepared from blood, plasma, or serum.

[0234] 42. Use according to any one of items 38 to 41 for detecting α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion.

[0235] 43. Use as described in any one of items 38-42, wherein the immunoassay is an immunoassay for detecting a glycopeptide of formula Ib or a glycoprotein containing a glycopeptide of formula Ib.

[0236] 44. Uses described in any one of items 38 to 43 for distinguishing α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion from PSA or a subsequence thereof lacking the α-1,6-core-fucosylated portion.

[0237] 45. An in vitroimu noassay method for detecting α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion in a sample using an antibody defined in any one of items 1-28 and 35.

[0238] 46. ​​The method according to item 45, wherein the method is an IHC assay and the sample is a tissue slide.

[0239] 47. The method according to item 45, wherein the method is a serum immunoassay and the sample is a body fluid.

[0240] 48. The method according to item 47, wherein the bodily fluid is a blood sample, semen, or urine.

[0241] 49. The method described in item 47, wherein the body fluid is a blood sample, which is whole blood, serum, or plasma.

[0242] 50. The method according to any one of items 45 to 49, comprising (i) pre-treating a sample, and (ii) incubating the pre-treated sample with an antibody defined in any one of items 1 to 28 and 35.

[0243] 51. A method according to any one of items 45-50, for distinguishing α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion from PSA or a subsequence thereof lacking the α-1,6-core-fucosylated portion.

[0244] 52. A kit containing an antibody described in any one of items 1-28 and 35.

[0245] 53. An immunoassay kit, as described in item 52.

[0246] 54. A method for preparing a histochemical or cytochemical sample for microscopic analysis, comprising performing immunohistochemical or immunocytochemical staining using an antibody or antigen-binding fragment described in any one of items 1 to 28 and 35 as a primary antibody.

[0247] 55. Immunohistochemical or immunocytochemical staining, (a) Contacting the sample with the primary antibody under conditions sufficient to promote specific binding between the primary antibody and the glycopeptide of formula Ib or a glycoprotein containing the glycopeptide of formula Ib; and (b) Remove unbound primary antibody from the sample. The method described in item 54, including the method described in item 54.

[0248] 56. The method according to item 55, wherein the conditions reduce the binding of the primary antibody to the glycopeptide of formula IIb or a glycoprotein containing the glycopeptide of formula IIb.

[0249] 57. The method according to any one of items 54 to 56, wherein the primary antibody is conjugated to a detectable portion.

[0250] 58. The method according to item 55 or 56, further comprising (c) contacting a sample with a set of detection reagents suitable for depositing a detectable portion in close proximity to the primary antibody bound to the sample.

[0251] 59. The method according to item 57 or 58, wherein the detectable portion is a pigment, a fluorophore, a phosphorescent molecule, a luminescent molecule, or a mass tag.

[0252] 60.(c) is, (i) Conjugating a detectably labeled secondary antibody to the primary antibody; (ii) A secondary antibody conjugated to the primary antibody, and a tertiary antibody conjugated to the secondary antibody, wherein the tertiary antibody, or both the secondary and tertiary antibodies, are detectably labeled; (iii) Conjugating an epitope-tagged secondary antibody to the primary antibody, and conjugating a tertiary antibody specifically labeled for the epitope tag to the secondary antibody; (iv) A secondary antibody conjugated to an enzyme is bound to a primary antibody, and the signal transduction conjugate is reacted with the enzyme, wherein the signal transduction conjugate comprises an epitope tag and a potential reactive moiety, and the enzyme catalyzes the transformation of the potential reactive moiety into a reactive species that binds to the sample, and a tertiary antibody is bound to the epitope tag of the signal transduction conjugate that binds to the sample, wherein the enzymes of the secondary and tertiary antibodies are the same, and the enzyme is reacted with an additional reagent to result in the deposition of a detectable moiety on the sample; or (v) A secondary antibody conjugated to an epitope tag is bound to a primary antibody; a tertiary antibody conjugated to an enzyme is bound to the epitope tag; the sample is brought into contact with the signal transduction conjugate containing the epitope tag and a potential reactive moiety under conditions in which the enzyme catalyzes the conversion of the potential reactive moiety to a reactive species that binds to the sample; an additional tertiary antibody is bound to the epitope tag of the signal transduction conjugate that binds to the sample; and the enzyme is reacted with an additional reagent to cause deposition of a detectable moiety on the sample. The method described in item 58, which is a method of selection from.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that is specific to α-1,6-core-fucosylated prostate-specific antigen (PSA) or a partial sequence containing the α-1,6-core-fucosylated part thereof, wherein the partial sequence contains or consists of SEQ ID NO: 18, and the monoclonal antibody or antigen-binding fragment is (i) A heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 22, CDR-H2 having the amino acid sequence of SEQ ID NO: 23, and CDR-H3 having the amino acid sequence of SEQ ID NO: 24; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 34, CDR-L2 having the amino acid sequence of SEQ ID NO: 35, and CDR-L3 having the amino acid sequence of SEQ ID NO: 36; (ii) Heavy chain variable domains (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 26, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 34, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 38; (iii) Heavy chain variable domains (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 28, CDR-H2 having the amino acid sequence of SEQ ID NO: 29, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 39, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 40; (iv) A heavy chain variable domain (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 30, and CDR-H3 having the amino acid sequence of SEQ ID NO: 27; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 41, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO: 42; (v) Heavy chain variable domains (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 31, and CDR-H3 having the amino acid sequence of SEQ ID NO: 32; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 43, CDR-L2 having the amino acid sequence of SEQ ID NO: 44, and CDR-L3 having the amino acid sequence of SEQ ID NO: 45; or (vi) Heavy chain variable domains (VH) comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 25, CDR-H2 having the amino acid sequence of SEQ ID NO: 31, and CDR-H3 having the amino acid sequence of SEQ ID NO: 33; and A light chain variable domain (VL) comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 46, CDR-L2 having the amino acid sequence of SEQ ID NO: 37, and CDR-L3 having the amino acid sequence of SEQ ID NO:

40. A monoclonal antibody or its antigen-binding fragment, including the above.

2. The α-1,6-core-fucosylated prostate-specific antigen (PSA), or the partial sequence thereof containing the α-1,6-core-fucosylated form, (i) Glycopeptide of formula Ib or (ii) Glycopeptide of formula IV A monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising or consisting of the following.

3. The α-1,6-core-fucosylated PSA, or a partial sequence of the PSA containing the α-1,6-core-fucosylated portion, (i) PSA or a partial sequence thereof lacking α-1,6-core-fucose residues; and / or (ii) α-1,6-core-fucosylated glycan of formula (IIIb) A monoclonal antibody or antigen-binding fragment according to claim 1 or 2, which identifies the following.

4. The partial sequence of PSA lacking the α-1,6-core-fucose residue is (i) Sequence ID 18; or (ii) Glycopeptide of formula IIb The monoclonal antibody or antigen-binding fragment according to claim 3, comprising or consisting of the following.

5. (i) The α-1,6-core-fucosylated PSA or a partial fragment thereof containing the α-1,6-core-fucosylated PSA. The binding affinity of the antibody or fragment to the said antibody is (iiia) PSA lacking the α-1,6-core-fucose residue or a partial sequence of PSA lacking the α-1,6-core-fucose residue; and / or (iib) Core-fucosylated glycan of formula IIIb At least 10 times, at least 20 times, at least 50 times, or at least 100 times greater than its binding affinity to Hereinafter, the binding to (i) and ((iia) and / or (iib)) is carried out under the same conditions, as described in claim 3 or 4, for the monoclonal antibody or antigen-binding fragment.

6. The monoclonal antibody or antigen-binding fragment according to claim 5, wherein the binding affinity is determined as KD, and the antibody or fragment binds to the glycopeptide of formula Ib with a KD of 30 nM or less, 20 nM or less, or 11 nM or less.

7. (i) Heavy chain variable domains having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or at least 93% sequence identity with SEQ ID NO: 19; and (ii) Light chain variable domain having an amino acid sequence having at least 80%, at least 86%, at least 87%, at least 90%, or at least 96% sequence identity with SEQ ID NO: 20 A monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 6, comprising:

8. (i) the heavy chain or heavy chain variable domain of a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 7, and / or (ii ) Light chain or light chain variable domain of monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 7 A polynucleotide that codes for [something].

9. A vector comprising the polynucleotide described in claim 8.

10. A host cell comprising the polynucleotide described in claim 8 or the vector described in claim 9.

11. A method for producing a monoclonal antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising culturing a host cell according to claim 10, and isolating the antibody or antigen-binding fragment.

12. A composition comprising an antibody according to any one of claims 1 to 7 or an antibody obtainable by the method described in claim 11, a polynucleotide according to claim 8, a vector according to claim 9, or a host cell according to claim 10.

13. In a sample that is a tissue slide or body fluid, (i) To detect α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated form; and / or (ii) To distinguish α-1,6-core-fucosylated PSA or a subsequence thereof containing the α-1,6-core-fucosylated portion from PSA or a subsequence thereof lacking the α-1,6-core-fucosylated portion. The composition according to claim 12, which is a diagnostic composition for use in an in vitro assay.

14. A kit comprising an antibody according to any one of claims 1 to 7 or an antibody that can be obtained by the method described in claim 11.

15. A method for preparing histochemical or cytochemical samples for microscopic analysis, (i) an antibody or antigen-binding fragment according to any one of claims 1 to 7, or an antibody or antigen-binding fragment that can be obtained by the method of claim 11, (ii) The composition according to claim 12, or (iii) The kit according to claim 14 This includes performing immunohistochemical or immunocytochemical staining using the following method: A method wherein the antibody or antibody fragment, or a component of the antibody or antibody fragment, is a primary antibody.

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