Combotope Antibody Library

Abstract

The present invention provides a library of specific Tn- and STn antibodies, as well as a method for identifying specific antibodies that target Tn- and / or STn glycosylation sites of any selected glycoprotein, particularly those targeting cancer cells. The present invention further provides antibodies having combined specificity to both the glycan epitopes and peptide epitopes of the glycoprotein identified by the novel concepts proposed herein.

Description

[Technical Field]

[0001] The present invention relates to antibodies, antibody libraries, and methods for identifying antibodies, wherein the antibodies target any select protein site, particularly Tn- and STn glycosylation sites of protein sites associated with binding to cancer cell targets. Antibodies identified based on the novel concepts proposed herein have specificity for both the glycoepitope and the peptide backbone in glycoproteins that are bound to or have glycoepitopes on cancer cells.

[0002] The present invention provides a library of Tn- and STn-specific antibodies and a method for identifying specific antibodies, wherein the antibodies target the Tn- or STn glycosylation site of any selected glycoprotein, particularly those relevant to binding to cancer cell targets (such as tumor cells). The present invention further provides antibodies identified based on novel concepts proposed herein, wherein the antibodies possess combination specificity for both the glycan epitopes and peptide epitopes of the glycoprotein. [Background technology]

[0003] High-density layers of complex carbohydrate structures are present in almost all eukaryotic cells. Tumor cells exhibit altered glycosylation patterns on their cell surface that differ from those of healthy counterpart cells. Such glycosylational changes in cancer include increased sialylation, fucosylation, short, truncated O-glycans, and increased N-branching.

[0004] One of the key features of glycosylation changes in cancer is the presence of short, truncated O-glycans, which are so-called tumor-associated glycoglycan antigens (TACAs); namely, Tn antigens and T antigens, as well as their sialylated forms (STn and ST, respectively).

[0005] Tn and STn are not typically found in normal human or rodent tissues, but are highly expressed in many solid tumors / cancers. Therefore, Tn and STn are potential primary targets for immunotherapy and are also useful in diagnosing cancerous conditions.

[0006] Numerous cell surface glycoproteins undergo altered glycosylation and play a major role in cancer; such glycoproteins include, for example, CD43 and CD44, as well as the mucin family members MUC1, MUC4, and MUC6.

[0007] MUC1 is the most extensively studied mucin belonging to the mucin family and is present in many adenocarcinomas that express short, truncated O-glycans. In healthy cells, the MUC1 peptide core is significantly glycosylated, and therefore the core is covered with O-glycan moieties, protecting MUC1 from proteolytic cleavage enzymes. In adenocarcinomas, the MUC1 protein has shorter and less dense O-glycan side chains, exposing the core domain of the protein to the cell surface. This change in glycosylation of MUC1 exposes the epitopes MUC1-Tn and MUC1-STn to the immune system.

[0008] CD43 (leukosiarin) is a type I transmembrane sialycoglycoprotein that is abundant in hematopoietic cells such as lymphocytes, monocytes, granulocytes, natural killer cells, and platelets, with the exception of quiescent mature B cells and erythrocytes. Human CD43 protein has a mucinous extracellular domain rich in serine and threonine residues, and these residues enable high levels of O-GalNAc glycosylation, resulting in highly heterogeneous molecular weight. Multiple hematopoietic and non-hematopoietic cancers have been reported to exhibit different glycosylation patterns of CD43, including lung cancer, breast cancer, colon cancer, cervical cancer, and prostate cancer, most of which express CD43 in the early stages of tumor progression.

[0009] Known antibodies against Tn / STn include 5E5 (Macias-Leon et al., 2020; Tarp et al., 2007; Blixt et al., 2010), anti-CD43 (Blixt et al., 2012), 2D9 (Sorensen et al., 2006; Tarp et al., 2007; Blixt et al., 2010), and 5F7 (US Patent Application No. 11161911B2). Further known antibodies for Tn / STn include G2D11 (Persson et al., 2017), 3F1 (Kjeldsen et al., 1988), 83D4 (Oppezzo et al., 2004), 15G9 (Mazal et al., 2013), 1E3 (Li et al., 2009), and MLS128 (Yuasa et al., 2012); 16E12.1D9.1B11 (WO2023 / 034569A1), and 1A5-2C9 (US Patent Application No. 2022 / 057402A1); however, these antibodies do not demonstrate a binding contribution to peptide / protein carriers, or are unknown Tn-hapten conjugates; this will be discussed further later in this specification.

[0010] Monoclonal antibodies against Tn and STn antigens are particularly difficult and expensive to produce; furthermore, their specificity is often not characterized in detail, especially regarding whether these antibodies simultaneously recognize Tn / STn and protein backbone / carrier (which is necessary for good specificity and therapeutic applications).

[0011] The development of therapeutic antibodies can be achieved through multiple strategies. Current techniques (such as established hybridoma techniques) rely on animal immunity, inoculating animals with the target antigen to induce an immune response. Subsequently, B cells are isolated from the spleen of immunized animals (mice, rats, and rabbits) and fused with myeloma cells to create hybridoma clones for testing. This is a very monotonous, laborious, and time-consuming method, and often has low fusion efficiency. Furthermore, the effectiveness of this method is highly dependent on the immunogenicity of the antigen, and it has been shown that some antigens have low immunogenicity.

[0012] Phage display facilitates protein expression on the phage surface. This is a molecular technique using linear phages, in which exogenous DNA encoding a peptide is inserted into the genome of a non-lytic linear phage and expressed as a fusion protein with the phage coat protein without affecting phage infectivity. Phage display makes it possible to express a large repertoire of antibodies or parts thereof on the phage surface in order to select high-affinity conjugates for target purposes. [Overview of the Initiative]

[0013] This invention provides a novel antibody concept and technology for the rapid development of conbotope antibodies (Abs) targeting Tn- and STn glycosylation sites of any selected glycoprotein site, opening the way to a new generation of therapeutic opportunities in the treatment and diagnosis of cancer.

[0014] A novel structural and biochemical description of the specific recognition of Tn- and STn by the VH hypervariable region of antibodies, presented for the first time herein, is a combination of phage display screening for specific peptide recognition by the VL domain, which provides a combotope antibody with high specificity and high affinity for a desired biological glycoprotein target; this is due to the combined recognition of the Tn glycan epitope and / or STn glycan epitope with a protein backbone / carrier that associates with the Tn glycan epitope and / or STn glycan epitope.

[0015] Phage display is a rapid method for antibody development. The constructed Tn and STn template libraries determine the VH portion of the antibody that binds to the desired glycosylation, while the VL diversity determines the peptide backbone specificity. As disclosed in the Examples section of this specification, MUC1 was used in a proof-of-concept targeting both (Tn and STn) libraries to evaluate the functionality of these two libraries. Biopanning of these libraries was performed to isolate single-stranded variant fragments (scFv) with sequences identical or similar to known MUC1 antibodies. CD43 was a further primary target used to identify scFv for the Tn-CD43 peptide. In vitro specificity characterization of both Tn-MUC1 scFv and Tn-CD43 scFv demonstrated their potential therapeutic applications against cancer cell lines. Furthermore, STn-MUC1 scFv was identified.

[0016] Thus, two types of template libraries were constructed: a Tn-template library (see Examples 2-4) and an STn-template library (see Examples 5-6). In these libraries, the VH domain was pre-selected to have the desired glycosylation specificity, and the peptide backbone specificity of the VL domain was determined. The constructed library is a first library specifically designed for glycosylation targets. The constructed library is a first library specifically designed for specific glycosylation targets defined by certain glycoproteins having short, truncated O-glycans (Tn or STn) present on the surface of many cancer cells. The terms “tumor” and “cancer” are used synonymously throughout this disclosure. Any difference in meaning between these two terms is not applicable to the present invention.

[0017] In the first aspect, the present invention provides an antibody library for in vitro identification of specific antibodies that bind to tumor cells; Here, each antibody in the library includes (i) and (ii) below: (i) A first antibody domain that binds to the Tn- and / or STn-glycan epitopes of the glycoprotein of the tumor cell; and (ii) A second antibody domain selected from a repertoire of second antibody domains; Here, the repertoire of the second antibody domains comprises one or more second antibody domains that bind to the peptide epitopes of the glycoproteins of the tumor cells; Here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0018] In a preferred embodiment, the first antibody domain is a VH-domain, and the second antibody domain is a VL-domain. Therefore, in a preferred embodiment, the present invention provides an antibody library for in vitro identification of specific antibodies that bind to tumor cells; Each antibody in the library is: (i) A VH-domain that binds to the Tn- and / or STn-glycan epitopes of the glycoprotein of the tumor cell; and (ii) A VL domain selected from the repertoire of VL domains, Includes; Here, the repertoire of VL-domains includes one or more VL-domains that bind to the peptide epitopes of the glycoproteins of the tumor cells; Here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0019] In the second aspect, the present invention provides a nucleic acid library encoding the antibody library of the first aspect of the present invention.

[0020] In a third aspect, the present invention provides a method for identifying antibodies that target tumor cells, the method comprising the following steps (i) and (ii): (i) A step of preparing an antibody library according to the first aspect of the present invention; and (ii) A step of screening the library for the purpose of identifying one or more tumor-targeting antibodies, preferably one or more specific tumor-targeting antibodies.

[0021] In a fourth aspect, the present invention provides a method for identifying glycopeptide targets, wherein the targets (such as glycoprotein targets in cancer cells) include Tn and / or STn epitopes and peptide epitopes; The method includes the following steps (i) and (ii): (i) A step of preparing an antibody library according to the first aspect of the present invention; and (ii) Incubating the antibody library with a sample containing the glycopeptide target; (iii) A step of analyzing one or more antibody / peptide complexes obtained in step (ii) for the purpose of identifying the amino acid sequence of the peptide epitope of the glycopeptide target.

[0022] In the sixth aspect, the present invention provides a specific tumor cell-binding antibody; The antibody comprises (i) and (ii) below: (i) VH domains that bind to Tn- and / or STn-glycan epitopes of tumor cell glycoproteins; and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell; Here, if the antibody is not 5E5, 5F7, or 2D9, then the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0023] Preferably, in one embodiment, the antibody may be used in a method for treating and / or preventing cancer.

[0024] In another embodiment, the antibody may be used for in vivo or ex vivo diagnosis of cancerous conditions using cell samples.

[0025] For this purpose, after being identified, isolated and / or generated from a library as described herein and / or using the methods described herein, the antibodies of the present invention, as described herein, may be subjected to one or more of the techniques known herein for the purpose of improving one or more of the desired properties of the antibodies (such as improved affinity, improved potency, or reduced immunogenicity), and such improved antibodies form further embodiments of the present invention. For example, in a non-limiting example, the antibodies may be subjected to an affinity maturation technique known herein for the purpose of enhancing affinity and / or potency. Alternatively, the potential immunogenicity may be reduced or eliminated by a humanization technique known herein and / or a technique for identifying potential immunogenic epitopes, followed by a technique for removing the potential epitopes by one or more more suitable amino acid mutations (which can also be carried out by methods known herein). To improve and / or increase expression in a desired host cell or host organism used for expression and / or production, the amino acids of the antibody (or the sequence encoding such an antibody) may be subjected to, for example, a known technique, which is obvious to those skilled in the art and may include known techniques relating to codon optimization. As will be obvious to those skilled in the art, each of the above prior arts will require, or include, some degree of trial and error within the scope of the art of those skilled in the art.

[0026] The types of different antibodies / clones are preferably more than two (not limited to a specific number of types), and libraries as described herein usually contain at least 10 or more different antibodies / clones, at least 50 or more different antibodies / clones, etc., for example, at least 100 or more different antibodies / clones. As will be apparent to those skilled in the art, the upper limit of the library size as described herein is not critical and may be determined with emphasis on considerations such as the desired diversity and practical considerations such as library sizes that may have simplicity and / or convenience with respect to generation, manipulation, and screening. For example, without limitation, libraries as described herein contain 10 4 or more different clones, for example, 10 6 or more different clones (10 7 or more different clones, etc., and up to 10 8 , 10 9 , 10 10 or more different clones) and may be considered to be included. Preferably, as mentioned herein and for the purpose of providing a beneficial degree of diversity, libraries as described herein contain at least 1000 (10 3 ) types (at least 10000 (10 4 ) types, etc.), preferably at least 100000 (10 5 ) types (such as 10 6 types or more) of different sequences (i.e., antibodies having different VH / VL pairs).

[0027] Techniques for creating / constructing libraries of a size suitable for the purposes of the present invention will also be apparent to those skilled in the art based on the disclosure herein; for example, such techniques include those used in the following references: Ponsel et al., Molecules. 2011;16(5):3675-3700; Frenzel et al. (2014), "Construction of human antibody gene libraries and selection of antibodies by phage display": Article in "Human monoclonal antibodies", methods and protocols: 215-243; Hutchings et al., (2001), "Generation of naive human antibody libraries": Article in "Antibody engineering", Springer, pp 93-108; the review by Shim, BMB Rep. 2015;48(9):489-494; Mandrup et al., PLoS One, 8, (2013); Bai et al., PLoS ONE 2015, 10, e0141045; Hoet et al., Nat. Biotechnol 2005, 23, 344-348; Knappik et al., J. Mol. Biol. 2000, 296, 57-86; Kugler et al., BMC Biotechnol. 2015, 15, 10; Prassler et al., J. Mol. Biol. 2011, 413, 261-278; Soderlind et al., Nat. Biotechnol. 2000, 18, 852-856; Tiller et al., Int. J. Mol. Sci. 2022, 23, 6255; and Valadon et al., mAbs 2019, 11, 516-531. Other preferred techniques will be obvious to those skilled in the art.

[0028] Similarly, as will be apparent to those skilled in the art based on the disclosure herein, the preparation of such a library generally involves combining a VH sequence selected for its ability to specifically bind to a Tn epitope or STn epitope (such as the VH sequences described herein or referenced herein) with a repertoire of VL sequences that confer a desired library size and diversity to the library. For example, as further described herein, such a VL repertoire may be a collection of naive sequences (e.g., obtained from a naive library prepared from mouse B cells or human B cells), preimmunized sequences, and synthetic or semi-synthetic sequences.

[0029] Furthermore, such libraries may be in any preferred format, such as DNA, RNA, or protein, and may be in the form of libraries containing VH and VL sequences in a preferred format / vector that enables preferred expression or expression of the antibody. This may be, for example, a phage library or a yeast library, depending on the technique intended for screening the library. Preferred screening techniques (and preferred library formats to be used in such screening techniques) will be obvious to those skilled in the art, but include, for example, phage displays, ribosome displays, yeast displays, or displays (technologies and libraries) using preferred mammalian cell systems.

[0030] It should also be noted that the numbering of amino acid residues in VH and VL domains as described herein, and the definitions of CDRs for such VH and VL domains, generally use the Kabat numbering scheme unless otherwise specified (Kabat et al., "Sequences of Immunoglobulin Chains: Tabulation and Analysis of Amino Acid Sequences of Precursors, V-regions, C-regions, J-Chain and BP-Microglobulins," 1979, U.S. Department of Health, Education and Human Services, Public Health Service, National Institutes of Health (1979)). However, it will be obvious to those skilled in the art that other schemes exist (e.g., the Chotia, IMGT, and AbM schemes); and those skilled in the art will be able to apply these schemes to the VH and VL sequences described herein.

[0031] Furthermore, the terms “CDR,” “CDR1,” “CDR2,” and “CDR3” as used herein have their common meanings in the art and, unless otherwise explicitly stated, are equivalent to the definitions used by Kabat.

[0032] Furthermore, when comparing two amino acid sequences, the term “amino acid difference” as used herein refers to the insertion, deletion, or substitution of a single amino acid residue at a position in the first sequence compared to the second sequence. This is understood to mean that the two amino acid sequences may contain one or more such amino acid differences.

[0033] With respect to the present invention and the "Claims," ​​when referring to the binding ability of an antibody to an antigen, such binding is preferably specific binding; this is typically 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10-12 moles / liter or less, and more preferably 10 -8 ~10 -12 With a dissociation constant (KD) of moles / liter (i.e., 10 5 ~10 12 Liters / mol or more, preferably 10 7 ~10 12 Liters / moles or more, and more comfortably 10 8 ~10 12 The binding constant (KA) in liters / mol means that such an antibody binds to that antigen. 10 4 Any KD value greater than moles / liter (or 10 4 M -1 Any KA value lower than liters / mol is generally considered to indicate nonspecific binding. Preferably, the antibody of the present invention binds to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, and more preferably less than 10 nM (e.g., an affinity of less than 500 pM). Specific binding of the antigen-binding protein to the antigen or antigenic determinant can be determined by any suitable method known in itself; such methods include, for example, scatchard analysis and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assay), and variations different from those known in the art, as well as other techniques referred to herein.

[0034] It should be noted that, as is generally apparent to those skilled in the art, the VH and VL domains referred to herein are part of a VH / VL pair to form a complete binding site, and may also be essentially part of a VH / VL pair. For this reason, it will be apparent to those skilled in the art that, when not part of a VH / VL pair, it is not always practical, nor is it always possible, to determine whether an individual VH or VL domain is capable of binding to an antigen or epitope. Therefore, in this specification and the claims, when we say that a VH or VL domain can (specifically) bind to an epitope, antigen, or protein, there are two possible meanings; The first one is: If such a domain is separable (i.e., not part of a VH / VL pair), then that domain can bind (and / or such binding can be determined or measured); or, Secondly: Binding of such domains occurs (and / or can be determined or measured) when such domains are part of a suitable VH / VL pair as described herein (i.e., a VH / VL pair as present in the antibodies described herein).

[0035] In a further aspect, the present invention provides a method for producing antibodies against targets present on (and / or expressed on the surface of) cancer cells, wherein the targets are present / expressed on cancer cells in glycosylated form (and in particular as glycosylated forms containing, comprises, or express one or more Tn epitopes or STn epitopes as described herein). The method comprises at least the following steps: • The process of constructing or providing an antibody library (or a library of sequences encoding antibodies); Here, each antibody in the library contains a VH domain and a VL domain; Here, each such VH domain is selected to be capable of binding to (and preferably specifically to, as further defined herein) a Tn epitope or an STn epitope; Furthermore, the library preferably has at least 1000 (10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It includes (a collection or repertoire of) different VL domains (such as more than one type); Each of the VL domains is combined with the VH domain as part of an antibody obtainable from the library; The library has at least 1000(10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It contains / provides (an aggregate or repertoire of) different antibodies (i.e., antibodies with different VH / VL combinations), such as more than one type; • The process of screening the library against the target; and preferably a step of screening the library against the target, wherein the target is glycosylated, The screening process; More preferably, a step of screening the library against the target, wherein the target is glycosylated and the glycosylated form contains, comprises, or expresses one or more Tn epitopes or STn epitopes. The screening process; The steps of identifying, creating, and / or isolating one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, which is a glycosylated form and contains, comprises, or expresses one or more Tn epitopes or STn epitopes; and A process that involves one or more steps to selectively improve one or more desired properties of the antibody(s) / antibodies(s) obtained as described above (for example, by humanization, affinity maturation, removal of potential immunogenic epitopes, and / or optimization of the sequence for expression or production in a desired host cell or host organism).

[0036] Based on the disclosure herein, the library of the present invention is a suitable collection or repertoire of different VL sequences (e.g., at least 1000 (10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It will be apparent to those skilled in the art that a single VH sequence (i.e., selected to specifically bind to a Tn epitope or STn epitope, as further described herein) may be included in combination with a different VL sequence (e.g., more than one type). Other libraries may include two or more different VH sequences (each of which is selected to specifically bind to a Tn epitope or STn epitope) (these VH sequences are also suitably combined with a suitable collection or repertoire of VL sequences, as further described herein).

[0037] Suitable VH sequences that can be used to construct the libraries of the present invention as described herein will be obvious to those skilled in the art based on the disclosure herein. For example, suitable candidates for VH sequences that may be useful in constructing the libraries of the present invention include, but are not limited to, VH sequences present in and / or derived from antibodies prepared against glycosylated proteins present and / or expressed on the surface of cancer cells (especially when it is known that such proteins contain, comprise, or express Tn epitopes or STn epitopes). Such VH sequences may then be tested for their performance, i.e., their performance as VH sequences in constructing the libraries described herein. In the present invention, for example, the VH sequences present in the antibodies 5E5, anti-CD43, 2D9, 5F7, G2D11, 3F1, 83D4, 15G9, 1E3, MLS128, 16E12.1D9.1B11 and / or 1A5-2C9 (all listed above) may be tested for their ability as VH sequences in the libraries of the present invention (and consequently, antibodies prepared from such libraries); such VH sequences (or VH sequences containing CDRs present in these antibodies) may be used as VH sequences in the construction of libraries provided by the present invention.

[0038] The library of the present invention is preferably a library in which all or essentially all VH sequences present in the library (and / or the library used to construct the library) are capable of binding to either a Tn epitope or an STn epitope. Furthermore, the library of the present invention is also preferably a library in which at least 90% (at least 95%, etc.), more preferably all or essentially all antibodies obtained by the method described herein (i.e., screening of the library) contain VH sequences capable of binding to a Tn epitope or an STn epitope; even more preferably a library in which VH sequences confer to the antibody(s) obtained from the library the ability to specifically bind to an epitope or antigen (the Tn or STn portion thereof) containing a Tn epitope or an STn epitope.

[0039] The present invention also provides VH sequences that have been found to be particularly suitable for use as VH sequences in libraries provided by the present invention (and consequently in antibodies that can be prepared using such libraries). These are the VH sequence of SEQ ID NO: 1 (which can be used to prepare libraries specific to Tn epitopes / proteins containing Tn epitopes) and the VH sequence of SEQ ID NO: 28 (which can be used to prepare libraries specific to STn epitopes / proteins containing STn epitopes). The VH sequences (and other suitable VH sequences having the same CDR as the VH sequence of SEQ ID NO: 1 or SEQ ID NO: 28, respectively), libraries of the present invention containing and / or based on such VH sequences, methods for preparing such libraries, and antibodies identified, prepared, and / or isolated from such libraries form a more preferred embodiment of the present invention.

[0040] Further preferred VH sequences will be obvious to those skilled in the art based on the disclosure herein; such preferred VH sequences include, The following (i) and (ii) are listed: (i) Sequence ID: an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to 1; Preferably, an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; and preferably With respect to Sequence ID No. 1, an amino acid sequence containing amino acid residues H32, A33, H35, Y50, and S99 (these amino acid sequences support recognition of the mono-Tn epitope as referred to herein); Furthermore (ii) Amino acid sequences having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28; Preferably, an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; and preferably an amino acid sequence comprising amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103 with respect to SEQ ID NO: 28 (these amino acid sequences support recognition to a mono-STn epitope as referred to herein). Also, such VH sequences, libraries of the present invention comprising and / or based on such VH sequences, methods for creating such libraries, and antibodies identified and created using such libraries, and / or antibodies isolated from such libraries form a more preferred embodiment of the present invention.

[0041] Therefore, in a further aspect, the present invention provides a method for producing antibodies against targets present on (and / or expressed on the surface of) cancer cells, wherein the targets are present / expressed on cancer cells as glycosylated forms (and in particular as glycosylated forms containing, comprises, or expresses one or more Tn epitopes or STn epitopes as described herein). The method comprises at least the following steps: • The process of constructing or providing an antibody library (or a library of sequences encoding antibodies); Here, each antibody in the library contains a VH domain and a VL domain; Here, each such VH domain is: (i) Sequence ID: an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to 1; Preferably, an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; and preferably an amino acid sequence that includes amino acid residues H32, A33, H35, Y50, and S99 with respect to SEQ ID NO:1; and / or (ii) Amino acid sequences having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28; Preferably, an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; and preferably an amino acid sequence comprising amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103 with respect to SEQ ID NO: 28; And here the library preferably has at least 1000 (10 3 ) types (at least 10000 (10 4(Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It includes (a collection or repertoire of) different VL domains (such as more than one type); Each of the VL domains is combined with the VH domain as part of an antibody obtainable from the library; The library has at least 1000(10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It contains / provides (an aggregate or repertoire of) different antibodies (i.e., antibodies with different VH / VL combinations), such as more than one type; • The process of screening the library against the target; and preferably a step of screening the library against the target, wherein the target is glycosylated, The screening process; More preferably, a step of screening the library against the target, wherein the target is glycosylated and the glycosylated form contains, comprises, or expresses one or more Tn epitopes or STn epitopes. The screening process; The steps of identifying, creating, and / or isolating one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, which is a glycosylated form and contains, comprises, or expresses one or more Tn epitopes or STn epitopes; And optionally, A process involving one or more steps to improve one or more desired properties of the antibody (single) / antibody(s) obtained as described above (for example, by humanization, affinity maturation, removal of potential immunogenic epitopes, and / or optimization of the sequence for expression or production in a desired host cell or host organism).

[0042] In yet another aspect, the present invention provides a method for producing antibodies against targets present on (and / or expressed on the surface of) cancer cells, wherein the targets are present / expressed on cancer cells in glycosylated form (and in particular as glycosylated forms containing, comprises, or expresses one or more Tn epitopes or STn epitopes as described herein). The method comprises at least the following steps: • The process of constructing or providing an antibody library (or a library of sequences encoding antibodies); Here, each antibody in the library contains a VH domain and a VL domain; Here, each such VH domain has the amino acid sequence of SEQ ID NO: 1 and / or SEQ ID NO: 28; And here the library preferably has at least 1000 (10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It includes (a collection or repertoire of) different VL domains (such as more than one type); Each of the VL domains is combined with the VH domain as part of an antibody obtainable from the library; The library has at least 1000(10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6It contains / provides (an aggregate or repertoire of) different antibodies (i.e., antibodies with different VH / VL combinations), such as more than one type; • The process of screening the library against the target; and preferably a step of screening the library against the target, wherein the target is glycosylated, The screening process; More preferably, a step of screening the library against the target, wherein the target is glycosylated and the glycosylated form contains, comprises, or expresses one or more Tn epitopes or STn epitopes. The screening process; The steps of identifying, creating, and / or isolating one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, which is a glycosylated form and contains, comprises, or expresses one or more Tn epitopes or STn epitopes; And optionally, A process involving one or more steps to improve one or more desired properties of the antibody (single) / antibody(s) obtained as described above (for example, by humanization, affinity maturation, removal of potential immunogenic epitopes, and / or optimization of the sequence for expression or production in a desired host cell or host organism).

[0043] In yet another aspect, the present invention provides a method for producing antibodies against targets present on (and / or expressed on the surface of) cancer cells, wherein the targets are present / expressed on cancer cells in glycosylated form (and in particular as glycosylated forms containing, comprises, or expresses one or more Tn epitopes or STn epitopes as described herein). The method comprises at least the following steps: • The process of constructing or providing an antibody library (or a library of sequences encoding antibodies); Here, each antibody in the library contains a VH domain and a VL domain; Here, each such VH domain is: (i) CDR1 having an amino acid sequence DHAIH (SEQ ID NO: 155) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155); and (ii) CDR2 having an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from the amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156); and (iii) CDR3 having an amino acid sequence SLPGTFDY (SEQ ID NO: 157) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLPGTFDY (SEQ ID NO: 157), Includes; and / or Here, each such VH domain is: (i) CDR1 having an amino acid sequence DHAIH (SEQ ID NO: 155) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155); and (ii) CDR2 having an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from YISPGNDDIKYNEKFKG (SEQ ID NO: 156) or the amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156); and (iii) CDR3 having an amino acid sequence SLLALDY (SEQ ID NO: 158) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLLALDY (SEQ ID NO: 158), Includes; Here, the library preferably has at least 1000 (10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It includes (a collection or repertoire of) different VL domains (such as more than one type); Each of the VL domains is combined with the VH domain as part of an antibody obtainable from the library; The library has at least 1000(10 3 ) types (at least 10000 (10 4 (Types, etc.), preferably at least 100,000 (10 5 ) Types (10 6 It contains / provides (an aggregate or repertoire of) different antibodies (i.e., antibodies with different VH / VL combinations), such as more than one type; • The process of screening the library against the target; and preferably a step of screening the library against the target, wherein the target is glycosylated, The screening process; More preferably, a step of screening the library against the target, wherein the target is glycosylated and the glycosylated form contains, comprises, or expresses one or more Tn epitopes or STn epitopes. The screening process; The steps of identifying, creating, and / or isolating one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, preferably one or more antibodies (or sequences encoding such antibodies) that can specifically bind to the target, which is a glycosylated form and contains, comprises, or expresses one or more Tn epitopes or STn epitopes; And optionally, A process involving one or more steps to improve one or more desired properties of the antibody (single) / antibody(s) obtained as described above (for example, by humanization, affinity maturation, removal of potential immunogenic epitopes, and / or optimization of the sequence for expression or production in a desired host cell or host organism).

[0044] As further described herein, in the library of the present invention: If the VH sequence includes a CDR1 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155), such a CDR1 preferably includes amino acid residues H32, A33, and H35; If the VH sequence includes a CDR2 which is an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from the amino acid sequence YISPGNDDIKYNEKFKG (Sequence ID: 156), then such a CDR2 preferably includes amino acid residues Y50, S52, N55, and D57; and If the VH sequence includes a CDR3 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLPGTFDY (SEQ ID NO: 157), such a CDR3 preferably includes amino acid residue S99; Alternatively, if the VH sequence includes a CDR3 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLLALDY (SEQ ID NO: 158), such a CDR3 preferably includes amino acid residues S99, L101, A102, and L103.

[0045] It will be apparent to those skilled in the art, based on the disclosure herein, that in carrying out the present invention, a library is available comprising both one or more VH sequences selected to confer specificity to a Tn epitope (or a protein containing, comprises, or expresses a Tn epitope) and one or more VH sequences selected to confer specificity to an STn epitope (or a protein containing, comprises, or expresses an STn epitope). For example, such a library can be suitably constructed (or such a library may suitably contain both sequences) using both the VH sequence of SEQ ID NO: 1 (or a VH sequence based on the VH sequence of SEQ ID NO: 1 as further described herein) and the VH sequence of SEQ ID NO: 28 (or a VH sequence based on the VH sequence of SEQ ID NO: 28 as further described herein). Such libraries can be used, for example, to create and / or screen for antibodies expressed on cancer cells, regardless of whether the protein contains or expresses a Tn epitope, an STn epitope, or both. Accordingly, one aspect of the present invention relates to a library as described herein, comprising at least one VH sequence that confers specificity to a Tn epitope to an antibody obtainable from such a library, and further comprising at least one VH sequence that confers specificity to an STn epitope to an antibody obtainable from such a library. Furthermore, as further described herein, in such a library, the VH domain conferring specificity to the Tn epitope may be the VH domain of SEQ ID NO: 1 (or a variant thereof as described herein and / or a VH domain having a CDR based on the CDR of SEQ ID NO: 1 as also described herein), and the VH domain conferring specificity to the STn epitope may be the VH domain of SEQ ID NO: 28 (or a variant thereof as described herein and / or a VH domain having a CDR based on the CDR of SEQ ID NO: 28 as also described herein).

[0046] If the target contains or is expected to contain one or more Tn epitopes, and / or if it is desired to create / obtain an antibody that can (specifically) bind to the target when the target expresses a Tn epitope, it will be apparent to those skilled in the art that the method of the present invention typically involves using a library containing only one or more VH sequences that can specifically bind to a Tn epitope (and / or are selected based on their binding ability to specifically bind to a Tn epitope). Furthermore, as further described herein, in such a library, the VH domain conferring specificity to a Tn epitope may be the VH domain of SEQ ID NO: 1 (or a VH domain having a CDR based on its variant as described herein and / or the CDR of SEQ ID NO: 1 as also described herein); Furthermore, a library comprising only VH sequences that can specifically bind to Tn epitopes (and / or are selected based on their binding ability to specifically bind to Tn epitopes) forms a further embodiment of the present invention.

[0047] Similarly, if the target contains or is expected to contain one or more STn epitopes, and / or if it is desired to create / obtain an antibody that can (specifically) bind to the target when the target expresses an STn epitope, the method of the present invention typically involves using a library containing only one or more VH sequences that can specifically bind to an STn epitope (and / or are selected based on their binding ability to specifically bind to an STn epitope). Furthermore, as further described herein, in such a library, the VH domain conferring specificity to an STn epitope may be the VH domain of SEQ ID NO: 28 (or a VH domain having a CDR based on its variant as described herein and / or the CDR of SEQ ID NO: 28 as also described herein); Furthermore, a library comprising only VH sequences that can specifically bind to STn epitopes (and / or are selected based on their binding ability to specifically bind to STn epitopes) forms a further embodiment of the present invention.

[0048] In each of the libraries of the present invention as described herein, the VL sequences (assemblies or repertoires of different VL sequences) present in / can be included in the library may be provided and / or created by any known preferred method, for example, an assembly or repertoire of naive VL sequences (for example, derived from a naive library obtained from antibody sequences of mouse B cells or human B cells), a preimmuno-VL sequence, a synthetic VL sequence and / or a semi-synthetic VL sequence; or any combination thereof. Methods and techniques for providing such an assembly or repertoire of VL sequences (the sequences encoding them), and methods and techniques for suitably including them in libraries as described herein, will be obvious to those skilled in the art (for example, the following references: Ponsel et al.; Frenzel et al.; Hutchings et al., (2001); Shim; Mandrup et al.; Bai et al.; Hoet et al.; Knappik et al.; Kugler et al.; Prassler et al.; Soderlind et al.; Tiller et al.; and Valadon et al.; all cited above). In carrying out the present invention, these preferred techniques and other preferred techniques may be used or suitably adapted to provide a library in which one or more VH sequences, selected to specifically bind to a Tn epitope or an STn epitope, are suitably combined with the desired VL sequence. This provides a library of the present invention as further described herein, where the size and / or diversity of the library is provided primarily by the different VL sequences (set or repertoire) present in the library.

[0049] Accordingly, the libraries of the present invention may contain or include (together with libraries based on a collection or repertoire of naive VL sequences forming a preferred embodiment of the present invention) a collection or repertoire of naive VL sequences, a collection or repertoire of synthetic VL sequences, or a collection or repertoire of semi-synthetic VL sequences, in each case combined with VH sequences as further described herein. In this regard, the presence / use of immunorepertoires of VL sequences is not excluded from the present invention in its broadest sense, but it should be noted that the use of immunorepertoires is generally less desirable; for this reason, VL sequences derived from immunoVH / VL repertoires may often require pairing with specific VH sequences associated with that immunorepertoire to provide the specificity intended for the purposes of the present invention.

[0050] Furthermore, in antibodies obtainable from each of these libraries (i.e., by screening and selection as further described herein), if the VH sequence present in such antibodies confers specificity to Tn epitopes, the VL domain of such antibodies is preferably capable of binding to a portion of a peptide backbone associated with a Tn epitope to which the VH sequence in the antibody can bind at the intended or desired target. Similarly, in antibodies obtainable from each of these libraries (i.e., by screening and selection as further described herein), if the VH sequence present in such antibodies confers specificity to STn epitopes, the VL domain of such antibodies is preferably capable of binding to a portion of a peptide backbone associated with an STn epitope to which the VH sequence in the antibody can bind at the intended or desired target. In each case, the VH domain (or its sequence) preferably does not essentially contribute to the binding of the antibody to the peptide epitope (i.e., to the binding site of the target peptide backbone).

[0051] As further described herein, the methods and libraries provided by the present invention can be used to obtain, identify and / or create antibodies (or sequences encoding antibodies) against proteins / targets present / expressed in particular on cancer cells; in particular to obtain, identify and / or create antibodies (or sequences encoding antibodies) against proteins / targets present / expressed on glycosylated cancer cells; and even more particularly to obtain, identify and / or create antibodies (or sequences encoding antibodies) against proteins / targets present / expressed on glycosylated cancer cells, where the glycosylated type (as further described herein) comprises, contains, or expresses one or more Tn and / or STn epitopes.

[0052] Such proteins / targets, as well as the types of cancer cells on which they are expressed on the surface (and may be overexpressed compared to healthy cells and / or other cancer cells), and the types of cancer cells to which such proteins, targets, and cells are associated, are obvious to those skilled in the art, for example: EGFR, VEGFR, HER2, CD37, FLT3, FGFR, CD19, CD22, CD27, CD25, CD30, CD33, CD38, CD43, Mesothelin, PD-L1, CD44, Podocalyxin (TRA 1.60 / 80), CD133, CD90, CD326, Cripto-1, ABCG2, CD24, CD49, Notch2, CD146 (MUC18), CD1 0, CD117, CD26, CXCR4, CD34, CD271, CD13, CD56, CD105, LGR5, CD114, CD54, CXCR1, TIM-3, CD55, DLL-4, CD 96, CD29, CD9, CD166, CD44, ABCB5, Notch3, CD123, MUC1, MUC4, MUC13, MUC16, MUC17, MUC21, CD33, CD40, Examples include, but are not limited to, Rhodin 18, Integrin α-3, CK7, CK20, CXCR2, CXCR4, Integrin α-5, CA9, EPCAM, MET, MMP14, DDR1, NRP1, LAMP1-4, CD99, ALCAM, SDC1, 2, 3, 4, Syndecanes (1-4), ITA5, ROBO1, Nectin-4, Nectin-2, LRP1, CD70, Podoplanin, IGF-1R, ABCG2, IBT1, ALK, TEM1, HVEM, TERT, LYPD3, GPR56, IL6RA, DDR1, LIFR, GPR64, S1PR1-4, LRCHD2, NMB, Plexin 1 / 2, PTHR, Semaphorins, Slit3, TACD2, VGFC, VLDLR, VTN, LAG-3, Fn14, and S1PR3.

[0053] Generally, when it is desired to create antibodies to treat a specific type of cancer, a person skilled in the art in the field of oncology can select proteins or targets associated with cancer cells contained in that type of cancer, and then use antibodies created using the methods and libraries described herein against those targets to treat that type of cancer. Alternatively, a person skilled in the art can determine which (glycosylated) proteins are overexpressed in the cancer cells of that type, and then use the methods and libraries of the present invention to create one or more antibodies against such proteins. Furthermore, if it becomes possible for a person skilled in the art to create antibodies targeting proteins expressed on cancer cells obtained from a patient to be treated, and then use those antibodies to treat that patient, then the latter aspect of the present invention is considered to have applications in the realm of so-called "personalized medicine."

[0054] In one aspect of the present invention, the method and library of the present invention are used for the purpose of creating antibodies against proteins / targets belonging to the MUC family that are expressed (as glycosylated forms) on cancer cells.

[0055] The present invention also relates to a method for treating cancer, comprising administering to a patient in need one or more antibodies obtained from the library and / or using the method herein, in a therapeutically effective amount. In a particular aspect, the present invention further relates to a method for treating cancer, comprising administering to a patient with cancer one or more antibodies obtained from the library and / or using the method herein, in a therapeutically effective amount, wherein the antibodies target (glycosylated) proteins present in / expressed by cancer cells in the body of the patient being treated. The present invention also relates to antibodies for cancer treatment that are obtainable from the library and / or using the method herein. In each aspect, the antibodies used may be antibodies as further described herein. In a particular aspect, such antibodies target proteins / targets belonging to the MUC family (as further described herein).

[0056] With regard to the creation, selection, and / or use of specific antibodies as described herein for the treatment of specific types of cancer, it should be noted that in certain types of cancer, the proteins expressed on the surface of cancer cells express Tn antigens primarily or almost entirely, while in other types of cancer cells, the proteins expressing STn antigens primarily or almost entirely, are expressed, as described, for example, in Rome, TB et al., Brit J Canc 125, 1239-1250 (2021); Jiang, Y. et al., J Cell Mol Med 22, 4875-4885 (2018); and Tsuchiya et al., Breast Canc 6, 175-180 (1999).

[0057] For example, as Romer et al. have noted, Tn antigen expression is observed mainly in tumors of the breast, colon, and pancreas, while STn antigen is highly expressed in tumors of the colon and pancreas.

[0058] Accordingly, a further aspect of the present invention relates to the use of antibodies obtainable from the library and / or obtained using the methods described herein for the treatment of cancer, wherein the tumor cells and / or cancer types relating to the present invention express proteins containing or expressing the Tn antigen on their surface, and the antibodies relating to the present invention contain a VH domain that confers specificity to the Tn antigen; and / or the antibodies relating to the present invention are obtained from a library constructed using VH sequences that confer specificity to the Tn antigen as described herein. As will be apparent to those skilled in the art based on the disclosure herein, the VL sequences present in such antibodies are sequences as further described herein, and in particular may confer specificity to proteins (or their protein backbone) present or expressed on cancer cells containing or expressing the Tn antigen.

[0059] Another aspect of the present invention relates to the use of antibodies obtainable from the library and / or using the method herein, or the use of obtained antibodies for the treatment of cancer, wherein the tumor cells and / or cancer types relating to the present invention primarily express proteins containing or expressing STn antigens on their surface, and the antibodies relating to the present invention contain a VH domain that confers specificity to STn antigens; and / or the antibodies relating to the present invention are obtained from a library constructed using VH sequences that confer specificity to STn antigens as described herein. As will be apparent to those skilled in the art based on the disclosure herein, the VL sequences present in such antibodies may, as will be further described herein, confer specificity to proteins (or their protein backbones) present on or expressed on cancer cells containing or expressing STn antigens.

[0060] In a further aspect, the present invention relates to antibodies obtainable from and / or obtained using the libraries described herein and / or the methods described herein.

[0061] In a particular aspect, such an antibody contains as a VH domain the amino acid sequence that is the VH sequence of SEQ ID NO: 1, or a variant of the VH sequence of SEQ ID NO: 1 (preferably a variant as further described herein), or a VH sequence having a CDR identical to the CDR sequence present in the VH sequence of SEQ ID NO: 1, or a VH sequence having a CDR derived from the CDR sequence present in the VH sequence of SEQ ID NO: 1. The CDR sequence present in the VH sequence of SEQ ID NO: 1 is the following sequence: CDR1=DHAIH(array code:155); CDR2=YISPGNDDIKYNEKFKG(array code:156); CDR3=SLPGTFDY(Sequence ID: 157).

[0062] In another specific aspect, such an antibody contains as a VH domain the amino acid sequence that is the VH sequence of SEQ ID NO: 28, or a variant of the VH sequence of SEQ ID NO: 28 (preferably a variant as further described herein), or a VH sequence having a CDR identical to the CDR sequence present in the VH sequence of SEQ ID NO: 28, or a VH sequence having a CDR derived from the CDR sequence present in the VH sequence of SEQ ID NO: 28. The CDR sequence present in the VH sequence of SEQ ID NO: 28 is the following sequence: CDR1=DHAIH(array code:155); CDR2=YISPGNDDIKYNEKFKG(array code:156); CDR3=SLLALDY(Sequence ID: 158).

[0063] Therefore, in a further aspect, the present invention relates to antibodies, in particular antibodies obtainable or acquired using the methods described herein and / or obtainable or acquired from libraries such as those described herein, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: • CDR1 having an amino acid sequence DHAIH (SEQ ID NO: 155) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155); and CDR2 having an amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156) or an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) with respect to the amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156); and • CDR3 having an amino acid sequence SLPGTFDY (SEQ ID NO: 157) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLPGTFDY (SEQ ID NO: 157); And the VL domain hereof is a VL domain as further described herein, in particular a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of a cancer cell (and more particularly a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of a cancer cell, wherein the glycosylated protein contains or expresses a Tn antigen); and / or a VL domain that confers to the antibody the ability to specifically bind to a protein present or expressed on the surface of a cancer cell (wherein particularly the protein is glycosylated, and more particularly glycosylated to contain or express a Tn antigen).

[0064] As will be obvious to those skilled in the art based on the disclosure herein, in an antibody according to the above embodiment: If the VH sequence in the antibody includes a CDR1 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155), such a CDR1 preferably includes amino acid residues H32, A33, and H35; If, in the VH sequence of the antibody, CDR2 is an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from the amino acid sequence YISPGNDDIKYNEKFKG (Sequence ID: 156), then such CDR2 preferably comprises amino acid residues Y50, S52, N55, and D57; and If the VH sequence in the antibody includes a CDR3 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLPGTFDY (SEQ ID NO: 157), such a CDR3 preferably includes amino acid residue S99.

[0065] In certain, but non-limiting, embodiments of the present invention relate to antibodies, in particular antibodies obtainable or acquired using the methods described herein and / or obtainable or acquired from libraries such as those described herein, comprising a VH domain and a VL domain, wherein the VH domain comprises CDR1 having the amino acid sequence DHAIH, CDR2 having the amino acid sequence YISPGNDDIKYNEKFKG, and CDR3 having the amino acid sequence SLPGTFDY (SEQ ID NO: 157), where the VL domain is a VL domain as further described herein. and, in particular, a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of cancer cells (and, more particularly, a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of cancer cells, wherein the glycosylated protein contains or expresses a Tn antigen), and / or a VL domain conferring the antibody the ability to specifically bind to a protein present or expressed on the surface of cancer cells (wherein, in particular, the protein is glycosylated, and, more particularly, glycosylated to contain or express a Tn antigen).

[0066] In a further aspect, the present invention relates to antibodies, in particular antibodies obtainable or acquired using the methods described herein and / or obtainable or acquired from libraries as described herein, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: • CDR1 having an amino acid sequence DHAIH (SEQ ID NO: 155) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155); and CDR2 having an amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156) or an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from the amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156); and • CDR3 having an amino acid sequence SLLALDY (SEQ ID NO: 158) or an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLLALDY (SEQ ID NO: 158); and herein the VL domain is a VL domain as further described herein; in particular a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of a cancer cell (and more particularly a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of a cancer cell, wherein the glycosylated protein contains or expresses an STn antigen), and / or a VL domain that confers to the antibody the ability to specifically bind to a protein present or expressed on the surface of a cancer cell (wherein particularly the protein is glycosylated, and more particularly glycosylated to contain or express an STn antigen).

[0067] As will be obvious to those skilled in the art based on the disclosure herein, in an antibody according to the above embodiment: If the VH sequence present in the antibody includes a CDR1 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence DHAIH (SEQ ID NO: 155), such a CDR1 preferably includes amino acid residues H32, A33, and H35; If, in the VH sequence of the antibody, CDR2 is an amino acid sequence having three, two, or one (and preferably two or one, and most preferably one) amino acid differences (as defined herein) from the amino acid sequence YISPGNDDIKYNEKFKG (Sequence ID: 156), then such CDR2 preferably comprises amino acid residues Y50, S52, N55, and D57; and If the VH sequence present in the antibody includes a CDR3 which is an amino acid sequence having a single amino acid difference (as defined herein) from the amino acid sequence SLLALDY (SEQ ID NO: 158), such a CDR3 preferably includes amino acid residues S99, L101, A102, and L103.

[0068] In a further aspect, the present invention relates to antibodies, in particular antibodies obtainable or acquired using the methods described herein and / or obtainable or acquired from libraries as described herein, wherein the antibody comprises a VH domain and a VL domain; wherein the VH domain comprises CDR1 having the amino acid sequence DHAIH (SEQ ID NO: 155), CDR2 having the amino acid sequence YISPGNDDIKYNEKFKG (SEQ ID NO: 156), and CDR3 having the amino acid sequence SLLALDY (SEQ ID NO: 158); and wherein the VL domain comprises V as described herein further. An L domain, in particular a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of cancer cells (and more particularly a VL domain capable of binding to the peptide backbone of a glycosylated protein present or expressed on the surface of cancer cells, wherein the glycosylated protein contains or expresses an STn antigen), and / or a VL domain that confers to the antibody the ability to specifically bind to a protein present or expressed on the surface of cancer cells (wherein particularly the protein is glycosylated, and more particularly glycosylated to contain or express an STn antigen).

[0069] Antibodies according to the above embodiments may be antibodies as further described herein. Further aspects of the present invention relate to nucleotide sequences encoding antibodies according to the above embodiments; host cells and / or host organisms that can be expressed or used for the production of antibodies according to any of the above embodiments; pharmaceutical compositions comprising at least one antibody according to any of the above embodiments; and uses of antibodies according to any of the above embodiments, all of which are preferably as further described herein.

[0070] Description of the present invention Definitions and abbreviations Unless otherwise specified, the term "nucleic acid" as used herein includes both double-stranded and single-stranded nucleotide molecules. When nucleic acid sequences are shown, they are listed in the 5′ to 3′ direction unless otherwise specified.

[0071] The terms "homology," "similarity," or "sequence identity" relating to two types of proteins are determined by comparing their amino acid sequences and examining the conserved amino acid substitutions in the second protein sequence relative to the first protein sequence. Similarity may be determined by means well known in the art, such as the BLAST program (Basic Local Alignment Search Tool from the National Center for Biotechnology Information). Similarly, homology, similarity, or sequence identity between two types of nucleic acid sequences may be determined by means well known in the art, such as the BLAST program (Basic Local Alignment Search Tool from the National Center for Biotechnology Information).

[0072] An "amino acid residue substitution" at a specific location means replacing a native amino acid residue at that particular location with a different amino acid. In conservative amino acid substitutions, the substitution is made by replacing an amino acid with another amino acid that is similar in size and chemical properties, so that the substitution has no effect or only a minor effect on the protein's structure and function; on the other hand, in non-conservative amino acid substitutions, the amino acid is replaced with another dissimilar amino acid, which is likely to affect the protein's structure and function.

[0073] In this specification, the term "antibody" should be understood to include a protein having a Y-shape with two characteristic arms, typical of an antibody molecule, and one or more antibody fragments that possess the ability to specifically bind to an antigen. Examples of antibodies include: Monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, transplant antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fv(scFv) (containing fragments in which VL and VH are linked by a synthetic or natural linker using a recombination method that allows VL and VH to be created as a single protein chain so that a pair of VL and VH regions forms a monovalent molecule; including single-chain Fab and scFab), single-chain antibodies, Fab fragments (containing monovalent fragments containing VL, VH, CL, and CHI domains), F(ab′)2 fragments (bivalent fragments containing two Fab fragments linked by disulfide crosslinking at the hinge region) Examples include, but are not limited to, fragments (including fragments containing VH and CHI fragments), Fd fragments (including fragments containing VL and VH domains of a single arm of the antibody), single-domain antibodies (dAb or sdAb) (including fragments containing the VH domain), isolated complementarity-determining regions (CDRs), diabodies (including fragments containing a bivalent dimer that recognizes two different antigens, such as two VL domains and two VH domains bound to each other), fragments contained in a single monomer variable domain, disulfide-linked Fv (sdFv), intrabodies, anti-idiotype (anti-Id) antibodies, or their ab antigen-binding fragments.

[0074] As those skilled in the art would understand, the term "VL" refers to the antibody variable domain, or light chain; on the other hand, the term "VH" refers to the antibody variable domain, or heavy chain.

[0075] In this specification, the term "Tn antigen" refers to N-acetylgalactosamine (GalNAc), a monosaccharide structure linked to serine (Ser) or threonine (Thr) of the peptide backbone by a glycosidic bond (i.e., GalNAcα1-O-Ser / Thr). Its abbreviation (Tn) is derived from Thomsen-nouveau. The Tn antigen is expressed in most cancers. In this specification, the term "Tn-glycosylem epitope" (or "Tn epitope") refers to the GalNAc portion of the Tn antigen. The term "mono-Sn" refers to the Tn portion (i.e., single GalNAc). The term "bis-Tn" refers to two Tn portions (i.e., 2GalNAc).

[0076] The term “STn antigen” as used herein refers to a sialyl-Tn antigen formed by the extension of a Tn antigen having sialic acid (Neu5Ac(a2-6)GalNAc) and in which linkage to serine (Ser) or threonine (Thr) is maintained (i.e., Neu5Acα2-6GalNAcα1-O-Ser / Thr). Such STn antigens are also common in cancer tumor cells. Both Tn and STn may undergo further modifications such as phosphorylation, acetylation, methylation, and sulfonation. The term “STn-glycan epitope” (or “STn epitope”) as used herein refers to the Neu5Ac(a2-6)GalNAc portion of a Tn antigen. The term “mono-STn” refers to a single STn portion (i.e., a single Neu5Ac(a2-6)GalNAc). The term "bis-STn" refers to two STn parts (i.e., two Neu5Ac(a2-6)GalNAc).

[0077] The term "glycoprotein" generally refers to a protein containing oligosaccharide chains covalently bonded to amino acid side chains. In this specification, the term "glycoprotein" refers to a protein or peptide containing a sugar chain portion (preferably a Tn epitope or STn epitope) covalently bonded to an amino acid residue (such as serine, threonine, or tyrosine; or any unnatural amino acid derivative thereof, such as by substituting O with S) of the protein or peptide.

[0078] The term “combotope” as used herein refers to a combination of a glycan epitope and a peptide epitope recognized by an antibody. These two types of epitopes form a “combotope” which is a common epitope distinct from either of the two epitopes that constitute it. A combotope in which the peptide epitope associates with a Tn epitope or an STn epitope is suitable for the present invention; the peptide epitope may associate with one or two Tn epitopes or STn epitopes. The combotope may be continuous or discontinuous; that is, the glycan epitope may be directly bound to the peptide epitope by covalent bond (continuous), or the glycan epitope may be linked to an amino acid residue located upstream or downstream of the peptide epitope at 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues (discontinuous). Preferably, the sugar chain epitope is covalently bonded to the peptide epitope.

[0079] The glycan epitope may also be a combination of two Tn epitopes or two STn epitopes (referred to as bis-Tn and bis-STn), or it may be one Tn epitope and one STn epitope located on adjacent amino acids in the peptide sequence of the glycoprotein. "Adjacent" means separated by 0 to 2 amino acids.

[0080] Antibodies called "combotope binders" recognize and bind to combinations of both the sugar chain epitopes and peptide epitopes of the glycoprotein in question. Such antibodies are also called "combotope antibodies."

[0081] In this application, the term "hapten" refers to a glycan portion (Tn or STn) independent of the peptide / protein. Antibodies referred to as "hapten binders" bind to a Tn or STn epitope independent of the peptide / protein carrier; therefore, they are not specific to both the glycan epitope and the peptide epitope of the glycoprotein (i.e., they are not combotope binders).

[0082] In this specification, the expression "domain that binds to ~" should be understood to mean "a domain suitable for binding to ~," "a domain that can bind to ~," and / or "a domain prepared for binding to ~." [Brief explanation of the drawing]

[0083] [Figure 1] This is a schematic diagram of an embodiment of the present invention. A Tn-template antibody library for identifying Tn-combotope antibodies. Phage display of the scFv antibody library. Each scFv in the library has a Tn-binding VH domain. The library is screened for Tn-peptide-specific scFv by biopanning using Tn-peptides. [Figure 2] This is a schematic diagram of the preparation of the phage display library of the present invention. (1) Isolation of mRNA from mouse spleen. (2) cDNA synthesis by reverse transcriptase using a random hexamer. (3) PCR amplification from a cDNA template using a specific primer set for the purpose of obtaining the VH domain; also, PCR amplification from a cDNA template using a VL primer mixture for the purpose of obtaining the VL domain repertoire. (4) PCR assembly of the VL-domain repertoire and specific VH-domain using 5′ phosphorylated outer primers. (5) Rolling circle amplification; in this method, the phosphorylated scFv gene is linked as a circular DNA by ligation; the dsDNA is denatured; annealed with a random hexamer; and the circular fragment is amplified as a long linear concatemer with Phi29 polymerase. (6) The amplified elongated scFv gene is digested with the restriction enzyme sfiI, and then ligated and linked to the phagemide vector pAK100 treated with sfiI and rSAP. (7) Electroporation is used to introduce a pool of phagemids containing the scFv gene into E. coli cells TG1. (8) Amplification of bacterial libraries containing different phagemids and infection with the helper phage VCSM13 to generate complete phages expressing scFv in the pIII coat protein. [Figure 3]X-rays of G2D11 scFv with APGS*T*AP peptide (where * represents a GalNac residue) reveal interaction sites between VH and the glycan structure. Major interaction sites with two adjacent GalNac residues include His32H, Ala33H, His35H in CDR1; His40H, Ser52H, Asn55H, Asp57H in CDR2; and Ser99H in CRD3. [Figure 4] VH domain sequence alignment with G2D11 aligned with other known VH domains. Conserved amino acid residues are indicated by arrows (H32, A33, H35, Y50, S52, N55, D57, and S99). [Figure 5] Phage and sequence enrichment after each biopanning (1st, 2nd, and 3rd) for bis-Tn-MUC1. Phage enrichment for the bis-Tn-MUC1 target peptide was confirmed by ELISA of polyclonal phages. Bis-Tn MUC1 = peptide number 1 in Table 1; Tn MUC1 = peptide number 3 in Table 1; SA = negative control. [Figure 6] The expression of scFv expressed overnight in a 96-well format was subjected to His tag detection by dot blot analysis, and based on this, the expression of MUC1 scFv and selected clones was confirmed. [Figure 7] MUC1 monoclonal ELISA. Screening of monoclonal scFv using MUC1 target and control peptides. [Figure 8] Binding assay and kinetic affinity of MUC1 scFv. (A) Titration of scFv against fixed concentration MUC1 target peptide (peptide number 1 in Table 1). (B) Titration of scFv against fixed concentration IgA hinge region control peptide (peptide number 8). Each data point is the mean of three independent experiments. (C) Representative histograms of cell binding of 1.25 μg / ml A3, D2, and D3 scFv, as well as 5E5 mAb, to MDA-MB-231 WT and COSMC KO cells. Flow cytometry experiments were repeated three times. [Figure 9]MUC1 scFv titrations were performed on MUC1 peptides 2, 3, 4, and 5. MUC1 scFvs titrations were also performed on different MUC1 glycopeptides and non-glycosylated MUC1. Asterisks indicate glycosylation sites. [Figure 10] Biological evaluation of MUC1 scFv by flow cytometry. (A) Negative binding of MUC1 scFv to HEK293 cells as a negative control cell line. (B) MCF7 cells at a concentration of 1.25 μg / mL. (C) Representative example of concentration-dependent binding to MDA-MB-231 WT and COSMC KO cells. Shows scFv D3 at 4-fold dilution starting from 5 μg / mL. [Figure 11] X-ray structure of 5E5 scFv with APGST*AP peptide (asterisks represent GalNac residues). Tyr98L and the conserved Tyr100L are key amino acids in the recognition of the peptide backbone. [Figure 12] Heatmaps of the binding of scFv D3, scFv A4, scFv 5E5, scFv 2D9Chi, and scFv G2D11 to the Tn-glycopeptides in Table 5. The glycopeptides were printed on a microarray chip. For brevity, the heatmaps show amino acids 9-19 of the peptides in Table 5. The heatmaps are shown in relative fluorescence units (RFU). The Tn-glycosylation site is shown in bold and underlined. Ala substitutions are shown in bold. [Figure 13] Polyclonal phage enrichment between three selections for bis-Tn-CD43 target peptide (peptide number 9 in Table 1) and control peptide (peptide number 10 in Table 1). [Figure 14] CD43 monoclonal ELISA. Screening of monoclonal scFv against CD43 target and control peptides. [Figure 15]CD43 scFv binding assay. (A) Eight scFvs were titrated against the bis-Tn-CD43 target peptide (peptide number 9 in Table 1). (B) In ScFv titration against the IgA1 hinge region control glycopeptide (peptide number 8 in Table 1), A7 and D3 showed cross-reactivity to IgA, while A1 and F4 did not bind to IgA1 very strongly. Each dot represents the average value of three independent experiments. (C) Representative histograms of Jurkat cells tested with 1.25 μg / mL of A1, D7, H1, and H2 scFv before and after neuraminidase treatment. Flow cytometry experiments were repeated three times. [Figure 16] Biological evaluation of CD43 scFv by flow cytometry. Representative examples of concentration-dependent binding of A1 scFv to HEK293 and Jurkat cells before and after neuraminidase treatment. scFv was started at 5 μg / mL and diluted 4-fold. [Figure 17] X-ray structure analysis of CD43 with the GAS*T*GSP peptide reveals the importance of Tyr99L as a major interaction site with the peptide backbone. [Figure 18] Alignment of bis-Tn binder G2D11 VH and mono-Tn binder 3F1 VH, aligned to identify amino acid residues associated with conversion to anti-bis-STn. [Figure 19] Microarray data of mutants (M1-4) containing selected mutations of G2D11, 3F1, and VH-G2D11, wherein the data are microarray data of binding to glycopeptides 1 (bis-TnMUC1), 11 (bis-STnMUC1), 12 (mono-STnMUC1), and 4 (non-glycosylated control). [Figure 20] Microarray data of STnMUC1-D4, D3, C7 scFv, showing binding to glycopeptides 1 (bis-TnMUC1), 11 (bis-STnMUC1), and 12 (mono-STnMUC1). [Figure 21]ELISA titration screening of humanized scFv against coated Tn-MUC1 and other Tn-proteins. (A) D3L1H1scFv, (B) D3L1H2scFv, (C) D3L2H3scFv, (D) D3L3H4scFv, (E) D3L4H5scFv, and (F) mouse parent D3. MUC1 = mucin 1, MUC21 = mucin 21, GPNMB = transmembrane glycoprotein NMB, EGFR = epidermal growth factor receptor, VVL = Vicia Villosa lectin used to detect Tn on Tn-proteins. [Figure 22] (A) Schematic diagrams of bis TnMUC1, (B) mono Tn(Thr)MUC1, and (C) mono Tn(Ser)MUC1. [Figure 23] Elisa titration of different target Tn-peptides detected by mouse D3 (1 μg / mL) scFv on streptavidin-coated plates. Column 1: Biotin-2OEG-2OEG-HSSSTIPTPA (MUC13), Column 2: Biotin-2OEG-2OEG-HSSSTIPIPT (MUC13), Column 3: Biotin-2OEG-2OEG-SESTTNVNSL (MUC13), Column 4: Biotin-2OEG-2OEG-ITASSPNDGL (MUC13), Column 5: Biotin-2OEG-2OEG-MSPTTEDNQz (MYC13), Column 6: Biotin-2OEG-2OEG-DNQSSGPPTG (MUC13), Column 7: Biotin-2OEG-2OEG-LHNTSFCLCL(MUC13), Column 8: Biotin-2OEG-2OEG-YNSSTCKKGK(MUC13), Column 9: Biotin-2OEG-2OEG-IRSSSSNFLN(MUC13), Column 10: Biotin-2OEG-2OEG-CVASSLKCPD(MUC13), Column 11: Biotin-2OEG-2OEG-SITSTGLTSP(MUC4), Column 12: Biotin-2OEG-2OEG-APGSTAPPAH(MUC1). [Modes for carrying out the invention]

[0084] The present invention relates to a novel antibody concept technology for the simple and rapid development of antibodies targeting Tn- and STn glycosylation sites of any selected glycoprotein site. Specifically, the present invention relates to an antibody library capable of screening antibodies having improved specificity due to specificity to a combination of epitopes in the sugar chain portion of a glycoprotein and epitopes in the peptide backbone associated with the sugar chain epitope in the glycoprotein. Such combination epitopes are referred to as "combotopes". A non-limiting embodiment of the present invention is schematically shown in Figure 1.

[0085] This invention is particularly useful in the discovery of therapeutic and diagnostic agents for cancer.

[0086] [I] Conbotope antibody The present invention provides a combotope antibody that has high specificity and high binding efficiency to target glycopeptides due to their combined specificity for both glycan epitopes and peptide backbone epitopes associated with glycoprotein target glycan epitopes. In a preferred embodiment, the present invention provides a combotope antibody that targets tumor cells having glycoproteins on their cell surface.

[0087] In one aspect, the present invention provides an antibody that targets tumor cells, the antibody comprising two antibody domains; the first antibody domain binding to a glycosylation epitope of a glycoprotein of the tumor cell; and the second antibody domain binding to a peptide epitope of a glycoprotein of the tumor cell; the antibody is selected to specifically bind to both epitopes (as normal epitopes) compared to binding to only one of the epitopes.

[0088] In one embodiment, the first antibody domain is a VH domain, and the second antibody domain is a VL domain. In another embodiment, both the first and second antibody domains are VH domains, but they are distinct from each other.

[0089] In one preferred embodiment, the antibody disclosed herein is an scFv antibody comprising a VH domain and a VL domain, where both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form an antigen-binding structure. In one embodiment, the linker is (GGGGS) n It is selected from, where n is 1, 2, 3, 4, 5, or 6; for example, (GGGGS)4 or (GGGGS)5. (GGGGS) n Other linkers can be used as alternatives to the linker. The methods for selecting such linkers will be understood by those skilled in the art.

[0090] In one aspect, the present invention provides an antibody that targets tumor cells, but the antibody is: (i) A VH domain that binds to the glycosylation epitope of the glycoprotein of the tumor cell; and (ii) The VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; Here, the antibody is selected to specifically bind to both epitopes (as a normal epitope) compared to binding to only one of the epitopes.

[0091] In one embodiment, the present invention provides an antibody that targets a glycoprotein (such as a specific glycoprotein on tumor cells). The glycoprotein comprises a glycan epitope and a peptide epitope. The glycan epitope is a short, truncated O-glycan (such as Tn or STn). The peptide epitope is associated with the glycan epitope (either directly associated by chemical linkage or in close proximity to each other due to the structural arrangement of the glycoprotein). The antibody of the present invention is characterized by its binding ability to specifically bind to both epitopes (as normal epitopes) compared to binding to only one of the epitopes. In one embodiment, the antibody is: (i) (a) Suitable for binding to glycosylation epitopes of tumor cell glycoproteins; and (b) Not binding to the peptide epitope of the glycoprotein in the tumor cell, VH domains characterized by; and (ii) A VL domain characterized by being suitable for binding to the peptide epitope of the glycoprotein of the tumor cell, This includes, in one embodiment, the VL domain is further characterized by not binding to the glycan epitope of the glycoprotein. In a particular embodiment, the antibody binds only to a combination of glycan and peptide epitopes; that is, both epitopes require expression for the antibody to bind.

[0092] In one embodiment, the present invention provides an antibody that binds to tumor cells, wherein the antibody is: (i) VH-domains that bind to glycosylation epitopes of glycoproteins in tumor cells; and (ii) The VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; Here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; Furthermore, the antibody is specific to the combination of glycan epitopes and peptide epitopes on the glycoproteins of the cancer cells.

[0093] In one embodiment, the present invention provides an antibody that binds to tumor cells, wherein the antibody is: (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell; and (i) A VH-domain that binds to a glycosylation epitope of a glycoprotein of the tumor cell, and does not contribute to or inhibit the binding of the glycoprotein of the tumor cell to a peptide epitope, that is, a VH-domain whose binding is not affected by the presence of any peptide epitope. Includes; And here the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; Furthermore, the antibody is specific to the combination of the glycan epitope and the peptide epitope on the glycoprotein of the cancer cell.

[0094] In one embodiment, the present invention provides an antibody that binds to tumor cells, wherein the antibody is: (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell; and (i) A VH-domain that binds only to the glycosylation epitopes of the glycoproteins of the tumor cells, i.e., a VH-domain that does not contribute to or inhibit the binding of peptide epitopes of the glycoproteins of the tumor cells, Includes; And here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; And herein, the antibody is specific to the combination of the glycan epitope and the peptide epitope on the glycoprotein of the cancer cell.

[0095] As disclosed herein, the antibodies of the present invention are specific to a combination of a peptide epitope of a glycopeptide in a tumor cell (such as on the surface of the tumor cell) and a glycan epitope that is a short, truncated O-glycan. The term “specific” in this context refers to a specific antibody that is highly selective to a particular glycoprotein, exhibiting strong binding and recognition to that glycoprotein, even though it shows minimal or no binding to other types of glycoproteins. Specificity may be expressed by evaluating the binding affinity of the antibody to the glycoprotein using biophysical techniques that would be understood by those skilled in the art. The antibodies of the present invention recognize both the glycan portion and the peptide sequence of the glycoprotein, which makes them highly specific.

[0096] In one embodiment, the peptide epitope recognized by the VL domain is a portion of a specific glycoprotein present on the surface of a particular type of cancer cell.

[0097] Furthermore, the combotope antibody of the present invention is selected to be specific to the combined glycoprotein epitope, that is, to the glycan epitope (combotope) combined with the peptide epitope, but not to each of these epitopes individually.

[0098] In one embodiment, the present invention provides an antibody that binds to one or more tumor cells, wherein the one or more tumor cells include a short, truncated O-glycan epitope that associates with a glycan epitope, preferably a peptide epitope, and the antibody is specific to both the glycan epitope and the peptide epitope.

[0099] In one embodiment, the specific antibody does not specifically bind to combinations other than the particular combotope, nor does it bind to (or has low binding affinity to) the glycan epitope or the peptide epitope individually.

[0100] In one embodiment, the present invention provides an antibody that binds to tumor cells; Here, the tumor cells contain short, truncated O-glycans that associate with glycan epitopes, preferably peptide epitopes; Here, an antibody containing the following (i) and (ii) is specific to the combination of the glycan epitope and the peptide epitope: (i) A VH-domain that binds to the glycan epitope; and (ii) A VL domain that binds to the peptide epitope.

[0101] Preferably, the glycan epitope is a part of a glycoprotein, and the peptide epitope is a part of the same glycoprotein. The glycan epitope and peptide epitope of the tumor are preferably surface-exposed to facilitate recognition by the antibody.

[0102] In a preferred embodiment, the glycan epitope to which the VH-domain binds is selected from mono-Tn, bis-Tn, mono-STn, bis-STn, and / or a combination of mono-Tn and mono-STn. The glycan epitope may contain one or more adjacent short truncated O-glycans, i.e., Tn, Tn·Tn, STn, STn·STn, or consist of the same. Thus, in a preferred embodiment, the present invention provides an antibody that binds to tumor cells, wherein the antibody is: (i) VH-domains that bind to the monoTn, bisTn, monoSTn, bisSTn, and / or monoTn+monoSTn glycosylation epitopes of the tumor cells; and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; Here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; Here, the antibody is specific to the combination of glycosylation epitopes and peptide epitopes of the glycoproteins of the cancer cells.

[0103] As disclosed herein, the target peptide epitope of the cell is associated with the glycan epitope. The term “associated with” preferably means that the peptide epitope and the glycan epitope are a continuous, uninterrupted epitope, i.e., the glycan is directly bound to the peptide by chemical linkage (such as covalent linkage). In another embodiment, the peptide epitope and the glycan epitope may be discontinuous epitopes, where “associated with” also means that the peptide epitope and the glycan epitope are in close proximity to each other, but that proximity is due to a molecular structure arrangement that facilitates the proximity. Thus, a discontinuous epitope is an epitope in which the amino acids surrounding the associated glycan epitope are not part of the peptide epitope.

[0104] In one embodiment, the present invention provides a tumor cell-binding antibody, the antibody being: (i) A VH-domain that binds to a glycosylation epitope of the glycoprotein of the tumor cell (preferably, the glycosylation epitope is one or more short truncated O-glycans); and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; Here, the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein, but not to other combinations of epitopes or to any one of the epitopes alone.

[0105] In one embodiment, the present invention provides a tumor cell-binding antibody, the antibody being: (i) A VH-domain that binds to a Tn- and / or STn-glycan epitope of a glycoprotein of the tumor cell (preferably the glycan epitope is one or more short truncated O-glycans); and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell; Includes; Here, the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein, but not to other combinations of epitopes or to any one of the epitopes alone.

[0106] The sugar chain epitope is composed of one or more short truncated O-glycans, where the short truncated O-glycans are selected from Tn and STn.

[0107] A Tn epitope or STn epitope may be formed by one or more Tn or STn moieties. In one embodiment, the Tn epitope or STn epitope interacting with the VH domain is a single Tn moiety or a single STn moiety, respectively. In a preferred embodiment, the Tn epitope or STn epitope interacting with the VH domain is a single Tn moiety or a single STn moiety, and the Tn epitope or STn epitope is covalently bound to an amino acid residue, where the amino acid residue is part of a peptide epitope interacting with the VL domain.

[0108] In another embodiment, the Tn epitope or STn epitope is formed by two Tn moieties or two STn moieties, each of which interacts with the VH domain. In a preferred embodiment, the Tn epitope or STn epitope interacting with the VH domain consists of two Tn moieties and one STn moiety; the two Tn moieties or STn moieties are covalently bonded to two different amino acid residues, where the amino acid residues are part of the peptide epitope interacting with the VL domain. In such an embodiment, the two different amino acid residues are adjacent to each other. In another embodiment, the two different amino acid residues are separated by one, two, three, or four other amino acid residues.

[0109] As disclosed herein, the VL domain of the conbotope antibody binds to a peptide epitope of a glycoprotein in tumor cells. In one embodiment, the peptide epitope is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues, preferably 2, 3, or 4 amino acid residues, most preferably 4 amino acid residues. In one embodiment, the peptide epitope interacting with the VL domain is 2-4 amino acid residues, 4-6 amino acid residues, 6-8 amino acid residues, 8-10 amino acid residues, 10-12 amino acid residues (e.g., 2-12 amino acid residues, 2-10 amino acid residues, 2-8 amino acid residues, preferably 2-6 amino acid residues, most preferably 2-4 amino acid residues, etc.).

[0110] In the most preferred embodiment, the antibody of the present invention differs from the prior art antibodies 5E5, 5F7, and 2D9.

[0111] 5F7 is disclosed in U.S. Patent Application No. 11161911B2. 5E5 is disclosed in WO2008 / 040362 and U.S. Patent Application No. 2021060070A1, as well as in reference scientific literature: Macias-Leon et al., 2020; Tarp et al., 2007; and Blixt et al., 2010. 2D9 is disclosed in reference scientific literature: Sorensen et al., 2006; Tarp et al., 2007; and Blixt et al., 2010.

[0112] In one embodiment, the antibodies of the present invention differ from the prior art antibodies 5E5, 5F7, and 2D9; therefore, the antibodies of the present invention do not contain combinations of VL and VH domains as disclosed herein.

[0113] [Table 1]

[0114] In one embodiment, the amino acid sequence of the antibody of the present invention does not include a combination of amino acid sequences selected from SEQ ID NOs: 3+4, SEQ ID NOs: 5+6, and SEQ ID NOs: 7+8.

[0115] Preferably, the antibody of the present invention is a humanized antibody, such as one prepared by the method of Clavero-Alvarez et al. (2018). The "humanized" form of a non-human antibody may be a chimeric antibody containing the minimal sequence derived from the non-human antibody. A humanized antibody is generally a human antibody (recipient antibody) in which a selective residue in a non-human antibody (donor antibody) has been substituted. The donor antibody may be any suitable non-human (mouse, rat, rabbit, chicken, etc.) antibody; or it may be a non-human primate antibody having the desired specificity, affinity, or biological effect. With respect to the present invention, the donor antibody is preferably identified by screening the antibody library of the present invention. In some examples, a selective framework region residue of the recipient antibody is substituted with a corresponding framework region residue derived from the donor antibody. The humanized antibody may also contain residues that are not present in either the recipient antibody or the donor antibody. In some examples, these modifications are made to further enhance antibody performance. Those skilled in the art will be familiar with the method for converting the conbotope antibody of the present invention into a humanized conbotope antibody.

[0116] In a further aspect, the present invention provides nucleic acid sequences encoding antibodies according to the present invention as disclosed herein.

[0117] [Ii]Tn-combotope As disclosed herein, the VH domain of the conbotope antibody binds to the glycan epitope, preferably mono-Tn, bis-Tn, mono-STn, or bis-STN glycan epitope.

[0118] In one preferred scenario, the VH domain of the conbotope antibody binds to a Tn-glycan epitope (such as mono-Tn or bis-Tn).

[0119] The inventors unexpectedly discovered the following: Structural characterization in the presence of the bisTn-MUC1 peptide APGS*T*AP (where * represents the GalNAc moiety) revealed that the VH-domain of antibody G2D11 (SEQ ID NO: 1) recognizes two GalNAc moieties, but does not recognize the peptide sequence linking them (see Example 1). Sequence ID: 1 (VH-domain G2D11): QVQMQQSDAELVKPGASVKISCKASGYIFADHAIHWVKRKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS

[0120] The novel conbotope antibodies disclosed herein are based in part on these structural observations; namely, the observation that the VH chain of G2D11 supports recognition of the glycoside portion of the antigen, without being affected by / unaffected by the peptide portion of the glycoprotein, but does not bind to the peptide. Further development will identify the conbotope antibody as disclosed herein, where the VL-domain provides specific binding to the peptide epitope of the conbotope, while the VH-chain supports recognition of the glycosylation portion of the glycoprotein.

[0121] As disclosed in the background section, several anti-Tn antibodies are known in the art, many of which share the same germline sequence (at least essential sequence) as G2D11. However, it was not previously known that the VH domain of G2D11 provides exclusive support for the recognition of the Tn glycan epitope without binding to the peptide epitope. Therefore, while prior art anti-Tn antibodies may contain the same germline sequence as G2D11, which is important for the Tn-binding VH domain, they either have no binding contribution to the peptide / protein carrier or their binding contribution is unknown; thus, they are nonspecific Tn-binding antibodies and therefore not conbotope antibodies according to the present invention. On the other hand, the conbotopes of the present invention are screened using the antibody library of the present invention for the purpose of obtaining antibodies specific to a particular target glycoprotein, as further disclosed herein.

[0122] In one embodiment, the VH-domain of the conbotope antibody of the present invention is a G2D11-like VH-domain. In another embodiment, the amino acids of the VH-domain in the conbotope antibody of the present invention are similar to those of a G2D11-like VH-domain in their structural conformation.

[0123] G2D11 allows binding of any combination of Tn-Thr / Tn-Ser, Tn-Ser / Tn-Thr, Tn-Ser / Tn-Ser, and Tn-Thr / Tn-Thr. As shown in Example 1 of this specification, referring to SEQ ID NO: 1 (VH domain of G2D11), amino acid residues H32, A33, H35, Y50, and S99 are the key residues when the VH domain binds to one of the GalNAc moieties of bis-Tn-MUC1 on the peptide, while amino acid residues S52, N55, and D57 are the key residues when the VH domain binds to other GalNAc moieties of bis-Tn-MUC1 on the peptide.

[0124] In one embodiment, the VH-domain of the conbotope antibody of the present invention comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, and comprising amino acid residues H32, A33, H35, Y50, and S99 with respect to SEQ ID NO: 1. The conbotope antibody supports recognition of a mono-Tn epitope.

[0125] Therefore, in one embodiment, a conbotope antibody for targeting tumor cells is provided, comprising a VH domain and a VL domain; where the VH domain of the antibody is a Tn-binding domain, and the amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1; where the amino acid sequence comprises amino acid residues H32, A33, H35, Y50, and S99 with respect to SEQ ID NO: 1; and where the VL domain of the antibody binds to a peptide backbone epitope associated with a Tn-glycan on tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0126] In one embodiment, the VH-domain of the conbotope antibody of the present invention comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, and comprising amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1. The conbotope antibody supports recognition of a mono-Tn epitope.

[0127] Therefore, in one embodiment, a conbotope antibody targeting tumor cells is provided, comprising a VH domain and a VL domain; where the VH domain of the antibody is a Tn-binding domain, and the amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1, wherein the amino acid sequence comprises amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1; and where the VL domain of the antibody binds to a peptide backbone epitope associated with a Tn-glycan on tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0128] As shown herein, sequence alignments of the amino acid sequences of the VH-domains of selected Tn-binding mAbs, including the VH-domain of G2D11, revealed conserved amino acids related to binding to GalNac in the CDR1, CDR2, and CDR3 regions (see Example 1.2). Specifically, referring to SEQ ID NO: 1, amino acid residues H32, A33, and H35 of CDR1 should preferably be conserved in the VH domain of the present invention; further, referring to SEQ ID NO: 1, amino acid residues Y50, S52, N55, and D57 of CDR2 should preferably be conserved in the VH domain of the present invention; and further, referring to SEQ ID NO: 1, amino acid residue S99 of CDR3 should preferably be conserved in the VH domain of the present invention.

[0129] In one embodiment, the VH-domain of the conbotope antibody contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1, and includes amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1. The conbotope antibody also supports recognition of the bis-Tn epitope.

[0130] In one embodiment, the amino acid sequence of the VH-domain of the conbotope antibody has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, and includes amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1.

[0131] In one embodiment, the VH domain of the conbotope antibody contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1, and in paired alignment with SEQ ID NO: 1, the amino acid sequence of the VH domain contains the amino acid residues histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), and serine (S) at positions corresponding to amino acid positions H32, A33, H35, Y50, S52, N55, D57, and S99 of SEQ ID NO: 1, respectively. Pair sorting of sequences is performed using a scoring matrix: blosum62, a gap opening penalty of 10, and a gap extension penalty of 0.2.

[0132] In one embodiment, in paired alignment with SEQ ID NO: 1, the amino acid sequence of the VH-domain of the conbotope antibody includes the amino acid residues histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), and serine (S) at positions corresponding to amino acid positions H32, A33, H35, Y50, S52, N55, D57, and S99 of SEQ ID NO: 1, respectively, and the amino acid sequence of the VH-domain has sequence homology of at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.

[0133] Therefore, in a preferred embodiment, an antibody is provided that targets tumor cells and comprises a VH domain and a VL domain; where the VH domain of the antibody is a Tn-binding domain, and the amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, wherein the amino acid sequence comprises amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1; and where the VL domain of the antibody binds to a peptide backbone epitope associated with a Tn-glycan on tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0134] The conbotope antibodies of the present invention, such as those described herein (e.g., the Tn-combotope disclosed herein), contain improved binding affinity to a specific antigen epitope called a conbotope, which consists of two different epitopes on a glycoprotein. In some embodiments, the antibody contains a binding affinity (e.g., kD) of 100 nM to 1 pM (e.g., less than 100 nM, less than 10 nM, less than 1 nM, less than 100 pM, or even less than 10 pM).

[0135] In some embodiments, the conbotope antibodies of the present invention are used in the treatment of cancer. In some examples, the cancer is lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, or bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0136] In yet another aspect, this specification provides specific antibodies.

[0137] In one embodiment, the antibody is: (i) A VH-domain having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, comprising amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1; and (ii) A VL domain having one amino acid sequence selected from sequence numbers 9 to 21, Includes.

[0138] In one embodiment, the present invention provides an antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 1 and a VL domain having one amino acid sequence selected from SEQ ID NOs: 9 to 21.

[0139] In one embodiment, the present invention provides an antibody, wherein the antibody is: (i) A VH-domain having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, comprising amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1; and (ii) Sequence ID: A VL domain having one amino acid sequence selected from 9 to 21, Includes; Here, the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, multiple specific antibody, transplant antibody, human antibody, humanized antibody, synthetic antibody, chimeric antibody, camelized antibody, single-chain Fv(scFv), single-chain antibody, Fab fragment, F(ab′)2 fragment, Fd fragment, Fv fragment, single-domain antibody, isolated complementarity-determining region (CDR), diabody, fragment contained in a single monomeric variable domain, disulfide-linked Fv(sdFv), intrabody, anti-idiotype (anti-Id) antibody, or its ab antigen-binding fragment.

[0140] One embodiment is: (i) VH domain having amino acid sequence number:1; and (ii) Sequence ID: VL domain having one of the amino acid sequences from 9 to 21, Antibodies containing are used in cancer treatment. In some examples, the cancer is lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, or bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0141] Another embodiment is: (i) VH domain having amino acid sequence number:1; and (ii) Sequence ID: VL domain having one of the amino acid sequences from 9 to 21, Antibodies containing [the specified substance] are used to diagnose cancerous conditions. These antibodies may be used for in vivo diagnosis or for ex vivo determination of cancerous conditions in tissue or cell samples taken from patients.

[0142] The binding of a conbotope antibody to a cancer target for diagnosis can be observed or determined by known methods (e.g., by labeling the conbotope antibody and / or by using a labeled antibody for conbotope antibody binding). Sequence ID: 9 (VL domain D3-TnMUC1): DYKDIQMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPLTFGAGTKLEMKR Sequence ID: 10 (VL-domain A3-TnMUC1): DYKDIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGGGTKLEIKR Sequence ID: 11 (VL domain D2-TnMUC1): DYKDIQMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPLTFGGGTKLEIKR Sequence ID: 12 (VL domain Ori-TnCD43): DYKDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGTPYTFGGGTKLEIKR Sequence ID: 13 (VL domain H1-TnCD43): DYKDIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPWTFGGGTKLEIKR Sequence ID: 14 ​​(VL-domain A1-TnCD43): DYKDIVMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQSEDFGSYYCQHHYGTPYTFGGGTKLEIKR Sequence ID: 15 (VL domain F4-TnCD43): DYKDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPYTFGGGTKLEMKR Sequence ID: 16 (VL domain C5-TnCD43): DYKDVQMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQQHYSTPYTFGGGTKLEIKR Sequence ID: 17 (VL domain C5-TnCD43): DYKDIVMTQSHKFMSTSVGDRVSITCKASQDVGTAVAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPYTFGGGTKLEMKR Sequence ID: 18 (VL domain D3-TnCD43): DYKDIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPLTFGAGTKLEIKR Sequence ID: 19 (VL domain G3-TnCD43): DYKDVVMTQSQKFMSTSVRDRVSITCKASQNVGTAVAWYQQKPGQSPKLLIYSASYRYSGVPDHFTGSGSGTDFTLTISNVQSEDLAEYFCQQYYSYPYTFGGGTKLEIKR Sequence ID: 20 (VL domain D7-TnCD43): DYKDLVLTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPWTFGGGTKLEMKR Sequence ID: 21 (VL domain H2-TnCD43): DYKDIVMTQSPSSLAVSVGEKVTMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPYTFGGGTKLEIKR

[0143] Preferably, the selected antibody of the present invention is a humanized antibody as described above.

[0144] The following combinations of humanized VH and LV domains proved to be particularly interesting: D3VL1+D3VH1, D3VL1+D3VH2, D3VL2+D3VH3, D3VL3+D3VH4, and D3VL4+D3VH5. >D3VL1 (Sequence ID: 159) DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGAGTKLEMK >D3VH1 (Sequence ID: 160) EVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYNQKFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS >D3VL1 (Sequence ID: 159) DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGAGTKLEMK >D3VH2 (Sequence ID: 161) EVQLVQSGAEVKKPGSSVKVSCKASGYIFADHAIHWVRRAPGQGLEWIGYISPGNDDIKYNEKFKGRATLTADKSTSTAYMELSSLRSEDTAVYFCKRSLPGTFDYWGQGTTLTVSS >D3VL2(Sequence ID: 162) DIVMTQSPDSLAVSLGEKATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGGGTKVEIK >D3VH3 (Sequence ID: 163) EVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQKFQDKVTLTADKSASTAYMELSSLRSEDTAVYFCKRSLPGTFDYWGQGTTVTVSS >D3VL3 (Sequence ID: 164) DIQMTQSPSSVSASVGDRLTITCRSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLKPEDFATYYCQQYYSYPLTFGQGTKVEIK >D3VH4 (Sequence ID: 165) EVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQEFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS >D3VL4(Sequence ID: 166) DIQMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKVEIK >D3VH5 (Sequence ID: 167) EVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQEFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS

[0145] In one embodiment, the present invention provides a humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 160. In one embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 160 is used for the treatment of cancer, for example, solid tumor cancer. In another embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 160 is used for the diagnosis of cancerous conditions, for example, by in vivo diagnosis or by determining the cancerous condition in tissue or cell samples taken from a patient.

[0146] In one embodiment, the present invention provides a humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 161. In one embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 161 is used for the treatment of cancer, for example, solid tumor cancer. In another embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 159 and a VL domain having the amino acid sequence of SEQ ID NO: 161 is used for the diagnosis of cancerous conditions, for example, by in vivo diagnosis or by determining the cancerous condition in tissue or cell samples taken from a patient.

[0147] In one embodiment, the present invention provides a humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 162 and a VL domain having the amino acid sequence of SEQ ID NO: 163. In one embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 162 and a VL domain having the amino acid sequence of SEQ ID NO: 163 is used for the treatment of cancer, for example, solid tumor cancer. In another embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 162 and a VL domain having the amino acid sequence of SEQ ID NO: 163 is used for the diagnosis of cancerous conditions, for example, by in vivo diagnosis or by determining the cancerous condition in tissue or cell samples taken from a patient.

[0148] In one embodiment, the present invention provides a humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 164 and a VL domain having the amino acid sequence of SEQ ID NO: 165. In one embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 164 and a VL domain having the amino acid sequence of SEQ ID NO: 165 is used for the treatment of cancer, for example, solid tumor cancer. In another embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 164 and a VL domain having the amino acid sequence of SEQ ID NO: 165 is used for the diagnosis of cancerous conditions, for example, by in vivo diagnosis or by determining the cancerous condition in tissue or cell samples taken from a patient.

[0149] In one embodiment, the present invention provides a humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 166 and a VL domain having the amino acid sequence of SEQ ID NO: 167. In one embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 166 and a VL domain having the amino acid sequence of SEQ ID NO: 167 is used for the treatment of cancer, for example, solid tumor cancer. In another embodiment, the humanized antibody comprising a VH domain having the amino acid sequence of SEQ ID NO: 166 and a VL domain having the amino acid sequence of SEQ ID NO: 167 is used for the diagnosis of cancerous conditions, for example, by in vivo diagnosis or by determining the cancerous condition in tissue or cell samples taken from a patient.

[0150] [I.ii]STn-combotope As disclosed herein, the VH domain of the conbotope antibody binds to the glycan epitope, preferably mono-Tn, bis-Tn, mono-STn, or bis-STn glycan epitope.

[0151] In one preferred scenario, the VH domain of the conbotope antibody binds to an STn-glycan epitope (such as mono-STn or bis-STn).

[0152] The VH-domain of antibody G2D11 (SEQ ID NO: 1) was compared in structure to the VH domain of 3F1 (SEQ ID NO: 25) modified to the STn-binding VH-domain (SEQ ID NO: 28) (see Example 5). Compared to G2D11 (SEQ ID NO: 1), the STn-binding VH domain (SEQ ID NO: 28) has the following amino acid residue changes: I28T, A30T, P101L, delG102, T103A and F104L. SEQ ID NO: 28 (variant M2 of VH domain of G2D11: LAL-TFT) QSDAELVKPGASVKISCKASGYTFTDHAIHWVKRKPEQGLEWIGYISPGNDDIKYNEKFKGKATLTADKSSSTAYMQLNSLTSEDSAVYFCKRSLLALDYWGQGTTLTVSS

[0153] The novel STn-combotope antibodies disclosed herein are based on these structural modifications and further development as disclosed herein, and provide specific combotope recognition, where the VH-chain supports recognition of the STn sugar chain portion of the combotope of the glycoprotein antigen, while the VL-domain supports recognition of the peptide portion of the combotope of the glycoprotein antigen.

[0154] In one embodiment, the VH-domain of the combotope antibody of the present invention is a VH-domain like SEQ ID NO: 28. In one embodiment, the amino acids of the VH-domain of the combotope antibody of the present invention are similar to the VH-domain like SEQ ID NO: 28 in their structural conformations.

[0155] As shown in Example 5, amino acid residues T28, T30, L101, A102, and L103 with respect to SEQ ID NO: 28 are major amino acids for STn-specificity. They are necessary to accommodate sialyl; that is, it is necessary to create extra space to accommodate sialyl for STn binding.

[0156] [[ID=?]] In one embodiment, the VH-domain of the combinotope antibody comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; and comprises amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103 with respect to SEQ ID NO: 28. The combinotope antibody supports recognition of the mono-STn epitope.

[0157] Thus, in one embodiment, there is provided an antibody targeting tumor cells, the antibody comprising a VH domain and a VL domain; where the VH domain of the antibody is an STn-binding domain, and the amino acid sequence of the VH-domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; where the amino acid sequence comprises amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103 with respect to SEQ ID NO: 28; and where the VL domain of the antibody binds to the peptide backbone associated with the STn-glycan on the tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0158] In one embodiment, the VH-domain of the combinotope antibody comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; The conbotope antibody contains amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103 with respect to SEQ ID NO: 28. The conbotope antibody supports recognition of the mono-STn epitope.

[0159] Therefore, in one embodiment, an antibody that targets tumor cells is provided, comprising a VH domain and a VL domain; Here, the VH domain of the antibody is an STn-binding domain, and the amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; Here, the amino acid sequence comprises amino acid residues 28, T30, S52, N55, D57, L101, A102 and L103 with respect to SEQ ID NO: 28; The VL domain of the antibody binds to a peptide backbone associated with STn-glycans on tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0160] In one embodiment, the VH-domain of the conbotope antibody comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; The combotope antibody also contains amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103 with respect to Sequence ID No. 28. The combotope antibody also supports recognition against the bis-STn epitope.

[0161] In one embodiment, the amino acid sequence of the VH-domain of the conbotope antibody has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103.

[0162] In one embodiment, the VH domain of the conbotope antibody comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; In paired alignment with Sequence ID No. 28, the amino acid sequence of the VH domain contains the amino acid residues threonine (T), threonine (T), histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), serine (S), leucine (L), alanine (A), and leucine (L) at positions corresponding to amino acid positions T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 of Sequence ID No. 28, respectively. This paired sequence alignment is performed using the scoring matrix:blosum62, a gap opening penalty of 10, and a gap extension penalty of 0.2.

[0163] In one embodiment, in paired alignment with Sequence ID No. 28, the amino acid sequence of the VH-domain of the conbotope antibody includes the amino acid residues threonine (T), threonine (T), histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), serine (S), leucine (L), alanine (A), and leucine (L) at positions corresponding to amino acid positions T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 of Sequence ID No. 28, respectively; The amino acid sequence of the VH domain has an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably an amino acid sequence having at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28.

[0164] Therefore, in one embodiment, an antibody that targets tumor cells is provided, comprising a VH domain and a VL domain; Here, the VH domain of the antibody is an STn-binding domain, and the amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; Here, the amino acid sequence comprises amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to SEQ ID NO: 28; The VL domain of the antibody binds to a peptide backbone associated with STn-glycans on tumor cells. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0165] The conbotope antibodies of the present invention, as described herein (such as the STn-combotope disclosed herein), contain improved binding affinity to a specific antigen epitope (the conbotope) (compared to other epitopes on healthy or cancer cells). In some embodiments, the antibody contains a binding affinity (e.g., kD) of 100 nM to 1 pM (e.g., less than 100 nM, less than 10 nM, less than 1 nM, less than 100 pM, or even less than 10 pM).

[0166] In some embodiments, the antibodies of the present invention are used for cancer treatment. In some examples, the cancer is lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, or bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0167] Another embodiment is: (i) Sequence ID: VH domain having amino acid sequence 28; and (ii) Sequence ID: VL domain having one of the amino acid sequences from 9 to 21, Antibodies containing [the specified substance] are used to diagnose cancerous conditions. These antibodies may be used for in vivo diagnosis or for ex vivo determination of cancerous conditions in tissue or cell samples taken from patients.

[0168] The binding of a conbotope antibody to a cancer target for diagnosis can be observed or determined by known methods (e.g., by labeling the conbotope antibody and / or by using a labeled antibody for conbotope antibody binding).

[0169] In yet another aspect, the present invention discloses specific antibodies.

[0170] In one embodiment, the conbotope antibody of the present invention is: (i) A VH-domain having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO: 28, preferably at least 80% sequence identity to SEQ ID NO: 28, more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 28, and comprising amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 with respect to SEQ ID NO: 28; Comprising amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 with respect to SEQ ID NO: 28, A VH-domain; And (ii) A VL-domain having any one amino acid sequence selected from SEQ ID NOs: 22 - 24, Comprising.

[0171] In one embodiment, the present invention provides an antibody comprising a VH-domain having the amino acid sequence of SEQ ID NO: 28 and a VL-domain having any one amino acid sequence selected from SEQ ID NOs: 22 - 24.

[0172] In one embodiment, the present invention provides an antibody, where the antibody is: (i) A VH-domain having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence identity to SEQ ID NO: 28, preferably at least 80% sequence identity to SEQ ID NO: 28, more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 28, and comprising amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 with respect to SEQ ID NO: 28; And (ii) A VL-domain having any one amino acid sequence selected from SEQ ID NOs: 22 - 24, Comprising; Here, the antibody is a monoclonal antibody, polyclonal antibody, bispecific antibody, multiple specific antibody, transplant antibody, human antibody, humanized antibody, synthetic antibody, chimeric antibody, camelized antibody, single-chain Fv(scFv), single-chain antibody, Fab fragment, F(ab′)2 fragment, Fd fragment, Fv fragment, single-domain antibody, isolated complementarity-determining region (CDR), diabody, fragment contained in a single monomeric variable domain, disulfide-linked Fv(sdFv), intrabody, anti-idiotype (anti-Id) antibody, or its ab antigen-binding fragment.

[0173] In some embodiments; (i) Sequence ID: VH domain having amino acid sequence 28; and (ii) Sequence ID: VL domain having one amino acid sequence selected from 22 to 24, Antibodies containing are used to treat cancer. In some examples, the cancer is lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, or bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0174] Preferably, the selected antibody of the present invention is a humanized antibody as described above. Sequence ID: 22 (VL domain C4-STnMUC1): IVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPYTFGGGTKLEIKR Sequence ID: 23 (VL domain D3-STnMUC1): IVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYNSYPYTFGGGTKLEIKR Sequence ID: 24 (VL domain C7-STnMUC1): VVVTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPRTFGGGTKLEIKR

[0175] [I.iii] Tn-combotope or STn-combotope It may not be known whether the conbotope on the surface of the glycoprotein contains Tn or STn as short, truncated O-glycans, or whether a particular glycoprotein contains a mixture of these glycans. In such cases, it may be beneficial to use an antibody that binds to either the Tn epitope or the STn epitope in the conbotope. Therefore, in a particular embodiment of the present invention, the conbotope antibody comprises both a VH domain for the Tn epitope and a VH domain for the STn epitope. Such an antibody comprises a VH(Tn)-VL1 arm and a VH(STn)-VL2 arm, where the VL1 domain and the VL2 domain may be the same or different, and may bind to the same peptide epitope or to different peptide epitopes. The VH(Tn)-VL1 arm and the VH(STn)-VL2 arm may consist of two Fab arms in a normal antibody, and may be configured as F(ab)2, as a minibody, as a diabody, as a triabody, or as two scFv (e.g., scFv-Fc) linked in one molecule. In one embodiment, the VH(Tn) domain has the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof as defined above; on the other hand, the VH(STn) has the amino acid sequence of SEQ ID NO: 28, or a functional variant thereof as defined above.

[0176] [II] Antibody Library In one aspect, the present invention provides an antibody library for the in vitro identification of specific antibodies that bind to glycoproteins (such as glycoproteins on cancer tumor cells). Therefore, in one embodiment, the present invention provides an antibody library for the in vitro identification of specific antibodies that bind to one or more types of tumor cells.

[0177] As disclosed herein, tumor cells often contain Tn or STn glycosylated epitopes on specific glycoproteins on the surface of the cancer cells. Specifically, tumor cells often contain short, truncated O-glycans on their surface, for example, exposing Tn and / or STn epitopes on specific glycoproteins on the surface of the cancer cells. The antibody library of the present invention facilitates the identification of antibodies with improved specificity to tumor cells by having antibodies that are specific to both the glycan epitope (Tn and / or STn) and the peptide epitope of the protein backbone associated with the glycan (epitope). Specifically, such improved antibodies contain a VH domain that efficiently binds to the glycan epitope of the glycoprotein and a VL domain that efficiently binds to the glycoprotein peptide epitope associated with the glycan epitope, so that the antibodies are specific to the combination of the glycan epitope and peptide epitope of the glycoprotein (a combination epitope called a "combotope").

[0178] As described above, the VH-domain of antibody G2D11 (SEQ ID NO: 1) was found to specifically recognize the glycan epitope of the glycoprotein epitope bis-Tn on MUC1 through structural characterization, but did not recognize the peptide sequence of the said glycoprotein epitope (see Example 1).

[0179] Based on these structural observations, the antibody library of the present invention was conceptualized, in which each antibody in the library contains a pre-selected specific VH chain that supports recognition of the sugar chain portion of the conbotope antigen, while its VL domain is variable (containing one or more VL domains specific to a specific peptide sequence of a particular glycoprotein), creating a library capable of screening for specific conbotope antibodies, the VL domain supporting recognition of the peptide epitope within the glycoprotein conbotope.

[0180] Based on the binding and structural data presented herein (i.e., key amino acids for Tn and STn binding in combination with the generic VH sequence of specific G2D11), the antibody library of the present invention can be used to identify specific glycoprotein conbotope antibodies, where the VL domain is specific to the selected peptide epitope (i.e., glycoprotein).

[0181] In one aspect, the present invention provides an antibody library in which each antibody in the library comprises two types of antibody domains: (i) A first antibody domain that binds to a glycosylation epitope of a glycoprotein on cancer cells (i.e., Tn, bis.Tn, STn, or bisSTn); and (ii) A second antibody domain selected from the repertoire of antibody domains, Includes; Here, the repertoire of the antibody domain is: It comprises one or more domains that bind to the peptide epitope of the glycoprotein; Herein, the library is intended for the in vitro identification of specific antibodies from the library that target cancer cells; And here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein; that is, the specific antibody is a combotope antibody.

[0182] In one aspect, the present invention provides an antibody library in which each antibody in the library comprises two types of antibody domains: (i) A first antibody domain that binds to the glycosylation epitope of a glycoprotein on tumor cells; and (ii) A second antibody domain selected from the repertoire of antibody domains, Includes; Here, the repertoire of antibody domains includes one or more domains that bind to the peptide epitopes of the glycoproteins of the tumor cells; Here, the library is for in vitro identification of specific antibodies that target tumor cells from the library; And here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein; that is, the specific antibody is a combotope antibody.

[0183] In one embodiment, the first antibody domain is a VH domain, and the second antibody domain is a VL domain. In another embodiment, both the first and second antibodies are VH domains, but they are different from each other.

[0184] In one aspect, the present invention provides an antibody library, where each antibody in the antibody library is: (i) A VH-domain that binds to a glycosylation epitope of a glycoprotein on the tumor cell; and (ii) A VL domain selected from the repertoire of VL domains, Includes; Here, the repertoire of VL-domains includes one or more VL-domains that bind to the peptide epitopes of the glycoproteins of the tumor cells; The library is intended for in vitro identification of specific antibodies that bind to tumor cells; Here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein (a combination epitope called a "combotope").

[0185] In one embodiment, the present invention provides an antibody library for in vitro identification of specific antibodies that bind to tumor cells; Here, each of the antibodies in the library is: (ii) A VL domain selected from the VL domain repertoire; and (i) A VH-domain that binds to a glycosylation epitope of a glycoprotein of the tumor cell, and which does not contribute to or inhibit the binding of a peptide epitope of a glycoprotein of the tumor cell. Includes; Here, the repertoire of VL-domains includes one or more VL-domains that bind to peptide epitopes on glycoproteins of tumor cells; Here, the specific antibody is specific to a combination of the glycosylation epitope and peptide epitope of the glycoprotein, which is called a "combotope"; in other words, the specific antibody is a combotope antibody.

[0186] The VH chain of each antibody encoded in the library is pre-selected for the desired glycotype specificity, while its VL chain is selected from a repertoire of LV domains to determine its peptide backbone specificity, thereby determining its glycoprotein specificity.

[0187] In one embodiment, the VH chain of each antibody encoded in a pre-selected library relating to the desired glycotype specificity does not inhibit peptide backbone specificity; on the other hand, the VL chain's peptide backbone specificity is determined by selection from a repertoire of VL domains, thereby determining the glycoprotein specificity.

[0188] The structural analysis disclosed herein (Example 1) provides clear evidence that the VH domain of G2D11 does not interact with the peptide / protein carrier at all, and that all interaction of the antibody with the peptide / protein, as exemplified by antibodies obtained from the library of the present invention (e.g., antibodies Tn-MUC1 and Tn-CD43; see Examples 3 and 4), is due to the contribution of the VL chain.

[0189] In one preferred embodiment, the peptide epitope and the glycan epitope are a continuous, undisrupted conventional epitope, i.e., the glycan is in close proximity to the peptide by being chemically linked (e.g., by covalent bonds). In another embodiment, the peptide epitope and the glycan epitope may be discontinuous conventional epitopes, where “associated” means that the peptide epitope and the glycan epitope are in close proximity to each other, but by a molecular structure arrangement that facilitates the formation of such conventional epitopes.

[0190] The antibodies used herein are selected from monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, transplant antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain Fv(scFv), single-chain antibodies, Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, single-domain antibodies, isolated complementarity-determining regions (CDRs), diabodies, fragments contained in a single monomeric variable domain, disulfide-linked Fv(sdFv), intrabodies, anti-idiotype (anti-Id) antibodies, and their ab antigen-binding fragments.

[0191] In one preferred embodiment, the antibody encoded in the antibody library of the present invention is scFv, where the VH-domain is linked to the VL-domain.

[0192] In one preferred embodiment, the library disclosed herein comprises an scFv antibody comprising a VH domain and a VL domain, where both domains are located within a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH domain and the VL domain so that the scFv can form a desired structure for antigen binding. In one embodiment, the linker is (GGGGS) n It is selected from, where n is 1, 2, 3, 4, 5, or 6. (Example: GGGGS) n Many other linkers can be used as alternatives to this one. Those skilled in the art will understand how to select such linkers.

[0193] In one embodiment, the VH domain of each antibody in the antibody library specifically binds to one or more glycan epitopes selected from Tn and / or STn. In another embodiment, the VH domain of each antibody encoded in the antibody library specifically binds to one or more Tn moieties (such as mono-Tn epitopes or bis-Tn epitopes). In yet another embodiment, the VH domain of each antibody encoded in the antibody library specifically binds to one or more STn moieties (such as mono-STn epitopes or bis-STn epitopes). In yet another embodiment, the VH domains of some anti-antibodies among the antibodies encoded in the antibody library specifically bind to one or more Tn moieties, while the VH domains of other antibodies encoded in the antibody library specifically bind to one or more STn moieties. In yet another embodiment, the antibody contains two different VH domains, namely, one VH domain for Tn or bis-Tn, and the other VH domain for STn or bis-STn.

[0194] As disclosed above, in each antibody in the antibody library, the VH domain is specified to specifically bind to a particular glycan epitope characteristic of cancer cells. On the other hand, the VL domains of the antibodies in the antibody library differ from antibody to antibody, constituting a repertoire of VL domains that recognize different peptides in the target glycosylated protein. This generates a repertoire of VL domains, which are then screened for the identification of conbotope antibodies that have specificity for glycoproteins on tumor cells, due to the VH domains that bind to glycan epitopes of glycoproteins on tumor cells and the VL domains that bind to peptide epitopes of glycoproteins on tumor cells.

[0195] In one embodiment, the repertoire of VL domains is made from a naive immune repertoire, an immune repertoire obtained by immunizing a suitable animal, or a repertoire created by synthesis.

[0196] In one embodiment, the repertoire of VL-domains encoded in the antibody library is a naive immunological repertoire derived from an animal (such as a mouse or human). Other suitable animals are pigs, rats, dogs, horses, and rabbits, which are known to those skilled in the art.

[0197] In one preferred embodiment, the antibody library is a phage display library.

[0198] [II.i] Tn-Template Antibody Library This specification provides a Tn-template antibody library in which the first domain of each antibody in the library is a Tn-binding domain, while the second domain is selected from a repertoire of antibody domains that include one or more domains that bind to a peptide epitope on a target glycoprotein associated with the Tn epitope, as disclosed above.

[0199] This specification provides a Tn-template antibody library in which the VH domain of each antibody in the library is a Tn-binding VH domain, while the VL domain is selected from a repertoire of VL domains, which include one or more VL domains that bind to a peptide epitope on a target glycoprotein associated with the Tn epitope, as disclosed above.

[0200] In one embodiment, the present invention provides a mono-Tn template antibody library, where the VH domain of each antibody in the library is a mono-Tn-binding VH domain, while the VL domain is selected from a repertoire of VL domains, each containing one or more VL domains that bind to a peptide epitope on a target glycoprotein associated with the Tn epitope (as described above).

[0201] In one embodiment, the present invention provides a bis-Tn template antibody library, where the VH domain of each antibody in the library is a bis-Tn-binding VH domain, and the VL domain is selected from a repertoire of VL domains, which include one or more VL domains that bind to a peptide epitope on a target glycoprotein associated with the Tn epitope (as described above).

[0202] In a preferred embodiment, the present invention provides an antibody library which may be used for selecting antibodies that bind to mono-Tn and bis-Tn epitopes, wherein the VH domain of each antibody in the library is a mono- and bis-Tn-binding VH domain, while the VL domain is selected from a repertoire of VL domains which includes one or more VL domains that bind to a peptide epitope on a target glycoprotein associated with the Tn epitope (as described above).

[0203] In one embodiment, the VH-domain of each antibody in the Tn-template antibody library is a G2D11-like VH-domain (SEQ ID NO: 1). In one embodiment, the amino acids of the VH-domain of each antibody in the library are similar to the G2D11-like VH-domain in their structural conformation.

[0204] In one embodiment, the VH-domain of the Tn template antibody library comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; and comprises amino acid residues H32, A33, H35, Y50, and S99 with respect to SEQ ID NO: 1.

[0205] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided, wherein each antibody in the antibody library comprises a VH domain and a VL domain; Here, the VH domain of each antibody is a Tn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, and comprises amino acid residues H32, A33, H35, Y50, and S99 with respect to SEQ ID NO: 1; And here the VL domain is selected from the repertoire of VL domains; Herein, the repertoire includes at least one VL domain that binds to a peptide backbone epitope associated with a Tn-glycan on the tumor cell. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0206] In one embodiment, the VH-domain of the Tn template antibody library comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; and comprises amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1.

[0207] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided: Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is a Tn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology with respect to SEQ ID NO: 1, and comprises amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1; Here, the VL domain is selected from a repertoire of VL domains, the repertoire comprising at least one VL domain that binds to a peptide backbone epitope associated with a Tn-glycan on the tumor cell. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0208] In one embodiment, the VH-domain of the Tn template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; And with respect to Sequence ID No. 1, it includes amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99.

[0209] Therefore, in a preferred embodiment, an antibody library is provided for selecting tumor-targeting antibodies; Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is a Tn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; and with respect to Sequence ID No. 1, it includes amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99; And here the VL domain is selected from the repertoire of VL domains; Herein, the repertoire includes at least one VL domain that binds to a peptide backbone epitope associated with a Tn-glycan on the tumor cell. The sequence of the VH domain is preferably a sequence that does not bind to the peptide epitope.

[0210] In one embodiment, the VH domain of the Tn template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 1; In paired alignment with Sequence ID: 1, the amino acid sequence of the VH domain contains the amino acid residues histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), and serine (S) at positions corresponding to amino acid positions H32, A33, H35, Y50, S52, N55, D57, and S99 of Sequence ID: 1, respectively. This paired sequence alignment is performed using the scoring matrix: blosum62, a gap opening penalty of 10, and a gap extension penalty of 0.2.

[0211] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided; Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is a Tn-binding VH domain, and the amino acid sequence of the VH-domain having SEQ ID NO: 1 in paired arrangement contains the amino acid residues histidine (H), alanine (A), histidine (H), tyrosine (Y), serine (S), asparagine (N), aspartic acid (D), and serine (S) at positions corresponding to amino acid positions H32, A33, H35, Y50, S52, N55, D57, and S99 of SEQ ID NO: 1, respectively; The amino acid sequence of the VH domain has sequence homology of at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% with respect to SEQ ID NO: 1, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%.

[0212] In one embodiment, the first antibody domain is a mono- or bis-Tn-conjugated VH domain; Here, the VH-domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 1, comprising amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1, preferably comprising amino acid residues H32, A33, H35, Y50, and S99 and amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1.

[0213] This Tn template library is useful for screening antibodies that bind to conbotopes containing Tn epitopes.

[0214] [II.ii] STn-Template Antibody Library In this specification, an STn-template antibody library is provided, wherein the first domain of each antibody in the library is an STn-binding domain, and the second domain is selected from a repertoire of antibody domains, each containing one or more domains that bind to a peptide epitope on a target glycoprotein associated with the STn epitope, as disclosed above.

[0215] In this specification, an STn-template antibody library is provided, where the VH domain of each antibody in the library is an STn-binding VH domain, and the VL domain is selected from a repertoire of VL domains, which include one or more VL domains that bind to a peptide epitope on a target glycoprotein associated with the STn epitope, as described above.

[0216] In one embodiment, the present invention provides a mono-STn template antibody library, where the VH domain of each antibody in the library is a mono-STn-binding VH domain, and the VL domain is selected from a repertoire of VL domains, each of which binds to a peptide epitope on a target glycoprotein associated with the STn epitope (as described above).

[0217] In one embodiment, the present invention provides a bis-STn template antibody library, where the VH domain of each antibody in the library is a bis-STn-binding VH domain, and the VL domain is selected from a repertoire of VL domains, each of which binds to a peptide epitope on a target glycoprotein associated with the STn epitope (as described above).

[0218] In one preferred embodiment, the present invention provides an antibody library which may be used to select antibodies that bind to mono-STn and bis-STn epitopes; where the VH domain of each antibody in the library is a mono- and bis-STn-binding VH domain, and the VL domain is selected from a repertoire of VL domains which includes one or more VL domains that bind to a peptide epitope on a glycoprotein of interest that is associated with the STn epitope (as described above).

[0219] As disclosed herein, the VH-domain of antibody 3F1 (SEQ ID NO: 25) efficiently binds to STn. In one embodiment, the VH-domain of each antibody in the STn-template antibody library is a 3F1-like VH-domain (SEQ ID NO: 25). In one embodiment, the amino acids of the VH-domain of each antibody in the library are similar to those of the 3F1-like VH-domain in their structural conformation.

[0220] A potential drawback of 3F1 is poor expression, but G2D11 is much more stable and easier to produce compared to 3F1. For example, G2D11 is expressed very well in Pichia pastoris yeast, while 3F1 is not expressed as well. Furthermore, there was a need for knowledge about the molecular basis of methods for converting anti-Tn to anti-STn.

[0221] By comparing the sequences of G2D11 and 3F1, we identified amino acid residues that affect Tn specificity and STn specificity, respectively. To obtain STn specificity, a modified G2D11 VH domain was created to simulate the 3F1 VH domain (see Example 5). This modified G2D11 VH domain is shown herein as Sequence ID: 28. Compared to G2D11 (Sequence ID: 1), the modified STn-binding VH domain has the following amino acid residue changes: I28T, A30T, P101L, delG102, T103A, and F104L. In one embodiment, the VH-domain of each antibody in the STn-template antibody library is a VH-domain similar to SEQ ID NO: 28. In one embodiment, the amino acids of the VH-domain of each antibody in the library are similar to SEQ ID NO: 28 in their structural conformation.

[0222] In one embodiment, the VH-domain of the STn-template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103.

[0223] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided; Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is an STn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, H32, A33, H35, Y50, S99, L101, A102, and L103; And here the VL domain is selected from the repertoire of VL domains; Herein, the repertoire includes at least one VL domain that binds to a peptide backbone epitope associated with STn-glycans on the tumor cell.

[0224] In one embodiment, the VH-domain of the STn-template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, S52, N55, D57, L101, A102, and L103.

[0225] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided; Here, each antibody in the antibody library contains both a VH domain and a VL domain; Here, the VH domain of each antibody is an STn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, S52, N55, D57, L101, A102 and L103; And here the VL domain is selected from the repertoire of VL domains; Herein, the repertoire includes at least one VL domain that binds to a peptide backbone epitope associated with STn-glycans on the tumor cell.

[0226] In one embodiment, the VH-domain of the STn-template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103.

[0227] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided; Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is an STn-binding VH domain, and the VH-domain comprises an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; And with respect to Sequence ID No. 28, it includes amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103; And here the VL domain is selected from the repertoire of VL domains, Herein, the repertoire includes at least one VL domain that binds to a peptide backbone epitope associated with STn-glycans on the tumor cell.

[0228] In one embodiment, the VH domain of the STn-template antibody library contains an amino acid sequence having at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28; In paired alignment with Sequence ID No. 28, the amino acid sequence of the VH domain contains amino acid residues Thr, Thr, His, Ala, His, Tyr, Ser, Asn, Asp, Ser, Leu, Ala, and Leu at positions corresponding to amino acid positions 28, 30, 32, 33, 35, 50, 52, 55, 57, 99, 101, 102, and 103 of Sequence ID No. 28, respectively. This paired sequence alignment is performed using a scoring matrix: blosum62, a gap opening penalty of 10, and a gap extension penalty of 0.2.

[0229] Therefore, in one embodiment, an antibody library for selecting tumor-targeting antibodies is provided; Here, each antibody in the antibody library contains a VH domain and a VL domain; Here, the VH domain of each antibody is an STn-binding VH domain, and the amino acid sequence of the VH domain in paired alignment with SEQ ID NO: 28 contains amino acid residues Thr, Thr, His, Ala, His, Tyr, Ser, Asn, Asp, Ser, Leu, Ala, and Leu at positions corresponding to amino acid positions 28, 30, 32, 33, 35, 50, 52, 55, 57, 99, 101, 102, and 103 of SEQ ID NO: 28, respectively; The amino acid sequence of the VH domain has at least 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, or 98% sequence homology to SEQ ID NO: 28, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence homology to SEQ ID NO: 28.

[0230] In one embodiment, the first antibody domain is a mono- or bis-STn-conjugated VH domain; And herein, the VH-domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28; And the amino acid sequence is: Regarding sequence number 28, (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, Contains the amino acid residues; Preferably, the amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to SEQ ID NO: 28.

[0231] The STn template library is useful for screening antibodies that bind to combotopes containing STn epitopes. The library may also be useful for screening antibodies that bind to Tn epitopes, or combotopes containing combinations of Tn and STn epitopes.

[0232] [III] Nucleic acid libraries encoding antibodies In one aspect, the present invention provides a nucleic acid library encoding an antibody library disclosed herein.

[0233] All features and embodiments of the antibody libraries disclosed in Section 2 also apply equally to the nucleic acid libraries encoding the antibodies disclosed in this Section.

[0234] In one embodiment, the present invention provides a nucleic acid library encoding antibodies; Here, each of the nucleic acids in the library is: (i) A first nucleic acid sequence encoding an antibody domain that binds to a glycosylation epitope of a glycoprotein in the tumor cell; and (ii) A second nucleic acid sequence selected from a repertoire of nucleic acid sequences, which includes one or more nucleic acid sequences encoding an antibody domain that binds to a peptide epitope of the glycoprotein of the tumor cell. Includes; This library is for in vitro identification of specific antibodies that specifically bind to tumor cells from the library; Here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; Here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0235] In one embodiment, the present invention provides a nucleic acid library encoding antibodies; Here, each of the nucleic acids in the library is: (i) A first nucleic acid sequence encoding a VH-domain that binds to a glycosylation epitope of a glycoprotein of the tumor cell; and (ii) A second nucleic acid sequence selected from a repertoire of nucleic acid sequences, which includes one or more nucleic acid sequences encoding a VL domain that binds to a peptide epitope of the glycoprotein of the tumor cell. Includes. This library is intended for in vitro identification of specific antibodies that specifically bind to tumor cells from the library; Here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; And here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0236] This specification provides a nucleic acid library comprising multiple nucleic acid sequences, wherein each nucleic acid sequence encodes an amino acid sequence that forms at least a portion of an antibody as described herein.

[0237] Specifically, the present invention provides a nucleic acid library encoding multiple antibodies, where each of the nucleic acid sequences encoding an antibody is: (i) A first nucleic acid sequence encoding a VH-domain that binds to a glycosylation epitope on the glycoprotein of one or more tumor cells; and (ii) A second nucleic acid sequence selected from a repertoire of nucleic acid sequences, which includes one or more nucleic acid sequences encoding a VL domain that binds to a peptide epitope of the glycoprotein of the tumor cell. Includes; This library is for in vitro identification of specific antibodies that specifically bind to tumor cells; Here, the peptide epitope of the glycoprotein of the tumor cell is associated with the glycan epitope of the glycoprotein of the tumor cell; And here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein.

[0238] In one embodiment, the nucleic acid library is 10 8 ~10 9 Non-identical clones within a range of types (at least 10) 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 Includes more than one type of non-identical nucleic acid, etc.

[0239] In a further embodiment, the first nucleic acid sequence and the second nucleic acid sequence are linked by a nucleic acid sequence encoding a peptide linker that links the encoded VH sequence to the encoded VL sequence. The peptide linker has been discussed above. Such linkers are generally known to those skilled in the art.

[0240] This specification provides further vector libraries, including nucleic acid libraries as described herein. Exemplary expression vectors for inserting the nucleic acid libraries disclosed herein may include eukaryotic expression vectors or prokaryotic expression vectors. Preferably, the nucleic acid library encoding an antibody is expressed using phage display technology.

[0241] This specification provides further cell libraries, including nucleic acid libraries as described herein.

[0242] [IV] Method for identifying a conbotope antibody using the antibody library of the present invention In a further aspect, the present invention provides a method for identifying antibodies that target tumor cells, the method comprising preparing an antibody library as disclosed herein and screening the library to identify one or more tumor-targeting antibodies.

[0243] In one embodiment, the antibody library is prepared as a phage display library, and the screening includes biopanning the antibody library with a specific tumor glycopeptide or intact glycoprotein.

[0244] In a further embodiment, the method further includes isolating a tumor-targeting specific antibody and optionally purifying the antibody. The isolation and purification of the antibody may be carried out by conventional methods understood by those skilled in the art.

[0245] In one embodiment, the process for identifying and isolating tumor-specific antibodies consists of the following steps: (a) the step of preparing an antibody library as disclosed herein; and (b) A step of identifying candidate antibodies specific to the target glycoprotein antigen from the library by a binding assay, Includes.

[0246] In one embodiment, the identification of candidate tumor cell-specific antibodies includes biopanning of an antibody library using (specific) tumor glycopeptides or tumor glycoproteins, preferably O-glycosylated peptides or proteins, of the tumor cells; Here, the glycosylation preferably consists of a short, truncated O-glycan (such as Tn-mucin) or other O-glycosylated proteins having a mucin-like motif; The peptide / protein is either a purified peptide / purified protein or expressed on the cell surface / tissue.

[0247] The antibody library may be a phage display library, a yeast display library, a ribosome display library, or the like, as would be understood by those skilled in the art.

[0248] In one preferred embodiment, the antibody library is a phage display library.

[0249] In one embodiment, the antibody library is a phage display library prepared by a method comprising the following steps: (1) Steps to isolate mRNA from the spleen (immunizing a donor animal (e.g., a mouse) with a glycoprotein or glycopeptide containing a short, truncated O-glycan Tn or STn on its surface for the purpose of preparing the Tn and STn binding domains; collecting the spleen from a naive donor animal to prepare the peptide binding domains); (2) A step of synthesizing cDNA from the mRNA; (3a) A step of amplifying the cDNA using a specific primer set for obtaining a first nucleic acid sequence encoding the VH domain; (3b) A step of amplifying the cDNA using a primer mixture for obtaining multiple nucleic acid sequences encoding the repertoire of the VL domain; (4) The step of assembling a second nucleic acid sequence derived from a first nucleic acid sequence encoding the VH-domain and a plurality of nucleic acid sequences encoding the repertoire of the VL-domain to create a concatenated construct; (5) Inserting the construct into a phagemid vector; (6) A step of introducing a phagemide vector containing the construct into Escherichia coli in order to prepare a bacterial library; (7) A step of infecting a bacterial library with phages in order to create a phage display library.

[0250] As a non-limiting example, such a phage display library may be prepared by the method shown in Figure 2; The method includes the following steps; (1) The process of isolating mRNA from mouse spleen; (2) A step of synthesizing cDNA using a random hexamer and reverse transcriptase; (3) A step of performing PCR amplification from a cDNA template using a specific primer set in order to obtain the VH domain, and a step of performing PCR amplification from a cDNA template using a mixture of VL primers in order to obtain a repertoire of VL domains; (4) A step of performing PCR assembly of the VL-domain repertoire and specific VH-domains using a 5′ phosphorylated outer primer; (5) A rolling circle amplification step in which the phosphorylated scFv gene is linked as a circular DNA by ligation, the dsDNA is denatured, random hexamers are annealed, and the circular fragment is amplified into a long linear concatemer by Phi29 polymerase; (6) A step of digesting the amplified elongated scFv gene with the restriction enzyme sfiI, and ligating the digested product to the phagemide vector pAK100 that has been treated with SfiI and rSAP; (7) A step of introducing a pool of phagemids containing the scFv gene into E. coli cells TG1 by electroporation; (8) A step of growing a bacterial library containing different phagemids and infecting it with the helper phage VCSM13 to produce a complete phage that expresses scFv on the pIII coat protein of the phage.

[0251] The selection of the antibody library (e.g., one derived from a phage display library) may be performed by multiple searches (panning) using biotinylated target antigens (e.g., bis-Tn-MUC1 glycoprotein) immobilized on streptavidin-coated magnetic beads. After each selection, the phages are eluted, amplified, and precipitated. If necessary, irrelevant phage antibodies may be removed by absorption against non-targets (negative conjugate), bare beads, plastics, proteins, peptides, or normal human cells. Sequencing (NGS) of the enriched phages after each panning provides a fingerprint of the VL-domain antibody sequence corresponding to the target antigen structure and peptide sequence. Polyclonal phage ELISA may be used to confirm enrichment of the target conjugate (e.g., bis-Tn-MUC1 target protein / peptide). The phage pool may then be converted to soluble scFv and expressed as individual scFv. The expression of scFv in the supernatant may be evaluated by dot blot analysis.

[0252] Screening for tumor-specific scFv clones in the phage library may be performed using ELISA binding assays, glycoprotein / peptide microarrays, and biolayer interferometry (OCTET) against target proteins / peptides and control proteins / peptides (non-target). Such screening can yield scFv antibodies that target selected glycoprotein antigens with high specificity and high affinity.

[0253] Binding of selected scFv to tumor cells expressing the target glycoprotein antigen (such as mammary cancer cell lines MCF7 and MDA-MD-231 COSMC KO) (FACS) may be used to further confirm tumor specificity.

[0254] In one embodiment, the present invention provides a method as disclosed herein, wherein the antibody library is a phage display library; and the screening includes performing biopanning of the antibody library with specific tumor glycopeptides (such as Tn-MUC1, Tn-CD43, Tn-MUC4, Tn-MUC16, Tn-MUC13). Several different Tn-tumor targets are disclosed, as a non-limiting example, in the review by Kudelka et al. (2015).

[0255] [V] Epitope target The present invention provides a method and antibody library for identifying specific antibodies against specific glycoside-peptide epitopes (combotopes) of specific glycoproteins (such as Tn, bis-Tn, STn, or bis-STn epitopes) on specific cancer cells.

[0256] This specification provides glycoside-peptide epitope-conjugated antibodies that can exert therapeutic effects by having the ability to specifically bind to specific glycoproteins, for example, specific glycoproteins on cancer cells. Preferably, the antibody library provided herein facilitates the identification of antibodies that may be used for the identification (diagnosis) or treatment of diseases or disorders (such as cancer).

[0257] This specification provides methods for treating proliferative disorders. Further, this specification provides methods for treating proliferative disorders, where the proliferative disorder is cancer, and the methods include identifying and isolating anti-cancer cell antibodies by screening the antibody library of the present invention to obtain antibodies having high specificity against cancer cells, and administering the antibodies identified in the manner described herein to subjects diagnosed with the cancerous disorder. Specific therapeutic methods include the use of the conbotope antibodies of the present invention by loading the specific antibodies onto natural killer (NK) cells to induce NK cells to target cells (e.g., cancer cells). Prior to loading the antibodies, NK cells may be collected from the patient to be treated or provided as donor NK cells. The loading may be in the form of the antibody(s) themselves, or in the form of a nucleotide sequence encoding the specific antibody / antibody. In another method, the specific antibody is linked to a cytotoxin or its non-toxic precursor or a similar cytotoxic effector molecule that induces cell death. Which effector molecules may be useful will be understood by those skilled in the art. This specification further provides therapies for proliferative disorders, where the cancer is selected from lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, and bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0258] Aspects of the present invention include administering one of the specific antibodies identified by the method described herein to subjects determined to have an abnormal / shortened O-glycosylation (e.g., a shortened O-glycosylation of the MUC1 protein) compared to a reference level (e.g., a level in non-cancerous cells). In one embodiment, the shortened O-glycosylation is selected from Tn and STn antigens (e.g., Tn-MUC1).

[0259] In one embodiment, the present invention provides a combotope antibody as disclosed herein for use in the treatment of diseases associated with abnormal / shortened O-glycosylation. In one embodiment, the present invention provides a combotope antibody as disclosed herein for use in the treatment of diseases associated with Tn and / or STn antigens. In a preferred embodiment, the present invention provides a combotope antibody as disclosed herein for use in the treatment of cancer.

[0260] In another aspect, the disclosure features a method comprising administering one of the specific antibodies identified by the method herein, or a composition containing such antibody (e.g., a cell composition, an antibody-drug conjugate, or an antibody-radioisotope conjugate), to a subject in need thereof, wherein the subject has, or has been identified or diagnosed as having, cancer cells (e.g., pancreatic cancer, epithelial cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, or epithelial adenocarcinoma) characterized by low glycosylation of peptide epitopes.

[0261] Other embodiments include using, for example, a test kit or as part of a test kit in a test for the presence of cancer in a subject.

[0262] In some embodiments, the library of the present invention comprises antibodies adapted to the species of the intended therapeutic target. Generally, these methods include “mammalization.” In some examples, the mammals are mice, rats, horses, sheep, cattle, primates (e.g., chimpanzees, baboons, gorillas, orangutans, monkeys), dogs, cats, pigs, donkeys, rabbits, and humans. Preferably, the antibodies are intended for human therapeutic targets and are therefore humanized.

[0263] [VI] Uses of identified antibodies Tumor-specific antibodies are used in immuno-oncology against targeted cancer cells to activate the immune system to attack these cells. These specific antibodies can act by directly binding to cancer cells to trigger an immune response, or by targeting molecules on cancer cells that suppress the immune response. This can lead to increased tumor cell death and / or slower tumor growth. Tumor-specific mAbs are often used in combination with other immunotherapies (such as immune checkpoint inhibitors or CAR-T cell therapy) to enhance the anti-tumor immune response. Tumor-specific monoclonal antibodies are also used in the form of antibody-drug conjugates (ADCs) to deliver cytotoxic drugs directly to cancer cells. The mAbs in the ADC are designed to recognize and bind to specific proteins on the surface of cancer cells, and upon binding, release the cytotoxic drug to kill the cancer cells. The advantage of using ADCs is that they allow for selective delivery of the drug to cancer cells while minimizing damage to healthy cells. Some examples of ADCs using tumor-specific mAbs include trastuzumab emtansine (T-DM1) for HER2-positive breast cancer and inotuzumab ozogamicin for acute lymphoblastic leukemia.

[0264] In one embodiment, the antibody of the present invention (i.e., an antibody identified using the antibody library of the present invention) is used to target cancer cells (such as activating the immune system that attacks cancer cells).

[0265] Accordingly, in one aspect, the present invention provides antibodies such as those disclosed herein for use in the treatment and / or prevention of cancer. In some examples, the cancer is lung cancer, head and neck squamous cell carcinoma, colorectal cancer, melanoma, liver cancer, classical Hodgkin lymphoma, kidney cancer, gastric cancer, cervical cancer, Merkel cell carcinoma, B-cell lymphoma, or bladder cancer. In one preferred embodiment, the cancer is a solid tumor.

[0266] In one embodiment, the antibody of the present invention is used in combination with other immunotherapies (such as immune checkpoint inhibitors and / or CAR-T cell therapy) to enhance the antitumor immune response.

[0267] In one embodiment, the antibody of the present invention is used in the form of an antibody-drug conjugate (ADC) (for example, to deliver a cytotoxic drug directly to cancer cells).

[0268] In another aspect, the present invention is a method for treating cancer; The present specification provides a therapy comprising administering a formulation containing at least one specific antibody, such as those disclosed herein, to a patient in need of such a therapy.

[0269] In one embodiment, the administered antibody is complexed with a cytotoxic moiety or loaded onto NK cells (such as in patients with NK- cells that express it on their cell surface).

[0270] Specific antibodies used in cancer treatment may be selected from an antibody list, but all antibodies are: (i) A VH-domain that binds to the Tn- and / or STn-glycan epitopes of the glycoprotein of the tumor cell; and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; And here the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein; This antibody list does not include 5E5, 5F7, or 2D9.

[0271] In one embodiment, the antibody administered to a patient in cancer treatment is: (i) A VH domain comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO:1, and containing amino acid residues H32, A33, H35, Y50, and S99 and / or S52, N55, and D57 with respect to SEQ ID NO:1; and (ii) Sequence ID: VL domain containing an amino acid sequence selected from 9 to 21, Includes.

[0272] In one embodiment, the antibody administered to a patient in cancer treatment is: (I) Containing an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28, with respect to SEQ ID NO: 28, the amino acid residues: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, VH domains including; and (II) Sequence ID: VL domain containing an amino acid sequence selected from 22-23, Includes.

[0273] In one embodiment, the antibody administered to the patient is: (i) A first VH domain comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO:1, and comprising amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO:1; and (ii) Sequence ID: The first VL domain containing an amino acid sequence selected from 9 to 21, including, or (I) A second VH domain comprising an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28, and containing amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to SEQ ID NO: 28; and (II) SEQ ID NO: A second VL domain containing an amino acid sequence selected from 22-23, Includes.

[0274] In another aspect, the present invention relates to a diagnostic method. Specifically, by targeting a Tn / STn combotope that is present on the surface of many cancer cells but rarely present on normal cells, a Tn or STn-binding monoclonal antibody, such as those disclosed herein, can be used as a tool for diagnosing cancer in a subject. The subject may be human or animal. The process is initiated by the administration of a Tn or STn-binding mAb designed to specifically recognize and bind to the Tn or STn-antigen. Upon administration, the mAb circulates within the body and binds to the Tn- or STn-antigen expressed on the surface of cancer cells. This binding can be detected and visualized using various imaging techniques, such as PET, MRI, or fluorescence imaging, depending on the label linked to the mAb. The presence and distribution of the Tn / STn-antigen-mAb complex in the body can then be analyzed to determine the presence, extent, and, if possible, type of cancer. This method provides a targeted approach to cancer diagnosis and potentially enables a more accurate understanding of the site and extent of cancer, which is important for early detection and effective treatment planning. The conbotope antibodies of the present invention may be used in such a diagnostic approach. Another approach to diagnosing cancer in a patient involves taking a tissue sample from a patient suspected of having a cancerous condition (for example, in the form of a biopsy of suspected cancerous tissue, or by excising all or part of cancerous tissue), then conjugating one or more conbotope antibodies of the present invention to the tissue sample, and determining the specific binding of the antibodies in a manner well known to those skilled in the art who are engaged in the art of identifying tissue antigen targets by immunodetection, thereby performing ex vivo diagnosis. Such diagnostic methods are generally known and routinely performed in hospitals worldwide.

[0275] [VII] Humanization of Antibodies As described above, the antibodies of the present invention are preferably humanized. Humanization of antibodies can be carried out in several different ways, as will be understood by those skilled in the art.

[0276] A non-limiting example of the humanization of such antibodies includes the following steps: (1) Step to identify mouse monoclonal antibodies: The first step in the humanization of mouse monoclonal antibodies is to identify antibodies with the desired specificity and affinity. This identification is typically performed by screening a large library of mouse monoclonal antibodies using techniques such as ELISA or flow cytometry. (2) Steps to analyze the structure of the antibody: Once a mouse monoclonal antibody with the desired specificity and affinity is identified, its structure is analyzed to identify regions crucial to its antigen-binding properties. These regions are typically located in the highly diverse variable regions of the antibody, which are essential for the recognition and binding of specific antigens. (3) Steps to select a human antibody framework: Human antibody frameworks are selected based on their structural similarity to other human antibody frameworks. This is important because it ensures that the humanized antibody retains the overall structure and stability of the original antibody. (4) Step of replacing the antigen-binding region: The mouse-derived antigen-binding region, which is also known as a complementarity-determining region (CDR), is replaced with a human-derived CDR while maintaining the overall structure of the antibody. This is carried out using genetic engineering techniques such as PCR, cloning, and site-directed mutagenesis. Often, it is necessary to substitute a small number of amino acids. Conservative substitutions can be performed without changing the properties of the antibody. For example, methods for selecting conserved amino acids in an antibody for substitution purposes, such as to humanize the antibody or to facilitate antibody synthesis and production, are understood by those skilled in the art. Substitution is achieved by altering the nucleotide code in the nucleotide sequence that codes for the antibody domain. (5) Steps for testing humanized antibodies: Once a humanized antibody is created, it is tested for its specificity, affinity, and functionality. This is typically done using techniques such as ELISA, flow cytometry, and Western blotting. The humanized antibody is also tested for immunogenicity, which is its ability to elicit an immune response in humans. If the humanized antibody is found to be safe and effective, further development can be pursued for its therapeutic use in humans. In the case of immunogenicity of a particular promising conbotope antibody, some of its amino acids may be substituted with a conservative counterpart, but it must be clearly demonstrated that the specificity and efficacy remain unchanged or are even improved.

[0277] [VIII] Methods for identifying glycopeptide targets In a further aspect, the present invention provides a method for identifying a glycopeptide target, the target comprising a Tn and / or STn epitope (e.g., a glycopeptide target in cancer cells). For example, the library of the present invention is used for the identification of such glycopeptide targets by immunoprecipitation and mass spectrometry. This approach involves incubating the phage display antibody library with a cell lysate or tissue sample and allowing the antibody to bind to its target protein. The antibody / protein complex is then isolated by immunoprecipitation and subjected to mass spectrometry to identify the protein. Another example is a protein microarray, which is an array of immobilized antibodies that can be used to identify the protein targets of antibodies. The target protein can be identified by incubating the phage display antibody library array with a cell lysate or tissue sample and detecting the binding.

[0278] Therefore, in one embodiment, the present invention discloses a method for identifying glycopeptide targets, the targets comprising Tn and / or STn epitopes and peptide targets (such as glycopeptide targets on cancer cells), the method comprising the following steps: (i) the step of preparing an antibody library as disclosed herein; and (ii) Incubating the antibody library with a sample containing the glycopeptide target; (iii) A step of analyzing one or more antibody / peptide complexes obtained in step (ii) in order to identify the amino acid sequence of the peptide epitope of the glycopeptide target. Includes.

[0279] In one preferred embodiment, the sample is a cell sample or a tissue sample (such as lysed cells or tissue). Glycoproteins may be isolated from lysed cells and used for screening. The antibody library is preferably prepared as a phage display library, and the glycopeptide targets may be identified by analysis of the antibody / glycopeptide complex using mass spectrometry or other similar methods as understood by those skilled in the art.

[0280] [IX] Determination of the specificity of the VH-domain and VL-domain As disclosed herein, the VH-domain of the conbotope antibody of the present invention binds to a glycosylation epitope (Tn and / or STn) of a glycoprotein in the cancer cell, while the VL-domain of the conbotope antibody binds to a peptide epitope of the same glycoprotein that associates with the glycosylation epitope.

[0281] Those skilled in the art can determine, by structural characterization such as using X-ray crystallography (as described in Example 1 of this specification), whether the VH-domain can have the characteristics of a glycan epitope-binding VH domain, and further, whether the VL domain can have the characteristics of a peptide epitope-binding VL domain.

[0282] Another method for identifying the VH domain is by sequence analysis (as in Example 3.3 described herein). Determining whether a sequence is a VH domain is done as follows: (a) To determine whether the amino acid sequence in question contains amino acid residue positions corresponding to H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to Sequence ID No. 1, that is, whether the amino acid sequence in question has the key residues necessary for the functionality of the Tn-binding VH domain, this may be done by aligning the sequence in question with Sequence ID No. 1 of the G2D11 VH domain; or, (b) To determine whether the amino acid sequence in question contains amino acid residue positions corresponding to T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102, and L103 with respect to SEQ ID NO: 28, that is, whether the amino acid sequence in question has the key residues necessary for the functionality of the STn-binding VH domain, this may be done by aligning the sequence in question with SEQ ID NO: 28. In order to function as a VH domain that binds to Tn and / or STn, at least three amino acids corresponding to the above amino acid residues must be present in the VH domain.

[0283] It is important that the VH-domain is versatile and does not contribute to or inhibit the binding of any peptide epitopes on the glycoprotein; this means that the binding of the VH-domain to the glycosylation epitope is not inhibited or affected by any peptide epitopes on the glycoprotein. Only in such cases can any VL domain selected for "clean" binding that does not inhibit the binding between the antibody's VH-domain and the peptide epitope be freely combined with the VH-domain. In other words, unwanted binding can distort the results, for example, in diagnostics or specific drug delivery targeting a particular tumor glycotype (Tn or STn) on a given protein.

[0284] The VL domain is identified by the method disclosed herein and is unique to each target. The identified VL domain is: (1) Evaluations are based on specificity; and (2) Common features of the CDRs are correlated with other VL-domain sequences. These features may be later confirmed by X-ray.

[0285] Examples Examples are described below to better illustrate the principles and practices of the embodiments disclosed herein to those skilled in the art, but should not be considered as limiting the scope of any of the claimed embodiments.

[0286] Unless otherwise specified, all chemicals were purchased from Merck (Germany). Unless otherwise specified, all buffers and culture media were dissolved in Milli-Q water (MQ) and autoclaved.

[0287] peptide Table 1 shows the peptides used for phage display selection, ELISA, and biolayer interferometry (BLI). CD43 and IgA were previously synthesized in-house using solid-phase peptide synthesis (SPPS), as described by Persson et al. (2016). The MUC1 peptide was either synthesized or purchased from Biosyntan (Germany).

[0288] [Table 2]

[0289] cell line All cell lines were maintained at 37°C in a humidified incubator with 5% CO2. MCF7, MDA-MD-231 WT, and COMSC KO cells were maintained in DMEM+GlutaMax (Gibco, 32430-027) supplemented with 10% FBS (Fischer Scientific, 11550356), 1% penicillin-streptomycin (Fischer Scientific, 15140122), and 1 mM sodium pyruvate (Gibco, 11360). MDA-MB-231 WT and COSMC KO cells were generously provided by Ulrich auf dem Keller. Jurkat cells were maintained in RPMI (Life Technologies, 32404014) supplemented with 10% FBS, 1% penicillin-streptomycin, and 2 mM L-glutamine (Sigma, G7513). HEK293 cells were maintained in Freestyle medium (Thermo Scientific, 15285885).

[0290] Broth medium, plasmid, and E. coli strain XL1-Blue electrocompetent cells were purchased from Agilent (Agilent, 200228). TG1 for phage display was kindly provided by Peter Kristensen of Aalborg University. The vectors, pAK100 phagemid and pJB33 expression vector, were kindly provided by Plunthum of the University of Zurich; both vectors are resistant to chloramphenicol antibiotics. E. coli TG1 and XL1-blue electrocompetent cells were cultured in 2x YT broth medium. Unless otherwise specified, liquid media were supplemented with 25 μg / mL chloramphenicol and 2% glucose.

[0291] The G2D11 VH chain and its variants in the pTwist vector belong to Twist Biosciences.

[0292] Software GraphPad prism 9 was used to create graphs, and Biorender was used to create images. CLC Main workbench 8.0 software was used for sequence alignment.

[0293] Example 1: Characterization of G2D11 G2D11 is a mouse-derived anti-Tn-scFv mAb. ScFv consists of a VH domain sequence number 1 and an LV domain sequence number 2, bound via the peptide linker (GGGGS) 4.

[0294] [1.1] Characterization of the structure Crystals of ScFv G2D11 were prepared using sitting drop technique and an appropriate precipitation solution. The structure of the obtained crystals was analyzed at a resolution of 1.9 Å to elucidate the density map (Figure 3). Despite the presence of two molecules weakly in contact with each other in the asymmetric unit, analytical ultracentrifugation revealed that this monomeric form behaves as a monomer regardless of the presence or absence of the bisTn-MUC1 peptide APGS*T*AP (where * represents the GalNAc moiety (SEQ ID NO: 55)). The glycopeptide is located in surface grooves formed by the light chain (L) and heavy chain (H) (hereinafter, VL and VH, respectively), and in particular, the two GalNAc moieties were recognized by residues in the three hypervariable regions of VH (Figure 3).

[0295] With the exception of OH6, all Ser-bonded GalNAc hydroxyl groups participated in hydrogen bonding. Specifically, the hydroxyl group OH3 was Ala33. H The NH group interacts with the OH4, and the OH4 is His32 H and Ser99 H It interacted with the side chain. The intraring oxygen of the sugar is Ser99 H It participated in hydrogen bonding with the carbonyl group of GalNAc. H The methyl group is involved in hydrogen bonding with the side chain, and is His50 H It participated in the CH-π stacking interaction with it.

[0296] When compared with a previously elucidated structure (Macias-Leon et al., 2020) using scFv-5E5 complexed with mono-Tn-MUC1 peptide (APGST*AP), it was revealed that these interactions in G2D11 are conserved. Furthermore, in the case of 5E5, Phe102 H The CH-π interaction between the Thr5 methyl group and the PT5 methyl group was also visualized (Macias-Leon et al., 2020). Phe102 helps to guide VH to one of the two GalNAcs. This suggests that Phe102 has poor flexible proximity to Tn in 5E5, is limited to Tn-Thr, and can only bind in a specific way. This further suggests that mono-Tn is preferred over bis-Tn in 5E5.

[0297] Surprisingly, Thr-bound GalNA was also closely recognized by scFv-G2D11. In this case, Ser52 H G2D11 participates in hydrogen bonding interactions with the carbonyl group, OH3, and OH4, while the side chains of Asn55 and Asp57 interact with OH4. G2D11 is more open and can more readily accommodate bonding to any combination of TnThr / TnSer, TnSer / TnThr, TnSer / TnSer, and TnThr / TnThr.

[0298] Recognition of G2D11 at the GalNac binding site is similar to that of the triple mutant (H32A) of the bis-Tn peptide. H -H35A H -S52A H ) is also a double mutant (S99A H -S52A H This was further supported by the fact that they were not joined (data not provided).

[0299] Interestingly, scFv-G2D11 did not recognize the peptide sequence, but Ala1 and Pro2 were surrounded by aromatic residues in the VL (Figure 3). In contrast, scFv-5E5 VL recognized Tyr98 L and hydrogen bonds between the Pro7 backbone, and Tyr100L The peptide was recognized by a CH-π interaction between it and Pro7 (Macias-Leon et al., 2020).

[0300] [1.2] Array sorting Using CLUSTALW, the G2D11 VH-domain was aligned with other known anti-Tn antibody VH-domains (using standard settings for multiple alignment parameters: scoring matrix: blosum62, gap opening penalty: 10, and gap extension penalty: 0.2). As indicated by the arrows in Figure 4, based on this alignment, conserved amino acid residues in the CDR1, CDR2, and CDR3 regions related to the functionality of the VH chain (i.e., GalNac binding) were identified.

[0301] Specifically, when referring to G2D11 (Sequence ID: 1), the VH domain preferably needs to preserve amino acid residues H32, A33, and H35 of CDR1, amino acid residues Y50, S52, N55, and D57 of CDR2, and amino acid residue S99 of CDR3.

[0302] Example 2: Conceptualization of a Tn-template phage display library Based on the X-ray structural data of the VH-domain and the observation that bis-Tn binding is mediated by the VH-domain (disclosed in Example 1), the antibody library was conceptualized as follows: In this antibody library, each antibody contains the VH chain of the previously identified scFv G2D11, which supports the recognition of the glycoside portion of the antigen, while the VL domain is variable, derived from naive mice, creating an scFv phage display library called a Tn-template library that enables screening for specific scFvs. The VL domain of this specific scFv supports the recognition of peptide antigens within the combotope.

[0303] [2.1] Construction of a phage display library Wild-type BALB / c mice were euthanized, their spleens were removed, and stored directly at -80°C with RNAlater RNA stabilization reagent until use. RNA was isolated from the spleen using an automated tissue disperser (gentleMACS Dissociator) and a miRNeasy kit (Qiagen) according to the manufacturer's instructions. 1 μg of RNA was used for cDNA synthesis using random hexamer primers (Fisher Scientific, 10609275) and SuperScript IV reverse transcriptase (Invitrogen, 18090010). The constant VH gene and VL antibody-specific gene were amplified by PCR using Q5 Hot Start High-Fidelity DNA polymerase (NEB M0494S). The primers used are listed in the sequence listing (SEQ ID NOs: 31-32). The VL and VH genes were gel-extracted and assembled with 5′ phosphorylated outer primers for rolling circle amplification in the next step. RCA improves restriction enzyme (SfiI) cleavage of the scFv gene. Assembled scFv fragments were subcloned into the Sfil digested phagemide vector pAK100 (NEB M0369) using electroligase over 16 hours at 16°C / 25°C. A phagemide pool containing diverse scFv fragments was introduced into XL1-Blue electrocompetent cells (Agilent, 200228) by electroporation. Cells were harvested in SOC medium, incubated at 37°C at 220 rpm for 1 hour, then seeded on selective medium agar plates and incubated overnight at 30°C. Colonies were scraped off with a 2xYT coolant, and stored at -80°C with 25% v / v glycerol added. For phage rescue, cells were infected with VCSM13 helper phage to obtain 10¹³ phages / mL, which were used for biopanning. The primers used are shown in the sequence listing (SEQ ID NOs: 33-49).

[0304] [2.2] Solid-phase selection, phage rescue, and preparation of scFv antibodies Three selections were performed using streptavidin M-280 dynabeads (Invitrogen, 11205D). Unless otherwise specified, all incubations were performed at room temperature with rotational shaking.

[0305] Beads were blocked for 1 hour with PBST containing 5% BSA (PBS containing 0.05% Tween20). After washing three times with PBST, a 100 nM biotinylated peptide (50 nM in the final wash) was diluted with PBST containing 3% BSA and conjugated to the beads for 2 hours. A phage library (10¹² phages / mL) was pre-selected against bare beads for 1 hour, followed by 2 hours of positive selection for the target antigen. Unbound phages were removed by washing three times with PBST containing 3% BSA, three times with PBST, and three times with PBS. Binding phages were eluted with 1 mg / mL of freshly prepared trypsin solution and infected exponentially growing E. coli TG1 at 37°C for 30 minutes. Infected bacteria were spread on agar plates and incubated overnight at 30°C. Colonies were scraped onto culture medium, homogenized, and a 1:1000 homogeneous mixture was inoculated into liquid medium and grown at 220 rpm at 37°C until an OD600 of 0.4-0.5 was reached. Next, VCSM13 helper phage (10⁹ phages / mL) was inoculated at 37°C for 30 minutes. To precipitate the bacteria and induce phage production, the precipitate was resuspended in glucose-free liquid medium supplemented with antibiotics and isopropyl β-D-1-thiogalactopyranoside (IPTG, diluted 1:1000). The culture was incubated overnight, then centrifuged to remove the bacterial precipitate, and phages were precipitated from the supernatant by adding ice-cold PEG / NaCl (20% w / v PEG6000, 2.5M NaCl) in a 1:4 ratio. After incubation on ice for 1 hour, the precipitated phages were settled by centrifugation at 10,800xg for 30 minutes, followed by centrifugation at 5,000xg for 5 minutes. The phage pellet was resuspended in 1 mL of cold PBS and further centrifugation was performed at 13,000xg for 10 minutes to remove cell debris. The concentration was measured spectroscopically at 269 / 320 nm according to the following equation: Phage particle count / ml = (A269-A320) x 6 x 10¹⁶ / number of bases per virus particle. The precipitated phages were used in subsequent selection rounds.

[0306] For STn library selection, peptides were immobilized on NHS beads (Fisher Scientific, 88827). First, the beads were washed once with ice-cold 1M hydrochloric acid (HCl). A 100 nM peptide was diluted in print buffer and incubated with the beads for 2 hours. After washing twice with 0.1 M glycine (pH=2), blocking was performed with 3M ethanolamine for 1 hour. Incubation, washing, and trypsin elution of the phage library were performed according to the above method.

[0307] [2.3] Subcloning, expression, and scFv screening After three selections, polyclonal phagemids containing different scFv fragments were purified using the GeneJet Plamsid Miniprep kit (Thermo Fischer, K0503) according to its protocol and digested with restriction enzyme SfiI at 50°C for 20 minutes. These were then ligated to a pJB33 expression vector using T4 electroligase at 65°C for 1 hour. Pools of these different constructs were introduced into XL1-Blue electrocompetent cells by electroporation, the cells were recovered in SOC medium, incubated at 37°C at 220 rpm for 1 hour, then spread onto agar plates and incubated overnight at 37°C. 62 colonies were individually picked and transferred to a U-bottom 96-well plate and incubated overnight at 37°C. The overnight culture was inoculated into glucose-free fresh medium and incubated at 37°C at 220 rpm for 4 hours before induction of IPTG (final concentration 0.5 mM). It was then incubated overnight at 30°C in a humidified incubator at 800 rpm. Cells were precipitated by centrifugation at 3,000xg for 10 minutes. ELISA for binding positivity was performed using the supernatant. For clonal sequencing analysis, plasmid DNA was purified from individual clones and sent to Macrogen for Sanger sequencing using custom-designed M13R primers. CLC Main Workbench 8.0 software was used for sequence alignment.

[0308] [2.4] Preparation and purification of soluble scFv The total periplasm protein extraction process was performed on ice for 1 hour, and all centrifugation was carried out at 4°C. Selected clones were transferred from glycerol stock to 2xYT medium supplemented with 2% glucose and 25 μg / ml chloramphenicol, and grown overnight at 37°C at 220 rpm. The cultures were diluted overnight in fresh medium and cultured to the exponential growth phase before IPTG induction. Subsequently, they were incubated overnight at 20°C at 220 rpm in the presence of 1 mM IPTG. Bacteria were recovered by centrifugation at 6,000xg for 10 minutes, and the precipitate was resuspended in an ice-cold solution of 100 mM Tris (pH 8) and 20% w / v sucrose containing an EDTA-free protease inhibitor cocktail (ThemroFischer, A32965). After centrifugation at 8,000xg for 10 minutes, the precipitate was resuspended in an ice-cold MQ solution containing 5 mM MgSO4. The pellet was centrifuged at 8,000xg for 10 minutes, the two fractions were pooled together, and centrifuged again at 12,000xg for 60 minutes to remove cell debris.

[0309] The pool fraction containing soluble scFv was filtered through a 0.45 μm filter membrane, and the supernatant was mixed with 4x equilibration buffer (100 mM Tris, 1.2 M NaCl, pH 8) in a 3:1 ratio (v / v). This mixture was incubated overnight at 4°C with nickel-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen, 30210) while rotating and shaking. The beads containing the scFv were precipitated by centrifugation at 1,000 x g for 2 minutes and loaded onto an affinity resin column (Thermo Scientific, 29920) pre-equilibriumated with 10 column volumes (CV) of 1X equilibration buffer. Unbound proteins were washed away with 10 column volumes of washing buffer (1x equilibration buffer containing 10 mM imidazole (pH 8)), and bound scFv was eluted with 0.2 column volumes of elution buffer (1x equilibration buffer containing 250 mM imidazole (pH 8)). The above elution process was repeated twice. The eluted scFv antibody was desalted using a Zeba desalting spin column (Fisher Scientific, 89892) according to the manufacturer's instructions, and the buffer was changed with PBS. Protein quantification was performed using a BCA protein assay kit (ThermoFischer, 23225) according to its protocol, and purity was evaluated by SDS-PAGE.

[0310] [2.5] Enzyme-linked immunosorbent assay (ELISA) Unless otherwise specified, all steps were performed at room temperature with shaking. All washing between steps was done using PBST (PBS containing 0.05% Tween20). Antigen immobilization in coating buffer (0.015M Na2CO3, 0.035M NaHCO3, pH 9.6) was performed using a 96-well Maxisorp plate (ThermoScientific, 10394751). The plates were coated overnight at 4°C with streptavidin (NEB N7021S) at a concentration four times lower than the antigen concentration. The plates were blocked for 1 hour with shaking using PLIP (0.5M NaCl, 0.003M KCl, 0.0015M KH2PO4, 0.0065M Na2HPO4.2H2O, 1% w / v BSA, 1% Tween20) as the blocking buffer. To detect the positive signal, 3,3′,5,5-tetramethylbenzidine (TMB, Fisher Scientific 12617087) chromophor was used as the substrate. The reaction was stopped with 0.5 M H2SO4, and the absorbance at 450 nm was measured using a VICTOR Nivo plate reader.

[0311] In the polyclonal phage ELISA, plates were incubated for 2 hours with polyclonal phages serially diluted in blocking buffer, followed by 1 hour incubation with a secondary antibody. Binding phages were detected using a 1:10000 dilution of mouse monoclonal anti-M13-HRP antibody (Nordic Biosite 58-11973-MM05T-H-100).

[0312] In monoclonal scFv ELISA, the antigen concentration was set to 50 nM. 50 μL of supernatant from the culture obtained by culturing each clone overnight was added to each well. In antibody titration ELISA, the antigen coating was fixed at a peptide concentration of 330 nM, and a 5-fold titration was performed starting with 300 nM scFv. Binding scFv was detected using mouse monoclonal anti-His HRP (C-terminus) (Invitrogen, 46-0707) at a 1:2000 dilution.

[0313] [2.6] Cell binding assay Cells were washed twice with FACS buffer (DPBS containing 0.1% w / v BSA (Sigma-Aldrich, D8537)) and then treated with 100 mU / mL Clostridium perfringens neuraminidase (Sigma, N5631) at 37°C for 30 minutes. After the two washes, the cells were resuspended in 100 μL of FACS buffer and transferred to a U-bottom 96-well plate. To evaluate the efficiency of the neuraminidase, cells were stained with 2.5 μg / mL PNA (B-1075), 0.4 μg / mL VVA (B-1235), 1 μg / mL SNA (B-1305), and MAL I (B-1315). To detect binding positivity to the target of interest, cells were stained on ice for 30 minutes with anti-MUC1 scFv antibodies at concentrations of 5, 1.25, 0.3, and 0.08 μg / mL and anti-CD43 scFv antibodies. Positive controls included 1 μg / mL rabbit anti-MUC1 (HMFG2) (Abcam, ab245693) and 1:20 CD43-FITC (Miltenyi, 130-097-360). Cells were washed twice and stained with 1:1000 streptavidin conjugated with Alexa Fluor 488 (Invitrogen, S32354) to detect lectin binding; stained with 1:1000 anti-His Alexa Fluor 647 conjugate (R&D IC050R) to detect scFv binding; and stained with goat anti-rabbit Alexa Fluor 647 (1:1000) on ice in the dark for 20 minutes. After washing the cells twice, they were analyzed using Miltenyi Biotech-MACS Quant 16. Data analysis was performed using FlowJo version 10. All lectins were purchased from Vector Biolabs.

[0314] [2.7] Biolayer Interferometry (BLI) Steady-state reaction kinetics were determined using the Octet Red96 system. Samples and buffers were dispensed into 96-well black flat-bottom polypropylene plates (Greiner Bio-One, 655209) with a final volume of 200 μL per well. All measurements were performed at 30°C with shaking at 1000 rpm. Before each assay, the high-streptavidin biosensor chip (SAX) (Sartorius, 18-5117) was pre-wetted with reaction rate buffer (DPBS supplemented with 0.1% BSA and 0.02% Tween20) after equilibration in reaction rate buffer for 60 seconds and at least 10 minutes. The biotinylated target glycopeptide in the reaction rate buffer was loaded onto the streptavidin biosensor chip for 300 seconds, followed by a further 100-second equilibration step. Various different concentrations of scFvs were bound for 300 seconds. Finally, dissociation was observed for 300 seconds using reaction rate buffer. The binding and dissociation reactions were processed using Octet software (version 12). The interferometric data were fitted to a 2:1 model to calculate affinity and rate constants.

[0315] [2.8] VL diversity sequencing To track phage enrichment in each panning iteration, the following LV diversity screening was performed. If scFv VL sequences associated with the target peptide appear more frequently (i.e., are enriched), then phages containing scFv are confirmed to bind to the selected target. These data are then used to compare the sequences of selected clones.

[0316] Oxford Nanopore Technology (ONT) was used for sequencing. After each selection, bacteria were scraped from the agar plate, and DNA was purified from the homogeneous suspension using the GeneJet Miniprep kit according to the manufacturer's protocol. For the non-selected library, DNA purification was performed from the homogeneous suspension of scraped bacteria using Nucleobond Xtra EF plasmid purification (MACHEREY-NAGEL GmbH & Co, 740422.50M) according to the manufacturer's protocol. The primer sets used are shown in the sequence listing (SEQ ID NOs: 50-53). 3 pg of plasmid DNA was used as input material. Nanopore sequencing and data analysis were performed according to a modified method of Karst et al. (2021) with the following modifications: DNA cleanup after early and late PCR was performed using 0.8x volume AMPure XP beads, and all DNA washing for purification was performed using 80% ethanol. DNA was quantified using the Qubit dsHS DNA assay (Thermo Fisher Scientific). After late PCR, a 1% agarose gel was used to confirm the accurate product size. The SQK-LSK110 ligation sequencing kit protocol was used for samples prepared for R9 flow cells, while the SQK-LSK114 ligation sequencing kit protocol was used for samples prepared for R10 flow cells. Flow cell and sequencing tests of the samples were performed over 72 hours using a MinION Mk1B instrument.

[0317] Example 3: MUC1 as a proof of concept MUC1 was used as a proof-of-concept for a constructed library to identify binders for MUC1. Novel scFvs and known mAbs were compared in terms of sequence and antibody activity, and the efficacy and functionality of both libraries were determined. After sequencing of the identified scFvs, VL chain analysis was performed, and their specificity was characterized by ELISA, cell binding assays, and kinetic studies using BLI.

[0318] [3.1] Phage selection Each antibody, containing a Tn template library with the G2D11 VH domain (SEQ ID NO: 1), was subjected to three selections with the target antigen; in these selections, target peptide 1 (see Table 1) was immobilized on streptavidin-coated beads. After each selection, the phages were eluted, amplified, and precipitated. The phage stocks after each selection were titrated and analyzed by polyclonal phage ELISA (Figure 5). In each selection using zero nonspecific binders for streptavidin, polyclonal phage ELISA showed enrichment of specific binders for the MUC1 target peptide. Interestingly, in the second and third selections, the binder for peptide 3 (see Table 1) was enriched, so this library can be used to identify binders for peptides containing one GalNac. Furthermore, nanopore sequencing was performed after each biopanning to confirm the enrichment of specific sequences for MUC1. Previous studies based on crystallographic analysis of 5E5 mAb (SEQ ID NO: 3+4) and other known mAbs indicate that the CDR3 sequence, and specifically the YXY motif of CDR3, is crucial for binding to the MUC1 peptide backbone. Based on these observations, the 20 most frequent CDR3 sequences were ranked. Sequence enrichment was also demonstrated (Table 2). Furthermore, the MUC1-specific YXY motif in the CDR3 sequences was identified.

[0319] [Table 3]

[0320] To isolate specific binders for each target, the DNA polyclonal phagemide pool was purified after the third selection; after subcloning the pools of different scFv genes into expression vectors, the expression of individual scFv was examined in a 96-well format. ScFv expression in the supernatant was evaluated by dot blot analysis (Figure 6). Sixty-one clones were selected and screened by monoclonal ELISA against target peptide 1 (see Table 1) and control peptides 4 and 8 (Figure 7). Peptide 4 was used to double-check that mAbs against the backbone could not be selected in the Tn template library. The control peptide used in this study is IgA, which is produced in mucosa and plays a crucial role in mucosal immunity (see Table 1). IgA has N- and O-linked glycosylation sites and is involved in several pathological conditions, including IgA deficiency and IgA nephropathy. Clones that did not respond to the control peptide were selected for further characterization. Furthermore, Sanger sequencing was used to obtain the sequences of the 61 clones mentioned above, and when aligned with the VL sequences of 5E5 and 2D9, differences in the CDR of the VL chains were revealed. The important binding features shown in Table 2 were also present in the VL sequences of 5E5 and 2D9, further supporting their importance in determining interaction with the peptide backbone and specificity.

[0321] [3.2] Expression and evaluation of soluble scFv Based on the specificity of monoclonal scFv ELISA and VL sequence comparison, 10 clones were selected and purified. Of these 10 clones, three clones (D3, A3, and D2) were fully evaluated and are shown in sequence numbers 9-11 of the sequence listing. For scFv, only their VL domains are listed. The VH domain of scFv is identical in all clones; namely, it is the VH of G2D11 (sequence number 1). The VL and VH are linked by peptide linker (GGGS) 5.

[0322] The selected clones described above were generated on a large scale for evaluation using ELISA, reaction kinetics, and cell binding assays. The scFv was expressed and purified by His-tag affinity purification. The purity of the scFv was checked by SDS-PAGE Coomazy analysis and Western blotting to confirm the presence of the His-tag. In titration ELISA, soluble scFv was screened for binding to fixed-concentration MUC1 target peptide 1 and control peptide 8 (see Table 1). The results are shown in Figures 8A and 8B. Furthermore, scFv was screened for cross-reactivity to other MUC1 peptides 2, 3, 4, and 5 (see Table 1). The results are shown in Figure 9. The library hypothesis that it is impossible to select only scFv for the peptide backbone was confirmed by the negative binding to non-glycosylated MUC1. scFv D5 showed mono-Tn-MUC1 glycopeptide binding at concentrations >10 nM. Interestingly, clone D5 binds to peptide 3; on the other hand, clones H3 and D3 also bind to peptide 3 only at high concentrations.

[0323] Clones that showed higher specificity for target peptide 1 and zero cross-reactivity to the IgA1 hinge region were selected for further evaluation. Clones A3 and D2 did not bind to the IgA1 hinge region at all, while D3 and H3 bound at high antibody concentrations. The remaining scFvs recognized IgA1 in addition to MUC1. Based on the scFv specificity of the titration ELISA, A3, D2, and D3 scFvs were selected and subjected to biological evaluation and reaction kinetic studies.

[0324] In the cell binding assay, two types of mammary cancer cell lines were used: MCF7 and MDA-MB-231 WT, as well as COSMC KO cells. COSMC KO refers to the knockout of the COSMC gene, a chaperone necessary to support the catalyst for transposition of Galb1-3 to the second-to-last sugar (GalNAc) of Tn. This glycosylation leads to the formation and subsequent elongation of Tn antigens. In cancer, this COSMC is a frequently observed phenomenon and is a result of the exposure of Tn and STn antigens on tumor proteins. mAb HMFG2 (anti-MUC1) was used as a positive control to confirm MUC1 expression on cells (data not shown); and 5E5 was used as a control mAb to confirm the presence of Tn glycosylation on MUC1. In this assay, when binding occurs, the main peak of the flow cytometry diagram shifts to the right compared to the control. All three scFv strains (A3, D2, and D3) showed positive binding to MDA-MD-231 COSMC KO cells but not to WT cells (Figure 8C). However, neither the selected MUC1 scFv clone nor the 5E5 mAb control showed any positive binding in MCF7 cells (Figure 10A). Neuraminidase treatment did not enhance the binding of the 5E5 control or the selected MUC1 scFv clones D3, D2, and A4 to MCF7 cells (data not shown).

[0325] To exclude nonspecific binding, scFv binding to HEK293 was tested. This was because HEK293 does not express endogenous MUC1. No binding was detected before or after neuraminidase treatment (Figure 10B).

[0326] To reinforce the concept of VL's contribution to specific combotope recognition, the binding affinity (KD) of G2D11 and the newly identified MUC1 scFv clone was determined by BLI. Biotinylated target peptide 1 was immobilized on a streptavidin sensor chip to determine the binding and dissociation rates necessary for calculating the dissociation constant. Only clone D3 showed higher binding affinity compared to G2D11 (Table 3).

[0327] [Table 4]

[0328] Interestingly, the curve fit the 2:1 binding model (heterogeneous binding model), but not the 1:1 binding model. Therefore, the calculation of the steady-state kinetics was impossible, and in fact, two types of KD values (i.e., KD and KD2) were obtained. One explanation for this is that VH-G2D11 binds to bis-Tn in two orientations that are more flexible with respect to binding to the Tn-structure, which is in contrast to 5E5, for example, which prefers mono-Tn linked to threonine (Thr), but is less so for the bis-Tn structure.

[0329] Based on the above, it can be concluded that the clones against the peptide bond (combotope) have enhanced affinity (p-nM) compared to G2D11 (60 nM). Specificity is improved and affinity is enhanced by the interaction of more antigen / antibody bindings.

[0330] [3.3] Sequence analysis Analysis of the individual scFv sequences revealed information regarding the CDR regions of the VL chain. scFv D3, D5, H3, like 5E5, share the MUC1-specific binding motif YSY in CDR3, and this motif is a requirement for peptide backbone binding interactions as shown previously by crystal analysis (Figure 11). More specifically, Y98 L and Y100 L contribute to the peptide binding. scFv A3 and D2 share the WNY motif, while scFv B5 and A2 have the SSY motif (Table 4). In all scFvs as well as the 5E5 mAb, Y100 L is conserved. There are several variations in CDR1 and CDR2, which may be related to the sequence and size of the target peptide (e.g., the W50 L residue in proximity to the peptide backbone).

[0331] [Table 5]

[0332] The acquired data correlates with currently known information (X-ray and specific interaction residues) obtained for mAb 5E5 as a reference. This demonstrates that the library concept herein allows for the enrichment and selection of sequences with the same characteristics as those obtained from immunized mice and hybridomas targeting the same antigen. This represents a significant advance and provides a rapid system without the use of animals. The approach also provides a very large number of further clones for evaluating similar or different VL-sequence options, which may be potentially better or potentially different binders. This method provides a relatively controlled and systematic approach (compared to hybridomas) for identifying a large number of evaluation candidates.

[0333] [3.4] Binding profiles of novel selected scFv compared to known anti-Tn antibodies The glycopeptides in Table 5 were printed onto a microarray chip, and the binding of scFv D3 and scFv A4 to these peptides was compared with the binding by scFv 5E5, scFv 2D9Chi, and scFv G2D11. 2D9Chi contains the 2D9 VL domain and G2D11 VH domain. The results are shown in Figure 12 (for brevity, the heatmap shows amino acids 9-19 of the peptides in Table 5).

[0334] [Table 6]

[0335] Substitution of Thr with Ala and the VTSA epitope were found to eliminate binding of svFV 5E5. The same epitope profile was shown for scFv D3. Mono-Tn was not sufficient for binding to scFv G2D11, but scFv G2D11 was found to bind to two adjacent Tn antigens. As disclosed above in this specification, the VH domain of scFv G2D11 is important for Tn binding. This binding test further revealed that the VH domain of G2D11 can support mono-Tn binding when peptide binding is involved (such as in the case of scFV D3; i.e., having the VH domain of G2D11 and the VL domain identified by library screening).

[0336] In contrast to scFV G2D11, which also binds to two adjacent Tn antigens in the Thr-Ser sequence, ScFv 2D9Chi binds to two adjacent Tn antigens only in the Ser-Thr sequence. ScFv A3 showed the same epitope recognition as 2D9Chi. Substitution of the Pro residue with Ala resulted in the loss of binding to the glycopeptide in all scFv molecules.

[0337] Example 4: Conceptual Evaluation - CD43 as the First Example Not only for MUC1, but also to evaluate the further potential efficacy of the Tn template library, we selected CD43 as the primary target for identifying the binder, following the same procedure.

[0338] Target peptide 9 (see Table 1) was immobilized on streptavidin beads, and three selections were performed. Polyclonal phage ELISA and nanopore sequencing were performed for efficient phage selection. Both polyclonal phage ELISA (Figure 13) and sequencing confirmed phage and sequence enrichment between each selection. However, in the case of CD43, it was unclear which CDR was important for peptide backbone binding, as in the case of MUC1 (Table 6), so the sequences were grouped based on combinations of CDR1, CDR2, and CDR3.

[0339] [Table 7]

[0340] Sixty-one clones were selected, and the binding of scFv to target peptide 9 and control peptides 7 and 10 (see Table 1) was evaluated by monoclonal scFv ELISA (results are shown in Figure 14); VL sequences were also obtained. Based on the results of the monoclonal ELISA, ten clones that showed high specificity to the target peptides and zero or low cross-reactivity were selected for further evaluation.

[0341] As the first step, the binding specificity of the 10 clones (referred to as ori, H1, -A1, F4, C5, A7, D3, G3, D7, and H2) was tested by titration ELISA. These 10 clones are listed in the sequence listing as SEQ ID NOs: 12-21. For scFv, only its VL domain is listed. The VH domain of scFV is identical in all clones; i.e., the VH of G2D11 (SEQ ID NO: 1). The VL and VH are linked via peptide linker (GGGS) 5.

[0342] With the exception of C5, which showed the lowest binding specificity, all scFvs exhibited high binding specificity to the CD43 target peptide (peptide number 9) (Figure 15A). Clones A7 and D3 showed high binding affinity to the IgA1 hinge region peptide; on the other hand, A1 and F4 showed some degree of cross-binding only at high concentrations (Figure 15B). The remaining scFvs showed no binding to the IgA1 peptide at all. scFvs A1, D7, H1, and H2 were selected for further evaluation in cell binding assays and kinetic studies by BLI.

[0343] For biological evaluation, leukemia Jurkat cells were selected; the reason is that leukemia Jurkat cells highly express Tn antigen at high levels because single nucleotide base deletions result in frameshift and truncation of the COSMC chaperone. In either case, the cells were treated with neuraminidase and then stained. All 4 clones were positive for Jurkat cell staining, and this staining was enhanced by neuraminidase treatment (Figure 15C). scFvs were further tested using HEK293 cells that do not normally express CD43 (Figure 16).

[0344] Finally, to confirm the contribution of VL in the peptide bond, kinetic studies by BLI were performed. As in the case of the MUC1 scFv clone, the binding curve was fitted to a 2:1 binding model (heterogeneous ligand) to obtain two KDs (Table 7).

[0345]

Table 8

[0346] Sequence analysis of the 9 selected scFvs provided information on the CRD1 and CDR3 regions of the VL chain as demonstrated by X-ray. X-ray experiments of ori-CD43 scFv (SEQ ID NO: 12) with the CD43-GAS*T*GSP peptide (where the asterisk represents the GalNac residue) (SEQ ID NO: 56) revealed that Y99 L is required for specific peptide backbone binding interactions (Figure 17). Interestingly, all scFvs except D3, D7, and H1 had Y99 L D3 scFv had L99 L while D7 and H1 had W99 L (Table 8). In contrast to D7 and H1, which specifically interact with the CD43 peptide and do not show cross-reactivity with the TnIgA hinge or TnMUC1, D3 with L99 L showed cross-binding to the IgA hinge region peptide. However, Y99 LscFv C5, which contains [the specified compound], also showed cross-reactivity with the control peptide.

[0347] [Table 9]

[0348] This example of CD43 is presented as a second target; although it has a different peptide sequence, it is still a mucin-like peptide sequence (characterized by high content of S, T, P, etc., among other amino acids related to O-glycosylation); The enrichment of VL-domain sequences with unique CDR fingerprints, particularly those with CDR1 and CDR3 CDR fingerprints, was demonstrated and confirmed by X-ray. Further CDR1 interactions, as shown by ELISA, further enhancement of specificity occurs (no cross-reactivity to either TnIgA or TnMUC1). This indicates that the antibody library concept of the present invention can also target other Tn-peptides / proteins with different peptide sequences, and that it is possible to obtain VL-domain sequences with corresponding signatures different from the above-mentioned VL-domain sequences targeting Tn-MUC1.

[0349] Example 5: STn-Template Library- [5.1] Alignment of 3F1 and G2D11 Antibody 3F1 is a known anti-STn antibody (Prendergast et al., 2017). By comparing the sequences of the G2D11 VH domain (SEQ ID NO: 1) and the 3F1 VH domain (SEQ ID NO: 25) (see Figure 18), we identified amino acid positions in the CDR1 and CDR3 domains of G2D11 that are potentially essential for STn glycan binding; specifically, we modified the amino acid residues of the G2D11 VH domain as follows: Modifications of I28T, A30T, P101L, delG102, T103A, and F104L are considered promising for converting the Tn glycan binding specificity of VH-G2D11 to STn glycan binding specificity.

[0350] [5.2] STn-binding G2D11 mutant The G2D11 VH-domain variant was prepared based on the amino acid positions identified above, which are potentially related to STn specificity: M1(LAL): P101L, delG102, T103A, and F104L; M2 (LAL-TFT): I28T, A30T, P101L, delG102, T103A, and F104L; M3 (LAL-TFT-G): I28T, A30T, D56G, P101L, delG102, T103A and F104L; and M4 (TFT-G): I28T, A30T, and D56G.

[0351] Figure 19 shows microarray data of glycopeptide binding for scFV G2D11, 3F1, and mutants (M1-4). Mutants M1-M4 are variants of scFV G2D11 containing the selected mutations in the VH domain mentioned above. In the LAL mutant M1, Tn binding was lost, but STn binding was not observed at all; on the other hand, in the LAL+TFT mutant M2, STn binding was acquired, and Tn binding was not observed at all. These VH-domain mutations are sufficient to convert Tn binding to STn binding. Note that LAL is essential for this conversion. In the absence of LAL mutations (only TFT mutations), Tn binding remains.

[0352] In other words, it was revealed that a combination of mutations in the VH chain, more precisely, a mutation from "IFA to TFT" in CDR1 and a mutation from "PGTF to LAL" in CDR3, causes a conversion of binding ability from the Tn glycan form to the STn glycan form (Figure 19).

[0353] When this is combined with the VH sequence alignment described above (Example 1.2), it can be seen that the amino acid residues of the VH chain required for binding to bis-STn O-glycan are as follows: When referring to sequence number 28, see T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103.

[0354] [5.3] STn Template Library - A second phage display library (referred to as the STn template library) was created by chain shuffling a pool of mutant VH strands (SEQ ID NO: 28) (i.e., the G2D11 VH domain containing mutations from IFA to TFT in CDR1 and from PGTF to LAL in CDR3) and naive VL domains derived from naive mice.

[0355] Example 6: MUC1 as proof of concept in an STn template library To investigate the potential of the STn template library, MUC1 was used as a proof-of-concept. Two peptides, namely target peptides 6 and 7 (see Table 1), were immobilized on NHS beads, and three selections were performed using the method described above. After each selection, nanopore sequencing was performed, and the VL diversity between the two target peptides was compared.

[0356] Tables 9 and 10 show the top 10 most enriched combinations and their percentages (%) for bis-STn-MUC1 and mono-STn-MUC1 in each trial. The specific MUC1-binding motif Tyr-X-Tyr can be identified in both selections. The use of an STn library is possible, and the generation of data / clones similar to those obtained for TnMUC1 is confirmed by VL-domain sequencing.

[0357] [Table 10]

[0358] [Table 11]

[0359] The enriched VL-domain sequence contains the same characteristics as those found in Tn-MUC1, further clarifying the fingerprint associated with the MUC1 peptide target.

[0360] Based on monoclonal scFv ELISA specificity and VL sequence comparison, three clones were selected and purified. These three clones (named C4, D3, and C7) are shown in sequence numbers 22-24 of the sequence listing. Only the VL domains of these scFvs are listed. The VH domain is identical in all scFV clones; i.e., the mutant VH chain of G2D11 (sequence number 28) (i.e., the G2D11 VH domain including the IFA to TFT mutation in CDR1 and the PGTF to LAL mutation in CDR3, as disclosed in Example 5). Its VL and VH are linked via the peptide linker (GGGS) 5.

[0361] Microarray analysis (Figure 20) shows that both bis-STn-MUC1 binders and mono-STn-MUC1 binders were obtained, but the Tn- binder was not.

[0362] Example 7: Kinetic affinity of MUC1-specific scFv and CD43-specific scFv Table 11 shows an overview of the kinetic affinities of MUC1-specific scFv and CD43-specific scFv identified using an antibody library according to the present invention.

[0363] [Table 12]

[0364] Example 8: Mono-Tn / STn scFv binder As can be seen from the polyclonalphage ELISA (Figure 5, Example 2), the enrichment of mono-Tn MUC1 conjugates by bis-Tn MUC1 biopanning indicates that it is also possible to pan the library using peptides linked to a single GalNac.

[0365] In fact, three selections were performed using MUC1 target peptides 2 and 3 (see Table 1). Although polyclonal phage ELISA did not show phage enrichment, sequencing of the selected clones revealed that specific MUC1 sequences were shared.

[0366] This is an important observation, and while not limited to theory, it can also be explained as follows: First, it is known that mono-Tn peptides do not bind to G2D11 (at least not in the absence of VL contribution; see Persson et al., 2017 in the references). This is supported by VH mutations (as in Example 1 above). G2D11 VH has high affinity (60 nM) for bis-Tn, in contrast to mono-Tn. As a result, panning with mono-Tn also results in a much lower degree of clone enrichment; this is because many of them are bis-Tn binders. However, as the quantity decreases, the quality improves, and the remaining binders are most likely to be combotope binders that include a VL peptide contribution that enhances binding affinity. This is an advantage, as it shows that stringency can be influenced by manipulating the binding strength of the VH domain or by removing one Tn, that Tn inhibitors can be provided, that VH can be mutated to reduce specificity for bis-Tn, and that other panning conditions are possible. Therefore, it is understood that the evaluated selected scFv clones contain a high proportion of the combotop binder and only a small proportion of the bis-Tn-hapten binder. This is also confirmed for mono-STn-MUC1 by the nanopore sequence enrichment phage data mentioned above. Conversely, the enrichment of the bis-Tn / STn binder contains a much larger proportion of the hapten binder and a smaller proportion of the combotop binder. In summary, the antibody library concept of the present invention can be used not only for bis-Tn / Stn-peptide binders but also for mono-Tn / STn-peptide binders, which greatly increases its practicality; this is because Tn / ST can be individual, bis, or in larger clusters.

[0367] Example 9: Humanized mouse mAb Humanized scFv is generated by humanizing the VL and VH immunoglobulin domains of mouse-derived anti-CD43. Humanization of VL and VH is performed in the scFv format as follows.

[0368] protocol (1) Identify the complementarity determination area (CDR). CDRs are the components of an antibody that interact with an antigen. We identify CDRs in mouse monoclonal antibodies. The VH and VL amino acid sequences of mouse anti-CD43 scFv obtained by phage display are numbered according to IGMT; and CDR residues defined by IMGT are identified. Amino acids involved in binding but not included in the CDR residues defined by IMGT are also included as CDRs. (2) Design a humanized version of the antibody. Computational modeling tools are used to design humanized versions of monoclonal antibodies. The goal is to maintain the antigen-binding specificity of the mouse antibody while substituting mouse-derived CDRs with human-derived CDRs. VH and VL sequences are generated from amino acid sequences containing three complementarity-determining regions (CDRs) within the VL and VH domains, with the CDRs masked. These sequences are used as input to identify similar frameworks from the human V gene (heavy chain, kappa, lambda) germline database in the Basic Local Alignment Search Tool (BLAST) algorithm (Altschul et al., 1997). Furthermore, amino acid sequences of framework 4 derived from the VH and VL of mouse anti-CD43 scFv are used to identify similar human J gene segments. Based on our own analysis of anti-CD43 sequence identity, individual and pairing frequencies of the V gene, and previous experience with the use of specific templates in conventional humanization, we select human V and J gene segments as template frameworks. To identify potential sites that could induce reverse mutations at the corresponding mouse amino acids, selected human V gene frameworks are compared to their respective mouse VH and VL sequences. To identify the most important (major) and less important (secondary) reverse mutations, a proprietary matching evidence rule is used to determine the importance of specific framework locations likely to maintain CDR conformation (and antigen-binding affinity). The crystalline molecular structure of mouse anti-CD3 is analyzed to investigate the degree of spatial clustering of the identified reverse mutations. Initial humanized VH and VL sequences are generated by creating constructs that linearly transplant mouse CDRs onto selected human germline templates. To reduce the number of potential reverse mutation variants containing humanized strands, apparent spatial clustering of reverse mutation sites is used by simultaneously introducing spatially clustering mutations. We will use AbYsis' online tool to evaluate the immunogenicity of the final humanized sequence. (3) Cloning humanized antibodies The heavy and light chain genes of humanized antibodies are cloned into an expression vector. These vectors are used to produce humanized antibodies in a suitable expression system (such as mammalian cells). The amino acid sequences of the humanized VH and VL chains are combined in a VL-VH orientation. The scFv sequence contains a (G4S)4 linker between the VL and VH chains and an exa-His tag at the C-terminus. The scFv protein sequence is back-translated to optimize the codons. The codon-optimized whole DNA sequence is modified and synthesized to include 5′ and 3′ adapters suitable for HiFi cloning in pET22b(+). The DNA sequence is synthesized as double-stranded fragments (gBlocks) using TwistBioscience. The pET22b(+) backbone is linearized by PCR, the product is treated with DnpI, and then cleaned with Monarch DNA & PCR Cleanup. The gBlocks are inserted into pET22b(+) using the NEBuilder® HiFi DNA Assembly Cloning Kit. DH5α-competent E. coli are transformed with the ligation mixture, and positive transformed cells are selected on LB agar plates supplemented with 100 μg / ml carbenicillin. Colonies of the putative clone are cultured to extract plasmid DNA, and the DNA is subjected to Sanger sequencing to identify the correct clone. Transient transfection of HEK2936E suspension cultures with a construct encoding scFv Using the 293 Fectin transformation reagent, DNA (250 μg) from each plasmid construct was individually transduced into 250 ml of HEK 293 6E cell culture (live cell density of 1.85 x 10⁶ cells / ml). The culture was shaken at 124 rpm in a 5% CO₂ incubator at 37°C. After 48 hours and 72 hours of incubation, 6.2 ml of tryptone (200 g / l) and 6.2 ml of 3 M fructose were added to the cultures, respectively. Starting 48 hours after transformation, the viability (%) and viable cell density (cells / ml) of each culture are measured every 24 hours using a Vi-Cell cell counter and a viability analyzer (Beckman Coulter). Once the culture reaches <70% viability, it is collected by centrifugation at 4415xg for 30 minutes at 4°C and filtered using a 0.22 pm Millipore filter. The supernatant is stored at 4°C until needed for protein purification. (4) Purify the humanized antibody. The above humanized antibody is purified using a single-step affinity protein purification method. From the obtained supernatant, the scFv protein is purified using the AKTA Express system (AKTA). The supernatant is loaded at 5 ml / min onto a 5 ml HisTrap Excel column that has been pre-equalized with buffer A (50 mM HEPES pH 7.5, 400 mM NaCl, 20 mM imidazole). The column is washed by flowing 2 column volumes of buffer A at 5 ml / min from loading until it returns to the baseline. The protein is eluted by step elution with 50% buffer B (50 mM HEPES pH 7.5, 00 mM NaCl, 1 M imidazole). 3 column volumes of 50% buffer B are retained on the column. During this step elution, a 0.5 ml fraction is taken for the purification of 88A, and then a 1 ml fraction is taken for the subsequent total purification. After continuing the elution process until returning to the baseline, a washing process is performed with 100% buffer B in a volume equivalent to 3 columns. A single peak at 280 nM is expected on the resulting chromatogram, indicating the elution of the target protein. The fraction corresponding to this peak is pooled and transferred to a 5000 MW cutoff centrifuge tube. To separate the purified protein from the imidazole present in buffer B, the sample is buffer-changed from buffer B to PBS (60 ml). The sample is concentrated until ≤1 ml. The concentration is measured using a nanodropper, and the purified protein is diluted with PBS to a final concentration of 1 mg / ml. The final protein product is aliquoted and stored at -80°C for subsequent use. A 5 pg sample of the purified sample from that batch is subjected to electrophoresis by SDS-PAGE under reducing conditions; this reveals the purity and precise molecular weight of the purified protein. (5) Characterizing humanized antibodies Confirm the binding specificity and affinity of humanized antibodies against target antigens. Test the potential immunogenicity and other properties of the antibodies (such as stability and solubility). • Analytical size exclusion chromatography (aSEC) for assessing the homogeneity of purified proteins. • Mass spectrometry or peptide mass fingerprinting to confirm protein identity • Viacore analysis to confirm the coupling

[0369] Example 10: Humanization of mouse antibody clone D3 The objective of this example was to prepare humanized clones of the mouse antibody scFv clone D3 (i.e., SEQ ID NO: 9 (VL domain) and SEQ ID NO: 1 (VH domain) linked via the peptide linker (GGGS) 4).

[0370] Software used: igblastp 1.14.0, MAFFT v7.490, Sequence Manipulation Suite (DOI: 10.2144 / 00286ir01), and BioLuminate (Schrodinger).

[0371] [10.1] Approach (1) A homology model of mouse parent D3 Fv was constructed using Protein DataBank ID 2GKI; this yielded the best template for combination VH / VL. Subsequently, by modeling the CDR loop using an insight-based approach, three different models were obtained, each with a different CDR template. Furthermore, we modeled the D3 Fv using Alphafold v2 (installed locally) from the very beginning. (2) Each model was evaluated based on rigorous visual inspection and established protein quality measures (such as Ramachandran plots). In particular, the model derived from 2GKI homology showed excellent parameters. This model was then used to confirm the importance of individual framework residues. (3) In searching the human germline sequence database using the BLASTP function incorporated into Bioluminate, the mouse parent D3 sequence was used. Furthermore, mature human antibody sequences were also searched. As a result, two germline sequences were obtained for VH: IGHV1-3 and IGHV1-69; and one germline sequence for VL: IGKV4-1. The obtained mature sequences are as follows: • Crystal structure of PfCSP peptide 21 associated with the vaccine-induced human antimalarial antibody m42.127, which has a 95.9% matching human germline template (PDB ID 7LKB). • Crystal structure of the antibody Beta-32 Fab, which has a 94.9% matching human germline template PDB ID 7PS3. Furthermore, PDB ID 4LLU (pertuzumab) was also considered, but there were more significant mutations and potential significant mutations that ultimately led to the abandonment of this template. (4) Using these templates, we performed direct transplantation of CDRs based on the IMGT definition, following bioluminate guidelines. For subsequent analysis, we constructed homology models using the obtained sequences. (5) The template sequences containing the transplanted CDRs were aligned, and residue-wise analysis was performed for potential structural issues. (6) Next, to ensure that the CDR conformation was similar to that of the mouse parent Fv, further (reverse) mutations were introduced. The humanization of the framework based on the IMGT definition was >80% (preferably >85%), and potential sequence loadings (e.g., post-translational modifications) were removed as much as possible. (7) For both VH and VL, further frameworks were designed based on the consensus in the template, which included the (reverse) mutations described above. (8) The sequence name was changed to D3VHx or D3VLx. (9) To allow testing of different combinations (i.e., different combinations of D3VHx + D3VLx), the humanized VH and VL sequences are listed individually. Furthermore, the scFv sequence D3LxHx containing the Gly-Ser linker is provided. Note that the signal peptide is not provided.

[0372] [10.2] Results - scFv array The following D3VHx+D3VLx combinations turned out to be particularly interesting: D3VL1+D3VH1, D3VL1+D3VH2, D3VL2+D3VH3, D3VL3+D3VH4, and D3VL4+D3VH5.

[0373] Therefore, the following humanized scFv sequences were found to be particularly suitable: (A concatenation using Gly-Ser has been proposed; (GGGGS)4 is shown, but (GGGGS)5 may also be used.) >D3L1H1scFv (Sequence ID: 168) DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGAGTKLEMKGGGGSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYNQKFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS >D3L1H2scFv (Sequence ID: 169) DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQAPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGAGTKLEMKGGGGSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGYIFADHAIHWVRRAPGQGLEWIGYISPGNDDIKYNEKFKGRATLTADKSTSTAYMELSSLRSEDTAVYFCKRSLPGTFDYWGQGTTLTVSS >D3L2H3scFv (Sequence ID: 170) DIVMTQSPDSLAVSLGEKATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGGGTKVEIKGGGGSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQKFQDKVTLTADKSASTAYMELSSLRSEDTAVYFCKRSLPGTFDYWGQGTTVTVSS >D3L3H4scFv (Sequence ID: 171) DIQMTQSPSSVSASVGDRLTITCRSSQSLLYSSNQKNYLAWYQQKPGKAPKLLIYWASSLQSGVPSRFSGSGSGTDFTLTISSLKPEDFATYYCQQYYSYPLTFGQGTKVEIKGGGGSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQEFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS >D3L4H5scFv (Sequence ID: 172) DIQMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSYPLTFGQGTKVEIKGGGGSGGGGSGG GGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYIFADHAIHWVRQAPGQRLEWIGYISPGNDDIKYSQEFQGRVTLTADKSASTAYMELSSLRSEDSAVYFCKRSLPGTFDYWGQGTTLTVSS

[0374] [10.3] Results - Elisa titration Elisa titration of humanized scFv was performed on coated Tn-MUC1 and other Tn-proteins. The results are shown in Figure 21. All humanized scFvs bound to Tn-MUC1 and, at high concentrations, also bound to the control protein, but did not bind at dilutions. D3L1H2scFv showed similar binding characteristics to its parent, D3TnMUC1.

[0375] [10.4] Results - Kd values ​​for D3L1H2 Kd measurements were obtained using a streptavidin-coated chip sensor (SAX) from Startorius, under the manufacturer's conditions and reagent kit. See Figure 22. The results are shown in Table 12.

[0376] [Table 13]

[0377] Example 11: Conjugation of mouse antibody scFv-clone D3 to different Tn-peptide targets Elisa titration of different target Tn-peptides detected using mouse D3 scFv on streptavidin-coated plates. The results are shown in Figure 23. Biotinylated Tn-MUC1 peptide and Tn-MUC13 peptide. TnMUC1 (column 12, Figure 23) and a specific monosaccharide peptide of TnMUC13 (column 6, Figure 23, having a sequence similar to MUC1) were positive; other peptides were negative.

[0378] Preferred Embodiments of the Invention [Preferred Embodiment 1] An antibody library for in vitro identification of specific antibodies that bind to tumor cells; Here, each antibody in the library is (i) A first antibody domain that binds to the Tn- and / or STn-glycan epitopes of the glycoproteins of the tumor cells; and (ii) A second antibody domain selected from a repertoire of second antibody domains, wherein the repertoire of second antibody domains includes one or more second antibody domains that bind to the peptide epitope of the glycoprotein of the tumor cell. Second antibody domain, Includes; Here, the specific antibody is specific to the combination of the glycoprotein's epitope and peptide epitope. Antibody library. [Preferred Embodiment 2] An antibody library according to preferred embodiment 1, wherein the glycan epitope is covalently linked to the peptide epitope. Antibody library. [Preferred Embodiment 3] An antibody library according to preferred embodiment 1 or 2, wherein the first antibody domain is a VH-domain and the second antibody domain is a VL-domain. Antibody library. [Preferred Embodiment 4] An antibody library according to any one of the preferred embodiments 1 to 3, wherein the first antibody domain is a VH-domain, the second antibody domain is a VL-domain, and the antibody in the library is scFv, wherein the VH-domain is linked to the VL-domain via a peptide linker. Antibody library. [Preferred Embodiment 5] An antibody library according to any one of the preferred embodiments 1 to 4, wherein the glycan epitope is selected from mono-Tn, bis-Tn, mono-STn, bis-STn, and a combination of mono-Tn and mono-STn. Antibody library. [Preferred Embodiment 6] An antibody library according to any one of claims 3 to 5, wherein the VH domain is a mono- or bis-Tn-binding VH domain, and wherein the VH-domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 1, and comprises amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1. Antibody library. [Preferred Embodiment 6] An antibody library according to any one of the preferred embodiments 1 to 5, wherein the first antibody domain is a mono- or bis-Tn-conjugated VH domain, and wherein the VH domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 1, and comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1. Antibody library. [Preferred Embodiment 7] An antibody library according to any one of claims 3 to 5, wherein the VH domain is a mono- or bis-STn-conjugated VH domain, wherein the VH-domain comprises an amino acid sequence having at least 90% sequence homology with SEQ ID NO: 28, and comprises amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to SEQ ID NO: 28. [Preferred Embodiment 7] An antibody library according to any one of the preferred embodiments 1 to 5, wherein the first antibody domain is a mono- or bis-STn-conjugated VH domain, and wherein the VH domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28, and with respect to SEQ ID NO: 28, the amino acid residues: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, Antibody library. [Preferred Embodiment 8] An antibody library according to any one of preferred embodiments 1 to 7, wherein the first antibody domain neither contributes to nor inhibits binding to a peptide epitope. Antibody library. [Preferred Embodiment 9] An antibody library according to any one of preferred embodiments 1 to 8, wherein the second antibody domain repertoire is created from a naive immune repertoire of VL domains, an immune repertoire of VL domains by immunization, or a VL domain repertoire created by synthesis; preferably, from a naive immune repertoire of VL domains from an animal (such as a mouse or human). Antibody library. [Preferred Embodiment 10] An antibody library according to any one of the preferred embodiments 1 to 9, wherein the antibody library is a phage display library. Antibody library. [Preferred Embodiment 11] A method for identifying antibodies that target tumor cells, wherein the method is: (i) A step of preparing an antibody library according to any one of the preferred embodiments 1 to 10; and (ii) A step of screening the library to identify one or more tumor-targeting antibodies, including, method. [Preferred Embodiment 12] A method for identifying antibodies that target glycoproteins, for example, glycoproteins on tumor cells, wherein the method is: (i) Steps to prepare an antibody library according to any one of the preferred embodiments 1 to 10; and (ii) A step of screening the library to identify one or more tumor-targeting antibodies, including, method. [Preferred Embodiment 13] A method according to a preferred embodiment 11 or 12, comprising biopanning an antibody library with a glycopeptide or glycoprotein of the tumor cells, preferably an O-glycosylated peptide or O-glycosylated protein (such as Tn-mucin or other O-glycosylated proteins having a mucin-like motif); Here, the glycopeptide or glycoprotein is used in a purified form, or the glycopeptide or glycoprotein is expressed on the cell surface or tissue. method. [Preferred Embodiment 14] A method according to any one of the preferred embodiments 11 to 13, wherein the antibody library is comprised of the following steps: (1) The process of isolating mRNA from the spleen; (2) A step of synthesizing cDNA from the mRNA; (3a) A step of amplifying the cDNA using a specific primer set in order to obtain a first nucleic acid sequence that codes for the VH domain; (3b) A step of amplifying the cDNA using a primer mixture for obtaining multiple nucleic acid sequences that code for the repertoire of the VL domain; (4) The step of assembling a first nucleic acid sequence that codes for the VH-domain and a second nucleic acid sequence derived from a plurality of nucleic acid sequences that code for the VL-domain repertoire, in order to create a concatenated construct of them; (5) Inserting the structure into a phazimid vector; (6) The step of introducing a phazimide vector containing the construct into Escherichia coli in order to create a bacterial library; (7) A step of infecting a bacterial library with phages in order to create a phage display library, A phage display library prepared by a method including, method. [Preferred Embodiment 15] A method for identifying glycopeptide targets, wherein the targets include Tn and / or STn epitopes and peptide epitopes (such as glycopeptide targets in cancer cells); The method involves the following steps: (i) A step of preparing an antibody library according to any one of the preferred embodiments 1 to 10; (ii) Incubating the antibody library together with a sample containing the glycopeptide target; and (iii) A step of analyzing one or more antibody / peptide complexes obtained in step (ii) in order to identify the amino acid sequence of the peptide epitope of the glycopeptide target. including, method. [Preferred Embodiment 16] It is a specific tumor cell-binding antibody; (i) A VH-domain that binds to the Tn- and / or STn-glycan epitopes of the glycoproteins of the tumor cells; and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Includes; Here, if the antibody is not 5E5, 5F7, or 2D9, then the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein. Specific tumor cell-binding antibody. [Preferred Embodiment 17] An antibody according to preferred embodiment 16; Here, the VH domain is: (i) Sequence ID: comprises a first amino acid sequence having at least 90% sequence homology to 1; Here, the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to SEQ ID NO: 1; or The VH domain is: (ii) Containing a second amino acid sequence having at least 90% sequence homology to Sequence ID No. 28; Here, the second amino acid sequence includes amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to Sequence ID No. 28. antibody. [Preferred Embodiment 18] An antibody according to preferred embodiment 16; Here, the VH domain is: (i) Sequence ID: comprises a first amino acid sequence having at least 90% sequence homology to 1; Here, the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1; or The VH domain is: (ii) Containing a second amino acid sequence having at least 90% sequence homology to Sequence ID No. 28; Here, the second amino acid sequence is the amino acid residue with respect to SEQ ID NO: 28: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, antibody. [Preferred Embodiment 19] An antibody according to any one of the preferred embodiments 16 to 18, wherein the VL domain comprises an amino acid sequence selected from SEQ ID NOs: 9 to 24. antibody. [Preferred Embodiment 20] An antibody according to any one of the preferred embodiments 16 to 19; The antibody comprises first and second antigen-binding fragments; The first and second antigen-binding fragments each comprise a first VH and VL domain and a second VH and VL domain, respectively; Here, the antibody has a first VH domain that binds to a Tn-glycan epitope and a second VH domain that binds to an STn-glycan epitope. [Preferred Embodiment 21] An antibody according to any one of the preferred embodiments 20, wherein the first VH domain comprises a first amino acid sequence having at least 90% sequence homology to SEQ ID NO: 1, wherein the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1; and thereafter the second VH domain comprises a second amino acid sequence having at least 90% sequence homology with SEQ ID NO: 28, and thereafter the second amino acid sequence comprises amino acid residues with respect to SEQ ID NO: 28: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, antibody. [Preferred Embodiment 22] An antibody according to any one of the preferred embodiments 16 to 21, for use in the treatment and / or prevention of cancer. [Preferred Embodiment 23] A method for treating cancer in a subject, comprising administering a preparation containing at least one antibody according to any one of the preferred embodiments 16 to 21 to a patient in need thereof. [Preferred Embodiment 24] An antibody according to any one of the preferred embodiments 16 to 21, used for diagnosing cancerous conditions in a subject. [Preferred Embodiment 25] A method for diagnosing a cancerous condition in a subject, comprising administering to the subject a preparation containing at least one antibody according to any one of the preferred embodiments 16 to 21, and detecting the presence of an antigen-antibody complex containing at least one antibody according to any one of the preferred embodiments 16 to 21 and Tn- and / or STn- antigens. References Blixt et al 2010. A High-throughput O-glycopeptide Discovery Platform for Seromic Profiling. J Proteome Res. 2010 October 1; 9(10): 5250-5261. doi:10.1021 / pr1005229. Blixt et al 2012. Analysis of Tn antigenicity with a panel of new IgM and IgG1 monoclonal antibodies raised against leukemic cells. Glycobiology. 2012 Apr;22(4):529-42. doi: 10.1093 / glycob / cwr178. Epub 2011 Dec 5. Clavero-Alvarez et al 2018. Humanization of Antibodies using a Statistical Inference Approach. Sci Rep. 2018 Oct 4;8(1):14820. doi: 10.1038 / s41598-018-32986-y. Karst et al. 2021. High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing. Nat Methods 18, 165-169 (2021). Kjeldsen et al 1988. Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2----6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope. Cancer Res. 1988 Apr 15;48(8):2214-20. Kudelka et al 2015. Simple Sugars to Complex Disease-Mucin-Type O-Glycans in Cancer. Adv Cancer Res. 2015; 126: 53-135. Doi: 10.1016 / bs.acr.2014.11.002. Li et al 2009. Resolving conflicting data on expression of the Tn antigen and implications for clinical trials with cancer vaccines. Mol Cancer Ther. 2009 Apr;8(4):971-9. doi: 10.1158 / 1535-7163.MCT-08-0934. PMID: 19372570; PMCID: PMC2752371. Macias-Leon et al 2020. Structural characterization of an unprecedented lectin-like antitumoral anti-MUC1 antibody. Chem Commun (Camb). 2020 Dec 8;56(96):15137-15140. doi: 10.1039 / d0cc06349e. Mazal et al 2013. Monoclonal antibodies toward different Tn-amino acid backbones display distinct recognition patterns on human cancer cells. Implications for effective immuno-targeting of cancer. Cancer Immunol Immunother. 2013 Jun;62(6):1107-22. doi: 10.1007 / s00262-013-1425-7. Epub 2013 Apr 21. PMID: 23604173. Oppezzo Pet al 2000. Production and functional characterization of two mouse / human chimeric antibodies with specificity for the tumor-associated Tn-antigen. Hybridoma. 2000 Jun;19(3):229-39. doi: 10.1089 / 02724570050109620. PMID: 10952411. Persson et al. 2016. A combinatory antibody-antigen microarray assay for high-content screening of single-chain fragment variable clones from recombinant libraries. PLoS One 11, (2016). Person et al. 2017. Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells. Glycobiology, 2017, vol. 27, no. 7, 635-645. doi: 10.1093 / glycob / cwx033. Prendergast et al 2017. Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity. MAbs. 2017 May / Jun;9(4):615-627. doi: 10.1080 / 19420862.2017.1290752. Epub 2017 Feb 22. Sorensen et al 2006. Chemoenzymatically synthesized multimeric Tn / STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance. Glycobiology. 2006 Feb;16(2):96-107. doi: 10.1093 / glycob / cwj044. Epub 2005 Oct 5. Tarp et al 2007. Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat. Glycobiology vol. 17 no. 2 pp. 197-209, 2007. doi:10.1093 / glycob / cwl061. Yuasa et al 2012. Construction and expression of anti-Tn-antigen-specific single-chain antibody genes from hybridoma producing MLS128 monoclonal antibody. J Biochem. 2012 Apr;151(4):371-81. doi: 10.1093 / jb / mvs007. Epub 2012 Feb 8. PMID: 22318767.

Claims

1. An antibody library for in vitro identification of specific antibodies that bind to tumor cells; Here, each antibody in the library is: (i) VH domains that bind to the Tn- and / or STn-glycan epitopes of the glycoproteins of the tumor cells; and (ii) A VL domain selected from the VL domain repertoire, Including; Here, the repertoire of VL-domains includes one or more VL-domains that bind to the peptide epitopes of the glycoproteins of the tumor cells; Here, the specific antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein. Antibody library.

2. An antibody library according to claim 1; Herein, the glycan epitope is covalently linked to the peptide epitope in this antibody library.

3. An antibody library according to claim 1 or 2, wherein the antibody in the library is scFv; wherein the VH-domain is linked to the VL-domain via a peptide linker.

4. An antibody library according to any one of claims 1 to 3; wherein the glycan epitope is selected from mono-Tn, bis-Tn, mono-STn, bis-STn, and a combination of mono-Tn and mono-STn.

5. An antibody library according to any one of claims 1 to 4; Here, the VH domain is a mono- or bis-TN-associated VH domain; And the VH-domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 1, and comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to SEQ ID NO: 1, Antibody library.

6. An antibody library according to any one of claims 1 to 4; Here, the VH domain is a mono- or bis-TN-associated VH domain; And herein, the VH-domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 1; Furthermore, with respect to Sequence ID No. 1, the amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 are included. Antibody library.

7. An antibody library according to any one of claims 1 to 4; Here, the VH domain is a mono- or bis-STn-associated VH domain; And herein, the VH-domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28; Furthermore, regarding sequence number 28, the amino acid residues are: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, Antibody library.

8. An antibody library according to any one of claims 1 to 4; Here, the VH domain is a mono- or bis-STn-associated VH domain; And herein, the VH-domain comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28; Furthermore, with respect to Sequence ID No. 28, the amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 are included. Antibody library.

9. An antibody library according to any one of claims 1 to 8; Here, the VH domain does not contribute to or inhibit the binding of any peptide epitope. Antibody library.

10. An antibody library according to any one of claims 1 to 9; Here, the repertoire of VL domains is a naive immune repertoire of VL domains, an immune repertoire of VL domains mediated by immunity, or a repertoire of VL domains created by synthesis; preferably, it is created from a naive immune repertoire of VL domains derived from animals (such as mice or humans). Antibody library.

11. An antibody library according to any one of claims 1 to 10; Here, the antibody library is a phage display library. Antibody library.

12. A nucleic acid library for coding an antibody library according to any one of claims 1 to 11.

13. A method for identifying antibodies that target glycoproteins: (i) A step of preparing an antibody library according to any one of claims 1 to 12; and (ii) A step of screening the library to identify one or more tumor-targeting antibodies, Methods that include...

14. A method according to claim 13; The process includes a step of biopanning an antibody library using glycopeptides or glycoproteins from the tumor cells, preferably O-glycosylated peptides or O-glycosylated proteins (such as Tn-mucin or other O-glycosylated proteins having a mucin-like motif); Here, the glycopeptide or glycoprotein is used in a purified form, or the glycopeptide or glycoprotein is expressed on the cell surface or tissue. method.

15. A method according to claim 13 or 14; Here, the antibody library is a phage display library; The phage display library is: (1) Steps to isolate mRNA from the spleen (2) A step of synthesizing cDNA from the mRNA; (3a) A step of amplifying the cDNA using a specific primer set for obtaining a first nucleic acid sequence that codes for the VH domain; (3b) A step of amplifying the cDNA using a primer mixture for obtaining a plurality of nucleic acid sequences that code for the repertoire of the VL domain; (4) Assembling the first nucleic acid sequence and the second nucleic acid sequence in order to create a concatenated construct of the first nucleic acid sequence coding the VH-domain and the second nucleic acid sequence derived from a plurality of nucleic acid sequences coding the repertoire of the VL-domain; (5) Inserting the structure into a phagemid vector; (6) The step of introducing a phagemide vector containing the construct into Escherichia coli in order to create a bacterial library; (7) A step of infecting a bacterial library with phages in order to create a phage display library, Prepared by a method including, method.

16. A method for identifying glycopeptide targets, wherein the targets include Tn and / or STn epitopes and peptide epitopes (such as glycopeptide targets in cancer cells); The method is: (i) A step of preparing an antibody library according to any one of claims 1 to 11; (ii) Incubating the antibody library together with a sample containing the glycopeptide target; and (iii) A step of analyzing one or more antibody / peptide complexes obtained in step (ii) in order to identify the amino acid sequence of the peptide epitope of the glycopeptide target. including, method.

17. A specific tumor cell-binding antibody; The antibody: (i) VH domains that bind to Tn- and / or STn-glycan epitopes of tumor cell glycoproteins; and (ii) A VL domain that binds to the peptide epitope of the glycoprotein of the tumor cell, Including; Here, if the antibody is not 5E5, 5F7, or 2D9, then the antibody is specific to the combination of the glycosylation epitope and peptide epitope of the glycoprotein. Specific tumor cell-binding antibody.

18. An antibody according to claim 17; Here the VH domain in question: (i) Sequence ID: comprises a first amino acid sequence having at least 90% sequence homology to 1; Here, the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to Sequence ID No. 1; or The VH domain in question is: (ii) comprising a second amino acid sequence having at least 90% sequence homology to Sequence ID No. 28; And here the second amino acid sequence is, with respect to SEQ ID NO: 28, an amino acid residue: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, antibody.

19. An antibody according to claim 17; Here the VH domain in question: (i) Sequence ID: comprises a first amino acid sequence having at least 90% sequence homology to 1; Here, the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, S52, N55, D57, and S99 with respect to Sequence ID No. 1; or The VH domain in question is: (ii) comprising a second amino acid sequence having at least 90% sequence homology to Sequence ID No. 28; Here, the second amino acid sequence includes amino acid residues T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103 with respect to SEQ ID NO: 28, antibody.

20. An antibody according to claims 17 to 19; Herein, the VL domain contains an amino acid sequence selected from SEQ ID NOs: 9 to 24, which is an antibody.

21. An antibody according to any one of claims 17 to 20; The antibody comprises first and second antigen-binding fragments; The first and second antigen-binding fragments each comprise a first VH and VL domain and a second VH and VL domain; Here, the first VH domain binds to the Tn-glycan epitope, and the second VH domain binds to the STn-glycan epitope. antibody.

22. An antibody according to claim 21; Here, the first VH domain comprises a first amino acid sequence having at least 90% sequence homology to SEQ ID NO: 1; Here, the first amino acid sequence comprises amino acid residues H32, A33, H35, Y50, and S99 and / or amino acid residues S52, N55, and D57 with respect to Sequence ID No. 1; And herein, the second VH domain comprises a second amino acid sequence having at least 90% sequence homology to SEQ ID NO: 28; And here the second amino acid sequence is, with respect to SEQ ID NO: 28, an amino acid residue: (i) T28, T30, H32, A33, H35, Y50, S99, L101, A102 and L103; (ii) T28, T30, S52, N55, D57, L101, A102 and L103; or (iii) T28, T30, H32, A33, H35, Y50, S52, N55, D57, S99, L101, A102 and L103, including, antibody.

23. An antibody according to any one of claims 17 to 22; Antibodies used for the treatment and / or prevention of cancer.

24. A method for treating cancer in a subject; A preparation comprising administering to a patient in need thereof a preparation containing at least one antibody as described in any one of claims 17 to 22, method.

25. An antibody according to any one of claims 17 to 22; Antibodies used for diagnosing cancerous conditions in the target population.

26. A method for diagnosing cancerous conditions in a subject; Administering a preparation containing at least one antibody according to any one of claims 17 to 22 to the subject, and detecting the presence of an antigen-antibody complex containing at least one antibody according to any one of claims 17 to 22 and Tn- and / or STn- antigen, including, method.

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