Combined members

Abstract

The present invention relates to a method for specifically binding Lewis Y (Le y ) Carbohydrate-specific binding members, such as antibodies and fragments thereof. Also disclosed are uses of such binding members in medicine and nucleic acids encoding such binding members.

Description

[0001] The present invention relates to a method for specifically binding Lewis Y (Le y ) Carbohydrate-specific binding members, such as antibodies and fragments thereof. Also disclosed are uses of such binding members in medicine and nucleic acids encoding such binding members.

[0002] Glycan structures are present on protein and glycolipid backbones, and can be overexpressed in large quantities in cancer due to changes in the expression of glycosyltransferases. Glycolipids are composed of lipid tails with carbohydrate heads, accounting for about 5% of lipid molecules in the outer monolayer. Glycolipids mainly have three series, globo lipids, neolacto lipids and ganglion lipids. Due to the high surface density, fluidity and association with membrane microdomains of glycolipids (all of which contribute to strong cell interactions), glycolipids are considered to be very good targets. However, generating anti-glycolipid antibodies is a challenging task because there is no help from T cells, and monoclonal antibodies (mAbs) usually have low affinity and IgM subclasses (Durrant, Nobleet al.2012, Rabu, McIntosh et al.2012). Although mAbs for glycans expressed on proteins overcome this problem, they present new challenges because mAbs rarely see only small glycans, but usually recognize glycans on specific proteins, thereby making expression very limited.

[0003] Only a limited number of antibodies that recognize glycans have been described. To date, several anti-Lewis (Le) carbohydrate antigen mAbs have been generated, but they are generally cross-reactive with a range of glycans expressed on normal tissues. Lewis carbohydrate antigens are formed by the sequential addition of fucose to oligosaccharide precursor chains on glycoproteins or glycolipids by the action of a group of glycosyltransferases (Yuriev, Farrugia et al. 2005). The antigens can be divided into two groups, type I (Le a and Le b ) and type II (Le x and Le y ). a and Le b The antigens are believed to be blood group antigens, and Le x and Le yIt is expressed only in certain epithelial cells (Scott, Geleick et al. 2000), is considered a tumor-associated marker (Soejima and Koda 2005), and represents an attractive target for cancer therapy (including antibody-based immunotherapy) (Scott, Geleick et al. 2000). Most normal tissues do not express Lewis Y, except for gastrointestinal epithelial cells, but at very low density (Schuster, Umana et al. 2005). However, it has been reported that Lewis Y (Fuca1-2Galb1-4(Fuca1-3)GlcNAc) is expressed at high density in nearly 70% of human epithelial cancers and is therefore an attractive candidate target for cancer immunotherapy. The recognition that Lewis Y is a good therapeutic target has led to a large number of anti-Le y Antibody production. Anti-Le y Some mAbs cross-react with other Lewis antigens, such as b / y mAb 692 / 29 has demonstrated antitumor responses against colon tumors in a xenograft model (Noble, Spendlove et al. 2013).

[0004] Several other combinations y mAbs are known in the art, but they cross-react with other related Lewis antigens. For example, EP-B-0285059 discloses an antibody BR-55, which cross-reacts with Lewis antigens. y and B-7-2. B-7-2 has also been shown to be associated with tumor cells (EP-B-0285059). US5869045 discloses an antibody BR-96, which reacts with Lewis y and Lewis x Both haptens bind. y and Lewis b Antibodies that bind to both Lewis antigens are known. Studies have shown that the C14 monoclonal antibody recognizes and binds to Lewis y and Lewis b (Extended and non-extended forms) antigens (Durrant at al., Hybridoma, 12, 647-660 (1996)). WO02 / 092126 discloses the same y and Lewis b Hapten-binding mAb.

[0005] Many studies have been conducted using mouse or chimeric anti-Le yClinical trials of antibodies (Scott, Geleick et al. 2000). Unfortunately, anti-Le y mAbs often cross-react with Lewis X and H II structures that are more commonly expressed in normal tissues (Furukawa, Welt et al. 1990, Yin, Finstad et al. 1996). y Antibodies have also been identified, but a consistent problem with these antibodies is that they are x and the degree of cross-reactivity of H2-type structures leading to hemagglutination of erythrocytes and gastrointestinal toxicity (Kitamura, Stockert et al. 1994, Pai, Wittes et al. 1996, Tolcher, Sugarman et al. 1999). y Good specificity of antibodies is important for successful clinical treatment.

[0006] In a first aspect, the present invention provides a method for binding Fuca1-2Galb1-4(Fuca1-3)GlcNAc(Le y ) is an isolated specific binding member that specifically binds.

[0007] The present inventors provide specific binding members that exhibit strong in vivo anti-tumor activity. The specific binding members of the present invention show strong immune-mediated cytotoxic activity against human colon cancer cells in vitro through antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). They also have the ability to directly induce cell death without the need for immune effector cells. By immunizing with tumor glycolipids instead of glycoproteins or whole cells, the inventors produced super-specific anti-Le that do not cross-react with any other polysaccharides. y mAbs. Examples of these mAbs are the IgG3k murine mAb referred to herein as "FG27.10" and the IgG1k murine mAb referred to herein as "FG27.18", as well as a hIgG1 chimeric antibody (referred to as "CH27") and a hIgG1 humanized antibody (referred to as "Hu27").

[0008] Compared with the previous anti-Le that cross-reacts with other glycans yIn contrast to the mAb studies, the specific binding members of the present invention have a very strict normal distribution and bind only to subsets of cells in the normal stomach, lung, tonsil, pancreas, and duodenum. In addition, they do not stain the colon, jejunum, breast, kidney, or ileum. They bind tightly to a variety of tumors, including 80% of gastric tumors, 100% of colorectal tumors, 95% of ovarian tumors, and 85% of breast tumors. The specific binding members of the present invention, such as FG27.10 / 18, bind only to Le y The inventors have shown that this specific binding member does not bind to the cell line Colo21. y / x mAb strongly binds, Le y / b mAb bound moderately. This suggests that Colo201 expresses Le x and Le b , but does not express Le y , and confirmed the specificity of the specific binding members of the invention. This specificity is reflected in the similar but different sequences of the FG27 heavy and light chain variable regions ( Fig.13a , Fig.13b ). With other cross-reactive Le y Compared to the mAbs, FG27 showed 16%-68% differences in the amino acid sequence CDR within the heavy chain, 4.3%-16% differences in the amino acid sequence of the CDR in the light chain, 8.6%-49.5% differences in the amino acid sequence of the heavy chain variable region, and 2%-24% differences in the amino acid sequence of the light chain variable region (see Table 4 herein). The greatest variation was within CDRH2, where 3 to 5 of the 8 amino acids varied in different mAbs.

[0009] The specific binding members of the invention may be linked to a lipid or protein backbone. The protein backbone may have a molecular weight of about 50 kDa-150 kDa as determined by SDS-PAGE.

[0010] The specific binding members of the present invention preferably comprise a member selected from the group consisting of Figure 1a or Figure 1b One or more binding domains of a binding domain having an amino acid sequence as shown in residues 27 to 38 (CDRH1), 54 to 65 (CDRH2) or 105 to 116 (CDRH3) of Figure 1a or Figure 1b Such specific binding members may additionally comprise a binding domain having an amino acid sequence as shown in residues 105 to 116 of the amino acid sequence of (CDRH3). Figure 1a or Figure 1bOne or two, preferably both, of the binding domains of residues 27 to 38 (CDRH1) and 56 to 65 (CDRH2) of the amino acid sequence shown in .

[0011] The binding member may comprise essentially Figure 1a or Figure 1b The amino acid sequence shown in 1 to 127 (VH).

[0012] The specific binding member may comprise a member selected from Figure 1c The binding member may comprise a binding domain having an amino acid sequence of residues 27 to 38 (CDRL1), 56 to 65 (CDRL2) or 105 to 113 (CDRL3) of Figure 1c Such specific binding members may additionally comprise a binding domain having an amino acid sequence as shown in residues 105 to 113 of the amino acid sequence of Figure 1c One or two, preferably both, of the binding domains of residues 27 to 38 (CDRL1) and 56 to 65 (CDRL2) of the amino acid sequence shown in .

[0013] The present invention includes specific binding members or combinations thereof comprising multiple binding domains having the same or different sequences. The binding domain or each binding domain can be carried by a human antibody framework. For example, one or more binding domains can replace the complementary determining region (CDR) of the entire human antibody or its variable region.

[0014] An isolated specific binding member of the invention comprises essentially Figure 1c The amino acid sequence shown by residues 1 to 124 (VL) of the amino acid sequence.

[0015] An isolated specific binding member of the invention may comprise a Figure 1a or Figure 1b One or more (preferably all) binding domains having an amino acid sequence as shown in residues 27 to 38 (CDRH1), 54 to 65 (CDRH2) or 105 to 116 (CDRH3) of Figure 1c One or more (preferably all) binding domains of the amino acid sequence shown as residues 27 to 38 (CDRL1), 56 to 65 (CDRL2) or 105 to 113 (CDRL3) of the present invention.

[0016] The binding member may comprise essentially Figure 1a or Figure 1bThe amino acid sequence of residues 1 to 127 (VH) of the amino acid sequence of Figure 1c The amino acid sequence shown by residues 1 to 124 (VL) of the amino acid sequence.

[0017] Once a single prototype mAb with the desired properties described herein is isolated, such as FG27 mAb, it is simple to generate other mAbs with similar properties using methods known in the prior art. For example, the method of Jespers et al. in 1994 (Jespers, Roberts et al. 1994) can be used to guide the selection of mAbs with the same epitope and thus similar properties as the prototype mAb. Using phage display, the heavy chain of the prototype antibody is first paired with a library of (preferably human) light chains to select polysaccharide-binding mAbs, and then the new light chain is paired with a library of (preferably human) heavy chains to select (preferably human) polysaccharide-binding mAbs with the same epitope as the prototype mAb.

[0018] The specific binding member can be an antibody or an antibody fragment Fab, (Fab')2, scFv, Fv, dAb, Fd or a diabody. The antibody can be a polyclonal antibody. The antibody can be a monoclonal antibody (mAb). The antibody of the present invention can be a humanized antibody, a chimeric antibody or a veneered antibody, or can be a non-human antibody of any species.

[0019] Murine antibodies or chimeric antibodies carry an increased risk of adverse anti-mouse antibody (HAMA) reactions in patients (Schroff et al. 1985; Azinovic et al. 2006; Miotti et al. 1999; D'Arcy and Mannik 2001). Therefore, most approved therapeutic mAbs are humanized antibodies or fully human IgG antibodies.

[0020] The specific binding members of the invention may comprise a Figure 2a The heavy chain of the amino acid sequence shown and having substantially the same Figure 2b The amino acid sequence of the light chain is shown.

[0021] The specific binding members of the invention may comprise a Figure 3a The heavy chain of the amino acid sequence shown and having substantially the same Figure 3b The amino acid sequence of the light chain is shown.

[0022] The specific binding members of the invention may comprise a Fig.33 The heavy chain of the amino acid sequence shown and / or having substantially the same Fig.33 The amino acid sequence of the light chain is shown.

[0023] The present invention also provides a binding member comprising Figure 1a or Figure 1b A VH chain having an amino acid sequence of residues 1 to 127 of Figure 1c The VL chain of an antibody containing the amino acid sequence of residues 1 to 124 competes for binding to y of polysaccharides.

[0024] The present invention includes the ability to y Specific binding members that specifically bind to and are at least 90%, at least 95%, or at least 99% identical in the VH and / or VL domains to the VH and VL domains of Figures 1, 2, or 3. The present invention includes specific binding members that are capable of binding to Le y Specific binding and Fig.33 The specific binding members of the invention are heavy chains and / or light chains that are at least 90%, at least 95% or at least 99% identical. Preferably, such antibodies differ from the sequences of Figures 1, 2 or 3 by a small amount of functionally insignificant amino acid substitutions (e.g., conservative substitutions), deletions or insertions.

[0025] The specific binding members of the invention may carry a detectable or functional label.

[0026] In other aspects, the invention provides isolated nucleic acids encoding specific binding members of the invention and methods of making specific binding members of the invention, the methods comprising expressing the nucleic acid under conditions that result in expression of the binding member and recovering the binding member. y An isolated nucleic acid that specifically binds to a specific binding member that is at least 90%, at least 95%, or at least 99% identical to a sequence provided herein.

[0027] The specific binding members of the present invention can be used for treating or diagnosing a method of a human or animal body, such as a method for treating a tumor of a patient (preferably a human), the method comprising administering an effective amount of a specific binding member of the present invention to the patient. The present invention also provides a specific binding member of the present invention for use in medicine, preferably for treating a tumor, and the use of a specific binding member of the present invention in the preparation of a drug for diagnosing or treating a tumor. The tumor can be a gastric tumor, a colorectal tumor, a pancreatic tumor, a lung tumor, an ovarian tumor or a breast tumor.

[0028] Antigens that bind to specific binding members of the invention are disclosed herein. Preferably, a Le that can be bound (preferably specifically bound) by a specific binding member of the invention can be provided. y . yCan be provided in an isolated form and can be used for screening to develop additional specific binding members. For example, a compound library can be screened for specific binding to Le y Specifically bind to the library member. y It can be on a lipid or protein backbone. When on a protein backbone, Le y It may have a molecular weight of about 50 kDa-150 kDa.

[0029] In other aspects, the present invention provides an isolated specific binding member (preferably the first aspect of the present invention) which is capable of binding to a protein containing Le y Glycans of ovarian cancer are used for the diagnosis or prognosis of gastric, colorectal, pancreatic, lung, ovarian and breast tumors.

[0030] The present invention also provides a method for diagnosing cancer, which comprises using the specific binding member of the present invention to detect Le y In this diagnostic method, the pattern of glycans detected by the binding member can be used to stratify treatment options for individuals.

[0031] These and other aspects of the invention are described in further detail below.

[0032] As used herein, "specific binding members" are members of a pair of molecules that have binding specificity to each other. The members of the specific binding pair can be of natural origin or produced in whole or in part by synthesis. A member of the pair of molecules has an area on its surface, which can be a projection or a cavity, which specifically binds to the specific space and polarity organization of another member of the pair of molecules, and is therefore complementary to it. Therefore, the right members have the characteristic of specific binding to each other. Examples of the types of specific binding pairs include antigen-antibody, biotin-avidin, hormone-hormone receptor, 5 receptor-ligand, enzyme-substrate. The present invention generally relates to antigen-antibody type reactions, although it also relates to small molecules bound to antigens defined herein.

[0033] As used herein, "treatment" includes any regimen that can benefit a human or non-human animal, preferably a mammal. Treatment may be of an existing condition, or may be preventative (prophylactic treatment).

[0034] As used herein, a "tumor" is an abnormal growth of tissue. It can be localized (benign) or invade nearby tissue (malignant) or distant tissue (metastatic). Tumors include tumor growths that cause cancer, including esophageal, colorectal, gastric, breast, ovarian and endometrial tumors, as well as cancerous tissues or cell lines, including but not limited to leukemia cells. As used herein, "tumor" also includes endometriosis within its scope.

[0035] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen binding site that specifically binds to an antigen, whether the molecule is natural or partially or completely synthetically produced. The term also encompasses any polypeptide or protein having a binding domain that is an antibody binding domain or homologous to an antibody binding domain. These antibodies may be from natural sources, or they may be partially or completely synthetically produced. Examples of antibodies of the present invention are: immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and isotype subclasses thereof; fragments comprising an antigen binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. Monoclonal antibodies may be referred to as "mAbs" herein.

[0036] Monoclonal antibodies and other antibodies can be used and recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. This technology may involve introducing DNA encoding the immunoglobulin variable region or CDR of the antibody into the constant region or constant region plus framework region of a different immunoglobulin. For example, see EP-A-184187, GB2188638A or EP-A-239400. Hybridomas or other cells that produce antibodies may undergo genetic mutations or other changes, which may or may not change the binding specificity of the antibody produced.

[0037] Since antibodies can be modified in a variety of ways, the term "antibody" should be interpreted as covering any specific binding member or substance of a binding domain with desired specificity. Therefore, the term covers antibody fragments, derivatives, functional equivalents and homologues, humanized antibodies of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether the polypeptide is natural or synthesized in whole or in part. Therefore, a chimeric molecule comprising an immunoglobulin binding domain or an equivalent fused with another polypeptide is included. The cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023. Humanized antibodies can be modified antibodies with the variable region of non-human (e.g., mouse) antibodies and the constant region of human antibodies. For example, a method for preparing humanized antibodies is described in U.S. Patent No. 5,225,539.

[0038] It has been shown that fragments of whole antibodies can perform the function of binding to antigen. Examples of binding fragments are: (i) Fab fragments consisting of VL, VH, CL and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of VL and VH domains of a single antibody; (iv) dAb fragments consisting of VH domains (Ward, Gussow et al. 1989); (v) isolated CDR regions; (vi) F(ab')2 fragments, i.e., bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird, Hardman et al. 1988, Huston, Levinson et al. 1989). al. 1988); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies", i.e. multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; (Holliger, Prospero et al. 1993)).

[0039] Diabodies are multimers of polypeptides, each polypeptide comprising a first domain comprising a binding region for an immunoglobulin light chain and a second domain comprising a binding region for an immunoglobulin heavy chain, the two domains being connected (e.g., via a peptide linker) but unable to associate with each other to form an antigen binding site: the antigen binding site is formed by the association of the first domain of one polypeptide within the multimer with the second domain of another polypeptide within the multimer (WO94 / 13804).

[0040] Where bispecific antibodies are used, these may be conventional bispecific antibodies which may be made in a variety of ways (Holliger and Winter 1993) (e.g., by chemical preparation or by hybrid hybridomas), or may be any of the bispecific antibody fragments described above. It may be preferred to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs may be constructed without the Fc region using only the variable domains, potentially reducing the effect of anti-idiotypic responses. Other forms of bispecific antibodies include the single chain "Janusins" described in (Traunecker, Lanzavecchia et al. 1991).

[0041] Bispecific diabodies, which are different from bispecific full antibodies, may also be useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificities can be easily selected using phage display technology (WO 94 / 13804) from libraries. If one arm of the diabody is to be kept constant, for example, specific for antigen X, a library can be created in which the other arm is varied and antibodies with appropriate specificity are selected.

[0042] "Binding domain" is a portion of a specific binding member that contains a region of specific binding to part or all of an antigen and complementarity. In the case where the binding member is an antibody or an antigen-binding fragment thereof, the binding domain may be a CDR. When the antigen is large, the antibody may only bind to a specific portion of the antigen, which is referred to as an epitope. The antigen binding domain may be provided by one or more antibody variable domains. The antigen binding domain may include an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0043] "Specificity" is generally used to refer to situations where one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner, and, for example, has less than about 30%, preferably 20%, 10% or 1% cross-reactivity with any other molecule. The term also applies, for example, to situations where an antigen binding domain is specific for a particular epitope carried by many antigens, in which case a specific binding member carrying the antigen binding domain will be able to bind to a variety of antigens carrying that epitope. Specific binding members of the invention may be able to bind to Le y Specifically binds in the sense that there is no detectable binding to any other antigen (eg, any other glycan) when binding is tested according to the protocol outlined in "Glycomic Analysis" in the Examples herein.

[0044] According to the present invention, "isolated" refers to the state in which the specific binding members of the present invention or the nucleic acid encoding such binding members will preferably be. When this preparation is carried out by recombinant DNA technology implemented in vitro or in vivo, members and nucleic acids will generally contain or substantially contain substances naturally associated with them, such as other polypeptides or nucleic acids found in their natural environment or the environment (e.g., cell culture medium) in which they are prepared. Specific binding members and nucleic acids can be formulated with diluents or adjuvants, but are still separated for practical purposes-for example, if used to coat microtiter plates for immunoassays, members will generally be mixed with gelatin or other carriers, or when used for diagnosis or treatment, members are mixed with pharmaceutically acceptable carriers or diluents. Specific binding members can be naturally glycosylated or glycosylated by heterologous eukaryotic cell systems, or they can be (e.g., if produced by expression in prokaryotic cells) unglycosylated.

[0045] "Substantially as shown in..." means that the amino acid sequence of the present invention will be identical or highly homologous to the amino acid sequence referred to. "Highly homologous" contemplates that 1 to 5, 1 to 4, 1 to 3, 2 or 1 substitutions may be made in the sequence.

[0046] The present invention also includes within its scope polypeptides having amino acid sequences as shown in Figures 1, 2 or 3, polynucleotides having nucleic acid sequences as shown in Figures 1 or 2, and sequences having substantial identity therewith (e.g., having at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 99% identity therewith). The percent identity of two amino acid sequences or two nucleic acid sequences is generally determined by aligning the sequences for optimal comparison purposes (e.g., gaps may be introduced in the first sequence for optimal alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. An "optimal alignment" is an alignment between two sequences that achieves the highest percent identity. Percent identity is determined by comparing the number of identical amino acid residues or nucleotides in the sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0047] The determination of percent identity between two sequences can be accomplished using mathematical algorithms known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul in 1990 (Karlin and Altschul 1990), which was improved by Karlin and Altschul in 1993 (Karlin and Altschul 1993). The algorithm was incorporated into the NBLAST and XBLAST programs of Altschul et al. in 1990 (Altschul, Gish et al. 1990). BLAST nucleic acid searches can be performed with the NBLAST program (score = 100, word length = 12) to obtain nucleic acid sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain gapped alignments for comparison purposes, Gapped BLAST can be used, as described by Altschul et al. in 1997 (Altschul, Madden et al. 1997). Alternatively, PSI-Blast can be used to perform iterative searches to detect long-range relationships (Id.) between molecules. When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm that can be used for sequence alignment is the algorithm of Myers and Miller in 1989 (Myers and Miller 1989). The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates this algorithm. Other algorithms known in the art for sequence analysis include ADVANCE and ADAM as described by Torellis and Robotti (Torelli and Robotti 1994), and FASTA as described by Pearson and Lipman (Pearson and Lipman 1988). In FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0048] The isolated specific binding member of the present invention is capable of binding to Le y Carbohydrate binding, Le y Carbohydrates can be y The ceramide may be on the protein portion. Figure 1a and Figure 1b Residues 105 to 116 of (CDRH3) and Figure 1c The binding domain of the amino acid sequence shown in residues 105 to 113 (CDRL3) can be carried in a manner that allows these regions to bind to Le y Carbohydrates are bound in the structure.

[0049] The structures used to carry the binding domains of the present invention generally have antibody heavy or light chain sequences or substantial portions thereof, wherein the binding domains are located in positions corresponding to the CDR3 regions of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and positions of immunoglobulin variable domains can be determined by reference to http: / / www.imgt.org / . Figure 1a and Figure 1b The amino acid sequence shown in residues 105 to 116 of can be carried as CDR3 in a human heavy chain variable domain or a substantial portion thereof, and is essentially as Figure 1c The amino acid sequence set forth at residues 105 to 113 may be carried as CDR3 in a human light chain variable domain, or a substantial portion thereof.

[0050] The variable domain can be derived from any germline or rearranged human variable domain, or can be a synthetic variable domain based on the consensus sequence of a known human variable domain. Recombinant techniques can be used to introduce the CDR3 source sequence of the present invention into a library of variable domains lacking a CDR3 region. For example, Marks et al. (Marks, Griffiths et al. 1992) described a method for producing an antibody variable domain library in 1992, wherein a common primer at or near the 5' end of the variable domain region is used in combination with a common primer in the third framework region of a human VH gene to provide a VH variable domain library lacking CDR3. Mark et al. (Marks, Griffiths et al. 1992) also described how to combine the library with the CDR3 of a specific antibody in 1992. Using similar techniques, the sequence of the CDR3 source of the present invention can be shuffled with a library of a VH or VL domain lacking a CDR3, and a shuffled complete VH or VL domain is combined with a similar VL or VH domain to provide a specific binding member of the present invention. The library can then be displayed in a suitable host system (e.g., the phage display system of WO92 / 01047) so that suitable specific binding members can be selected. The library can include 10 4 More than 10 individual members (e.g. 6 Up to 10 8 or 10 10 members).

[0051] A similar promiscuous or combinatorial technique was also disclosed by Stemmer in 1994 (Stemmer 1994), who described the technique in relation to β-lactamase genes, but noted that the method could be used to generate antibodies. Another alternative is to generate new VH or VL regions carrying the CDR3-derived sequences of the invention using, for example, random mutagenesis of the FG27 VH or VL genes to generate mutations throughout the variable domain. This technique was described by Gram et al. (Gram, Marconi et al. 1992) in 1992, using error-prone PCR.

[0052] Another method that can be used is to directly mutagenesis the CDR region of the VH or VL gene. Barbas et al. (Barbas, Hu et al. 1994) and Schier et al. (Schier, McCall et al. 1996) disclosed this technology in 1994. The substantial portion of the immunoglobulin variable domain will generally contain at least three CDR regions and their intervening framework regions. The portion may also include at least about 50% of one or both of the first framework region and the fourth framework region, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The additional residues at the N-terminal or C-terminal end of the substantial portion of the variable domain may be residues that are not normally associated with the naturally occurring variable domain region. For example, construction of specific binding members of the invention by recombinant DNA techniques may result in the introduction of N-terminal or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps, including the introduction of linkers to connect the variable domains of the invention to other protein sequences including immunoglobulin heavy chains, other variable domains (e.g., in the production of diabodies), or protein tags, as discussed in more detail below.

[0053] The present invention provides specific binding members comprising a pair of binding domains based on the amino acid sequences of the VL and VH regions substantially as shown in Figure 1, i.e., Figure 1a and Figure 1b Amino acids 1 to 127 (VH) and Figure 1c A single binding domain based on any of these sequences forms further aspects of the invention. Figure 1a and Figure 1b In the case of binding domains of the listed VH region amino acid sequences, such binding domains can be used as targeting agents, since it is known that immunoglobulin VH domains can bind to target antigens in a specific manner. In the case of any single-chain specific binding domain, these domains can be used to screen for complementary domains that can form dual domain specific binding members with in vivo properties as good or equal to those of the FG27 antibodies disclosed herein.

[0054] This can be achieved by a phage display screening method using a so-called hierarchical dual combinatorial approach as disclosed in WO92 / 01047, in which a single colony containing either H or L chain clones is used to infect a complete clone library encoding the other chain (L or H), and the resulting double-chain specific binding members are selected according to phage display technology (e.g., as described in this reference). This technology was also disclosed by Marks et al. (Marks, Griffiths et al. 1992) in 1992.

[0055] The specific binding members of the present invention may also comprise an antibody constant region or a portion thereof. Figure 1c The specific binding member of the VL region shown in can be connected to the antibody light chain constant domain at its C-terminus. Figure 1a and Figure 1b The specific binding members of the VH regions shown may be linked at their C-termini to all or part of an immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE and IgM) and any isotype subclass (particularly IgGl, IgG2 and IgG4).

[0056] The specific binding members of the invention can be used in methods for diagnosing and treating tumors in human or animal subjects.

[0057] When used in diagnostics, the specific binding members of the invention may be detectably labeled (e.g., radiolabeled, such as 131 I or 99 The invention also provides a method for labeling a specific binding member of the present invention with a Tc-labeled antibody, which can be attached to a specific binding member of the present invention using conventional chemical methods known in the field of antibody imaging. Labels also include enzyme labels, such as horseradish peroxidase. Labels also include chemical moieties, such as biotin, which can be detected by binding to a specific homologous detectable moiety (e.g., a labeled avidin).

[0058] Although the specific binding members of the present invention have themselves been shown to be effective in killing cancer cells, they can be additionally labeled with functional markers. Functional markers include substances designed to target the cancer site to cause its destruction. Such functional markers include toxins (e.g., ricin) and enzymes (e.g., bacterial carboxypeptidases or nitroreductases) that are capable of converting prodrugs into active drugs. In addition, the specific binding component can be linked to a chemotherapeutic or cytotoxic agent (e.g., maytansine (DM1 and DM4), onides, auristatins, calicheamicin, duocamycin, doxorubicin) or a radioactive marker (e.g., 90 Y or 131 I) or otherwise associated therewith.

[0059] In addition, the specific binding members of the present invention can be used alone or in combination with other treatments, and can be used simultaneously or sequentially, depending on the disease treated. Therefore, the present invention further provides a product containing the specific binding members of the present invention and an activating agent as a combined preparation for treating tumors simultaneously, individually or sequentially. Activating agents may include chemotherapeutic agents or cytotoxic agents, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin (oxaliplatin) and tamoxifen (tamoxifen), which may synergize with the binding members of the present invention. Other activating agents may include analgesics of suitable doses, such as nonsteroidal anti-inflammatory drugs (such as aspirin, paracetamol, ibuprofen or ketoprofen) or opiates (such as morphine), or antiemetics.

[0060] Although not wishing to be bound by theory, the ability of the binding members of the invention to synergize with an active agent to enhance tumor killing may not be due to an immune effector mechanism, but rather may be due to the binding member binding to the cell surface Le y A direct consequence of glycan binding. Cancer immunotherapy involving antibodies directed against immune checkpoint molecules has shown effectiveness against various malignancies and in combination with different immuno-oncology treatment modalities.

[0061] The specific binding members of the present invention will generally be applied in the form of a pharmaceutical composition, which may also include at least one component in addition to the specific binding members. In addition to the active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable excipient, diluent, carrier, buffer, stabilizer or other substances well known to those skilled in the art. This substance should be nontoxic and should not interfere with the effect of the active ingredient. The exact nature of the carrier or other substances will depend on the route of administration, which may be oral or injected (e.g., intravenous). It is expected that injection will be the main route of therapeutic administration of the composition, but catheters or other surgical tubes may also be used to deliver. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration. Powder formulations can be reconstituted into liquid formulations for use.

[0062] For intravenous injection or injection in the affected area, the active ingredient will be in the form of a pyrogen-free, pH-appropriate, isotonic and stable enterally acceptable aqueous solution. Those skilled in the art are well able to prepare suitable solutions using, for example, isotonic vehicles (e.g., sodium chloride injection, Ringer's injection, sodium lactate Ringer's injection). Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as needed.

[0063] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. Tablets can contain solid carriers, such as gelatin or adjuvants. Liquid pharmaceutical compositions generally include liquid carriers, such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, dextrose or other sugar solutions, or glycerol such as ethylene glycol, propylene glycol or polyethylene glycol can be included. When the formulation is liquid, it can be, for example, a physiological saline solution containing a non-phosphate buffer of pH 6.8-7.6, or a lyophilized powder. The composition can also be applied by microspheres, liposomes, other microparticle delivery systems or sustained-release formulations placed in certain tissues (including blood). Suitable examples of sustained-release carriers include semi-permeable polymer matrices in the form of shared articles (such as suppositories or microcapsules). Implantable or microencapsulated sustained-release matrices include polylactide (U.S. Pat. No. 3,773,919; EP-A-0058481), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman, Steber et al. 1983), poly(2-hydroxyethyl-methacrylate). Liposomes containing polypeptides are prepared by known methods: DE 3,218,121A; (Eppstein, Marsh et al. 1985); (Hwang, Luk et al. 1980); EP-A-0052522; EP-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541; JP-A-83-11808; U.S. Pat. Nos. 4,485,045 and 4,544,545. Typically, the liposomes are small (about 200 angstroms to 800 angstroms) unilamellar types, wherein the lipid content is greater than about 30 mol.% cholesterol, the selected ratio being adjusted to obtain an optimal polypeptide leakage rate. The composition can be administered topically to a tumor site or other desired site, or can be delivered in a manner that targets tumors or other cells.

[0064] The composition is preferably administered to an individual in a "therapeutically effective amount," which is sufficient to show benefit to the individual. The actual amount administered, the rate of administration, and the time course will depend on the nature and severity of the disease being treated. Treatment prescriptions, such as dosage decisions, are the province of general practitioners and other physicians, and generally require consideration of the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician. The compositions of the present invention are particularly directed to the treatment of existing tumors, particularly cancer, and to the prevention of recurrence of such conditions following initial treatment or surgery. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16 thedition, Oslo, A. (ed), 1980 (Remington 1980).

[0065] The optimal dosage can be determined by the physician based on a number of parameters including, for example, age, sex, weight, severity of the condition being treated, the active ingredient being administered, and the route of administration. In general, serum concentrations of polypeptides and antibodies that saturate the receptors are desirable. Concentrations in excess of about 0.1 nM are usually sufficient. For example, 100 mg / m 2 The antibody dose provided a serum concentration of approximately 20 nM that lasted for approximately eight days.

[0066] As a rough guide, the antibody dose that can be given weekly is 10 mg / m 2 Up to 300 mg / m 2 Equivalent doses of the antibody fragment should be used at more frequent intervals to maintain serum levels above the level required to treat Le y The concentration at which the carbohydrate can be saturated. The dosage of the composition will depend on the characteristics of the binding member, for example, its binding activity and plasma half-life in vivo, the concentration of the polypeptide in the preparation, the route of administration, the site and rate of administration, the clinical tolerance of the patients involved, the pathological conditions suffered by the patients, etc., which are completely within the technical scope of the physician. For example, a dose of 300 μg of antibody per patient per administration is preferred, although the dosage range may be about 10 μg to 6 mg per dose. Different doses are used during a series of sequential vaccinations; a practitioner may perform an initial vaccination and then use a relatively smaller dose of the antibody for boosting.

[0067] The present invention also aims to optimize immunization regimens for enhancing protective immune responses against cancer.

[0068] Binding members of the present invention can be generated in whole or in part by chemical synthesis. The binding members can be easily prepared according to the standard liquid established or the preferred solid phase peptide synthesis method, and the general description of the method can be widely obtained (for example, referring to JM Stewart and JD Young, 1984 (Stewart and Young 1984), M.Bodanzsky and A.Bodanzsky, 1984 (Bodanzsky and Bodanzsky 1984)); Or, binding members can be prepared in solution by liquid phase method or by any combination of solid phase method, liquid phase method and solution chemistry, for example, first complete each peptide part, then if necessary and suitable, after removing any blocking group present, introduce residue X by the reaction of each carbonic acid or sulfonic acid or its reactive derivative.

[0069] Another convenient way to produce a binding member according to the invention is by expressing a nucleic acid encoding the binding member using the nucleic acid in an expression system.

[0070] The present invention also provides isolated nucleic acids encoding specific binding members of the present invention. Nucleic acids include DNA and RNA. In preferred aspects, the present invention provides nucleic acids encoding specific binding members of the present invention as defined above. Examples of such nucleic acids are shown in Fig. 1 and Fig. 2. Those skilled in the art can determine that replacement, deletion and / or addition of such nucleic acids will still be provided for specific binding members of the present invention.

[0071] The present invention also provides a construct in the form of a plasmid, a vector, a transcription cassette or an expression cassette containing at least one of the above-mentioned nucleic acids. The present invention also provides a recombinant host cell comprising one or more of the above-mentioned constructs. As described above, the nucleic acid encoding the specific binding members of the present invention constitutes an aspect of the present invention; the method for producing the specific binding members also constitutes an aspect of the present invention, and the method includes expressing by the encoding nucleic acid. By culturing a recombinant host cell containing the nucleic acid under appropriate conditions, expression can be conveniently achieved. After production by expression, any suitable technology can be used to separate and / or purify the specific binding members, and then use as appropriate.

[0072] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines that can be used in the art to express heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many other cells. A common preferred bacterial host is Escherichia coli (E. coli). The expression of antibodies and antibody fragments in prokaryotic cells (e.g., E. coli) is well known in the art. For a review, see Plückthun, 1991 (Pluckthun 1991). Those skilled in the art may also choose to use expression in eukaryotic cell culture to generate specific binding members, and recent reviews are shown, for example, in Reff, 1993 (Reff 1993), Trill et al., 1995 (Trill, Shatzman et al. 1995).

[0073] Suitable vectors containing appropriate regulatory sequences can be selected or constructed, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. As appropriate, the vector can be a plasmid, a virus, such as a phage or a phagemid. For more details, see, for example, Sambrook et al., 1989 (Sambrook 1989). Ausubel et al., 1992 (Ausubel 1992) describes in detail many known techniques and protocols for nucleic acid manipulation in, for example, preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis.

[0074] Therefore, another aspect of the present invention provides a host cell containing nucleic acids disclosed herein. In another aspect, a method is provided comprising introducing such nucleic acids into host cells. Any available technique can be used for introduction. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia virus, or for insect cells, baculovirus). For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophage. Expression of the nucleic acid can be initiated or allowed after introduction, for example, by culturing host cells under expression conditions of the gene.

[0075] The nucleic acid of the invention can be integrated into the genome (eg chromosome) of the host cell. Integration can be promoted by including sequences that promote recombination with the genome according to standard techniques.

[0076] The present invention also provides a method, which comprises using the construct as described above in an expression system to express the specific binding member or polypeptide as described above.

[0077] The preferred features of each aspect of the invention apply mutatis mutandis to each other aspect.The prior art documents mentioned herein are incorporated herein to the maximum extent permitted by law.

[0078] The inventors unexpectedly discovered that the mAb FG27 family recognizes Le y In one aspect, the present invention provides a method for inducing non-apoptotic direct cell death in the absence of immune effector cells. y Drugs that bind to the glycans of β-catenin and induce non-apoptotic cell death.

[0079] FG27 mAb induces cell membrane damage, leading to cell clumping, loss of microvilli, uptake of small molecular weight dyes and pore formation. Over time, cells form larger pores and lyse in a mechanism similar to oncosis. This is similar to many mAbs that recognize other glycans (Hellstrom, Garrigues et al. 1990, Chou, Takematsu et al. 1998, Zhong, Manzi et al. 2001, Alvarez-Rueda, Leprieur et al. 2007, Loo, Pryer et al. 2007, Zhang, Zhang et al. 2010). FG27 mAb also exhibits strong in vitro cytotoxic activity by ADCC and CDC. FG27 (0.1 mg intravenous injection (iv) twice a week for 9 weeks) was administered to mice with established metastases in the liver and peritoneal cavity, resulting in complete eradication of tumors and cure in about 30% of mice. The potential of FG27 mAb to eradicate established tumors without the need for concomitant chemotherapy suggests that they have the potential to be used as a therapeutic agent for Le-expressing y Potential as a single therapeutic agent for the treatment of human solid tumors.

[0080] The invention will now be described in the following non-limiting examples and accompanying drawings, in which:

[0081] Figure 1a : The complete amino acid and nucleotide sequence of mouse FG27.10 IgG3 heavy chain. The numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 1b : Complete amino acid and nucleotide sequence of mouse FG27.18 IgG1 heavy chain. Numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 1c : Complete amino acid and nucleotide sequences of mouse FG27.10 and FG27.18 kappa chains. Numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009).

[0082] Figure 2a : The complete amino acid and nucleotide sequence of the mouse FG27 heavy chain variable region chimerized with the human IgG1 heavy chain constant region. The numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 2b: The complete amino acid and nucleotide sequence of the mouse FG27 kappa chain variable region chimerized with the human kappa chain constant region. The numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009).

[0083] Figure 3a : The complete amino acid and nucleotide sequence of the humanized FG27 heavy chain variable region. The numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009). Figure 3b : Complete amino acid and nucleotide sequence of the humanized FG27 kappa chain variable region. Numbering refers to the standardized IMGT system for antibody sequence numbering (Lefranc, Giudicelli et al. 2009).

[0084] Figure 4 : Binding of FG27.10 and FG27.18 to ST16, HUVEC and PBMC. For ST16 and HUVEC, the negative control for this assay was NSO supernatant, and for PBMC, the negative control was cells alone; the positive control was anti-sialylated diLe recognizing HLA a mAb, 505 / 4 or W6 / 32.

[0085] Figure 5 :FG27.10 and FG27.18 are combined with glycolipid extracts from colorectal cell lines (C170), ovarian cell lines (OVCAR-3, OVCAR-4, OVCA433, OAW28, OAW42) and gastric cell lines (ST16, MKN45, AGS). Glycolipids extracted from a series of cancer cell lines are spread on ELISA plates. Cells and glycolipids are then incubated with FG27.10 or FG27.18 supernatants. Anti-IgG-HRP / TMB is used to detect the combination with glycolipids.

[0086] Figure 6 : Binding of FG27.10 to HSA-conjugated Lewis antigens (a, b, x, and y) was evaluated by ELISA. 505 / 4 (anti-sialylated di-Lewis a )、FG88.2(Le a-x )、FG88.7(Le a-x ) and 225-Le(Le b ) was used as a positive control and mouse HRPO was used as a negative control. Antibody activity was measured by absorbance at 405 nm.

[0087] Figure 7:The binding of FG27.10 and FG27.18 was screened against the Consortium for Functional Glycomics glycan array, which consists of 610 mammalian glycan targets. y / b ) and d) BR96 (Le y / x ) are compared between the fine specificities; where a = Le a , b=Le b , y=Le y , x-Le x , D-=Di, T-=Tri, S-=sialic acid, Ex-=extension, Indicates polysaccharides containing mannose, yx = Le y -Le x Please note that the glycan numbers for each mAb may be different due to screening mAbs on different versions of glycan arrays.

[0088] Figure 8 : FG27 and Le attached to total protein or lipid extracted from a panel of tumor cell lines y The combination of FG27 and Le y Western blot detection of binding with lysates (1 × 10 5 The total glycoproteins or glycolipids extracted from 100% lysine glycolipids (100% lysine glycolipids) were analyzed.

[0089] Fig. 9 :A series of cell lines with anti-Le y and Le b Comparison of mAbs. SC101 (692 / 29) and BR96 were titrated at 0.1 μg / ml to 30 μg / ml in a range of cell lines (ST16, HT29, C170, LoVo, Colo201, OVCAR-3, AGS), with FG27 supernatant diluted from neat to 1:30. Binding was probed with anti-IgG-FITC and analyzed by flow cytometry.

[0090] Fig.10 :Use 692 / 29(Le y / b )、BR96(Le y / x ) and FG27(Le y ) Representative IHC images of normal esophagus, stomach, duodenum, ileum, colon, pancreas, lung, and kidney stained with .

[0091] Fig.11a: In vivo antitumor activity of FG27. The percentage of tumor growth during the study is shown, in which the C170HM2 bioluminescent mouse tumor model was used to evaluate the antitumor activity of murine FG27 mAb compared to vehicle only control (PBS). In this model, bioluminescence represents viability. Number of groups n≥9; the study was terminated on day 32. Treatment with FG27 resulted in a significant reduction in bioluminescent tumor burden by the last day of the study compared to vehicle control (p=0.014). Fig.11b : Injection site tumor burden assessed by BLI, showing a significant reduction in injection site tumor growth after FG27 treatment (p=0.05).

[0092] Figure 12: In vivo anti-tumor activity of FG27; percentage of tumor growth is shown, where the C170HM2 bioluminescent mouse tumor model was used to evaluate the anti-tumor activity of FG27 compared to a positive control mAb and a vehicle-only control (PBS). Bioluminescence represents tumor cell viability in this model. Number of groups n≥8; FG27 treatment significantly reduced the percentage of tumor growth at day 51 compared to the treatment control (p=0.009). Figure 12b : Analysis by log-rank Mantel-Cox test showed that the survival rate of the FG27 treated group was significant compared to the vehicle only control (p=0.035). Treatment was stopped at day 120.

[0093] Fig.13a : The amino acid and nucleotide sequences of the mouse FG27 heavy chain variable region were compared with BR96, H18A, Hu3S193 and SC101, and the antibody sequences were numbered using the standardized IMGT system (Lefranc, Giudicelli et al. 2009). Fig.13b : The amino acid and nucleotide sequences of the mouse FG27 kappa chain variable region were compared with BR96, SC101, H18A, and Hu3S193, and the antibody sequences were numbered using the standardized IMGT system (Lefranc, Giudicelli et al. 2009).

[0094] Fig.14 Chimeric IgG1 (5 μg / ml) and IgG2 (5 μg / ml) mAbs were evaluated by indirect immunofluorescence and flow cytometry analysis. y Binding of the tumor cell line AGS was compared with the FG27.10 (5 μg / ml) and FG27.18 (5 μg / ml) mouse versions of the antibodies. Anti-HLA mAb W6 / 32 (1 / 100) was included as a positive control.

[0095] Fig.15a: PI uptake by ST16 cells mediated by the mouse IgG3 mAb FG27.10, but not the IgG1 variant FG27.18, induced efficient uptake of PI even at 4 °C. Fig.15b : PI uptake mediated by FG27.10 and FG27.18 on a panel of tumor cell lines (AGS, Colo201, C170, C170HM2, MCF7, OVCA4, LoVo, MKN45, 791T and Skov3). Fig.15c : PI uptake mediated by FG27.10, FG27.18, CH27 IgG2, and CH27 IgG1 on C170 cells.

[0096] Fig.16 : Internalization data of FG27.10, FG27.18 and CH27 at a series of concentrations (0.01nM to 30nM) on a group of cancer cell lines AGS, MCF7, H322 and C170. Cell internalization of mAbs was observed using 75ng / well saporin-conjugated goat anti-mouse (ATSbio, IT#48) and anti-human IgG (ZAP) antibodies (F'ab fragments), and cell internalization of mAbs was assessed by inhibiting thymidine uptake (added within the last 24 hours of incubation). Control wells consisted of cells cultured with culture medium only, cells cultured with irrelevant positive control IgG3 mAb FG88, and cells cultured with the second conjugate only.

[0097] Fig.17a : Example of FG27.10 mIgG3 mediated ADCC / CDC. Target cells were incubated with PBMC isolated from human blood (ADCC), or with complement (CDC), with FG27.10 supernatant or irrelevant IgG. Error bars representing the standard deviation of triplicate wells are presented, but the error bars may be obscured by the data points. Fig.17b : ADCC of MCF7 tumor cell line incubated with FG27.10, FG27.18, 692 / 29, CH27IgG1 and CH27 IgG2. These are results from one experiment but represent 2 replicates. Fig.17c : Average killing percentage of CDC after incubation of ST16 tumor cell line with FG27.10, FG27.18, 692 / 29, CH27 IgG1 and CH27 IgG2 with human serum for 4 hours. Herceptin was included as a control. The assay was performed with serial dilutions of 3μg / ml, 1μg / ml, 0.3μg / ml and 0.1μg / ml, and each dilution was incubated in triplicate. Fig.17d: Average percent killing of AGS cells by CDC after incubation for 4 hours with FG27.10, 692 / 29, CH27 IgG1 and CH27 IgG2 with human serum. Serial dilutions of 3 μg / ml, 1 μg / ml, 0.3 μg / ml and 0.1 μg / ml were assayed, and each dilution was incubated in triplicate.

[0098] Fig.18 : Gibbs analysis of FG27 heavy chain I: Sequence identity. Structurally important residues are highlighted: proline residues are in white, cysteine ​​residues are in black, and asparagine residues are in grey.

[0099] Fig.19 : FG27 heavy chain Gibbs analysis II: identity and similarity scores, Neighboring residues and CDR loop lengths. Cysteine ​​residues are highlighted in white.

[0100] Fig. 20 : Germline analysis of human heavy chain donor sequences.

[0101] Fig.21 : Structurally important residues for the FG27 heavy chain humanization strategy are highlighted: proline residues are marked in white, cysteine ​​residues are marked in black, and asparagine residues are marked in grey. Residues marked in grey and italics indicate back translation of mouse residues.

[0102] Fig. 22 : Gibbs analysis of 27 kappa light chain I: Sequence identity. Structurally important residues are highlighted: proline residues are in white, cysteine ​​residues are in black, and asparagine residues are in grey.

[0103] Fig.23 : 27κ light chain Gibbs analysis II: identity and similarity scores, Contiguous residues and CDR loop lengths.

[0104] Fig.24 : Germline analysis of human kappa light chain donor sequences.

[0105] Fig.25 : FG27 kappa light chain humanization strategy. Structurally important residues are highlighted: proline residues are marked in white, cysteine ​​residues are marked in black. Residues in bold represent back translations of mouse residues.

[0106] Fig.26: Antibody binding to Lewis Y-HSA antigen was compared for antibody combinations denoted LewRHA with LewRKA, LewRKB, LewRKC or LewRKD, and LewRHB with LewRKA, LewRKB, LewRKC or LewRKD, which included a cFG27 positive control antibody and a c1210 negative control antibody.

[0107] Fig. 27 : Thermal stability of LewRHA with LewRKA, LewRKB, LewRKC or LewRKD, and LewRHB with LewRKA, LewRKB, LewRKC or LewRKD, including the cFG27 positive control antibody.

[0108] Fig.28 :Lewis y Thermal drift analysis of FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27.

[0109] Fig.29 : BiaCore analysis of antibody affinity between FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27.

[0110] Fig.30 : Non-specific protein-protein interactions (CIC) measured by cross-interaction chromatography for FG27 humanized variants hLewAB, hLewAC, hLewAD, hLewBC and control mouse FG27.

[0111] Fig.31 : Binding of FG27.10, FG27.18, ch27IgG1 and the humanized variant hLewAC to a panel of cell lines.

[0112] Fig.32 : i27G1 significantly increased proliferation inhibition on MCF7 (Fig. i) and AGS (Fig. ii) compared to 27hIgG1. Significant increase in functional affinity (SPR) of i27G1 compared to 27hIgG1 (Fig. iii). i27G1 maintained equivalent ADCC (Fig. iv) and CDC activity (Fig. v) on MCF7. Significance compared to the parental construct was inferred from two-way ANOVA (direct cytotoxicity) or one-way ANOVA (functional affinity) using Dunnett correction for multiple comparisons.

[0113] Fig.33 : The amino acid and nucleic acid sequences of each light and heavy chain of the 'i27G1' antibody. Example

[0114] method

[0115] Combined with tumor cell lines: 1×10 5 Cancer cells were incubated with 50 μl of primary antibody at 4 °C for 1 hour. The cells were washed with 200 μl RPMI 10% newborn calf serum (NBCS: Sigma, Poole, UK) and spun at 1000 rpm for 5 minutes. The supernatant was discarded and 50 μl FITC-conjugated anti-mouse IgG Fc-specific mAb (Sigma; 1 / 100 in RPMI 10% NBCS) was used as a secondary antibody. The cells were incubated at 4 °C in the dark for 1 hour, then washed with 200 μl RPMI 10% NBCS and spun at 1000 rpm for 5 minutes. After discarding the supernatant, the cells were fixed with 0.4% formaldehyde. The samples were analyzed on a Beckman coulter FC-500 flow cytometer (Beckman Coulter, High Wycombe, UK). WinMDI 2.9 software was used to analyze and plot the raw data.

[0116] Binding to blood: 50 μl of healthy donor blood was incubated with 50 μl of primary antibody at 4 ° C for 1 hour. The blood was washed with 150 μl RPMI 10% NBCS and spun at 1000 rpm for 5 minutes. The supernatant was discarded and 50 μl FITC-conjugated anti-mouse IgG Fc-specific mAb (1 / 100 in RPMI 10% NBCS) was used as a secondary antibody. The cells were incubated at 4 ° C for 1 hour in the dark, then washed with 150 μl RPMI 10% NBCS and spun at 1000 rpm for 5 minutes. After discarding the supernatant, 50 μl / well Cal-Lyse (Invitrogen, Paisley, UK) was used, followed by 500 μl / well distilled water to lyse the red blood cells. Subsequently, the blood was spun at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were fixed with 0.4% formaldehyde. Samples were analyzed on a FC-500 flow cytometer (Beckman Coulter). To analyze and plot raw data, WinMDI2.9 software was used.

[0117] Plasma membrane glycolipid extraction: Cell pellets (5×10 7Cells) were resuspended in 500 μl of mannitol / HEPES buffer (50 mM mannitol, 5 mM HEPES, pH 7.2, all from Sigma) and passed through 3 needles (23G, 25G, 27G), 30 pulses per needle. 5 μl of 1M CaCl2 was added to the cells and passed through 3 needles, 30 pulses per needle, as described above. The sheared cells were incubated on ice for 20 minutes and then spun at 3000g for 15 minutes at room temperature (RT). The supernatant was collected and spun at 48000g for 30 minutes at 4°C and the supernatant was discarded. The pellet was resuspended in 1 ml of methanol, then added to 1 ml of chloroform and rolled and incubated at RT for 30 minutes. The sample was then spun at 1200g for 10 minutes to remove precipitated proteins. The supernatant containing plasma membrane glycolipids was collected and stored at -20°C.

[0118] Glycomic Analysis: To elucidate the fine specificity of the FG27 mAb, the antibody was labeled with FITC and sent to the Functional Glycomics Consortium (http: / / www.functionalglycomics.org / static / consortium / resources / resourcecoreh8.shtml]) where it was screened against ≥600 natural and synthetic glycans. Briefly, synthetic glycans and mammalian glycans with amino linkers were printed onto glass microscope slides activated with N-hydroxysuccinimide (NHS) to form amide bonds. This work was performed by Core H of the CFG at Emory University. FG27 was tested at 1 μg / ml in PBS at room temperature. Briefly, the antibody samples were applied to the printed surface of the microarray and incubated for 1 hour in a humidified chamber. The slides were then rinsed 4 times with PBS, and then fluorescently labeled (Alexa Fluor 488) anti-rabbit IgG was added and incubated for 1 hour. Slides were then rinsed four times in PBS, and fluorescence was measured using a Perkin-Elmer Microarray XL4000 scanner and analyzed using Imagene software (BioDiscovery).

[0119] Further details of the scheme used are given below:

[0120] Glycan Binding Assays Using Unlabeled Monoclonal Antibodies

[0121] 1. Introduction:

[0122] 1.1. The main purpose of Core H is to determine the binding specificity of glycan binding proteins (GBPs) to various organisms submitted by researchers using printed glycan microarrays.

[0123] 2. Reference:

[0124] 2.1. www.functionalglycomics.org

[0125] 3. Required materials:

[0126] 3.1. Glycan-printed slides (Core D), printed on the side of the slide with the white etched barcode and black logo - do not touch this area

[0127] 3.2. Cover glass (Fisher scientific, 12-545F)

[0128] 3.3. Humidified slide handling chamber (Fisher Science, NC9091416), or a homemade system using a petri dish with a wet paper towel on the bottom of the chamber

[0129] 3.4. 100 ml Coplin jar for washing slides

[0130] 3.5.Tris-HCl (Fisher scientific, BP152-1)

[0131] 3.6.NaCl (Fisher scientific, S271-3)

[0132] 3.7.CaCl2 (Fisher scientific, C79-500)

[0133] 3.8.MgCl2 (Fisher scientific, BP214-500)

[0134] 3.9. Potassium dihydrogen phosphate (Fisher scientific, P285-3)

[0135] 3.10.dH20

[0136] 3.11. Cyanine 5-Streptavidin (Zymed 43-4316)

[0137] 3.12. Appropriate secondary antibodies, fluorescently labeled (if available)

[0138] 3.13.BSA (Fisher scientific, Bp1600-100)

[0139] 3.14.Tween-20 (EMD Biosciences, 655205)

[0140] 3.15. Sodium azide (Fisher scientific, S227-500)

[0141] 3.16. ProScanArray Scanner (Perkin Elmer)

[0142] 4. Buffer:

[0143] 4.1.TSM=20mM Tris-HCl, pH 7.4 150mM NaCl, 2mM CaCl2, 2mM MgCl2

[0144] 4.2.TSM Wash Buffer (TSMW) = TSM Buffer + 0.05% Tween-20

[0145] 4.3.TSM binding buffer (TSMBB) = TSM buffer + 0.05% Tween 20 + 1% BSA

[0146] 5. Solution:

[0147] 5.1. Make working stocks of wash buffers (TSM, TSM wash buffer, and HO) or collect reagents and bring them to room temperature (if they were in the refrigerator).

[0148] 5.1.1. Buffer (A) TSM: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2

[0149] 5.1.2. Buffer (B) TSM wash buffer (TSMW): TSM buffer + 0.05% Tween-20

[0150] 5.1.3. Buffer (C) TSM binding buffer (TSMBB): TSM buffer + 0.05% Tween 20 + 1% BSA

[0151] 5.1.4.dH2O

[0152] 5.2. Prepare 100 μl of sample by diluting the antibody in TSMBB or appropriate binding buffer to a final concentration of 5 ug / ml to 50 ug / ml or the appropriate concentration required for the assay, depending on the characteristics of the antibody.

[0153] 5.3. Take out the slides from the desiccator and label the slides with the sample name outside the black mark and near the barcode.

[0154] 5.4. Hydrate the slides by placing them in a glass Coplin jar containing 100 ml TSMW for 5 minutes.

[0155] 5.5. Remove excess liquid from the slide by placing the slide vertically to drain the liquid.

[0156] 5.6. Carefully apply 70 μl of the sample (see 5.2) between the black marks near the left edge of the slide.

[0157] 5.7. Slowly place the coverslip onto the slide, trying to avoid bubbles forming in the sample under the coverslip. If necessary, remove any bubbles by gently tapping the coverslip with a pipette tip or slowly lifting one side of the coverslip. Make sure the coverslip is between the black marks.

[0158] SDS-PAGE and Western Blot Analysis: In brief, analyze 10 5 or 10 6 FG27 binding of individual cell equivalents (cell lysates, plasma membrane lipid extracts). Tumor cell plasma membrane lipid extracts and cell lysates were reduced with dithiothreitol (DTT; Pierce Biotechnology, ThermoFisher, Loughborough, UK), and SDS-PAGE was performed using NOVE×4% to 12% Bis-Tris gel (Invitrogen), and then transferred to Hybond-P PVDF membrane (GE Healthcare, Amersham, UK) at 30V using 1x transfer buffer (20x, Invitrogen) and 20% (v / v) methanol for 1 hour. The membrane was blocked with 0.05% (v / v) Tween-PBS solution of 5% (w / v) skim milk powder for 1 hour, and then probed with a primary antibody diluted in Tween-PBS, 2% BSA for 1 hour. Primary antibody binding was detected using a biotin-conjugated anti-mouse IgG Fc-specific secondary antibody (Sigma; 1 / 2000 dilution in Tween-PBS, 2% BSA) for 1 h and visualized using IRDye 800CW Streptavidin (LICOR Biosciences, UK; 1 / 1000 dilution in Tween-PBS 2% BSA).

[0159] Immunohistochemistry evaluation of FG27: To determine the therapeutic value of FG27, it was screened by immunohistochemistry (IHC) on gastric, colorectal, ovarian, and breast tumor tissue microarrays.

[0160] Methods: Immunohistochemistry was performed using the standard avidin-biotin peroxide method. Paraffin-embedded tissue sections were placed on a 60°C heat block to melt the paraffin. Tissue sections were dewaxed with xylene and rehydrated through graded alcohols. Sections were then immersed in 500 ml of citrate buffer (pH 6) and heated in a microwave oven (Whirlpool) for 20 min to retrieve antigens. Endogenous peroxidase activity was blocked by incubating tissue sections with endogenous peroxidase solution (Dako Ltd, Ely, UK) for 5 min. Normal porcine serum (NSS; Vector Labs, CA, USA; 1 / 50 PBS) was added to each section for 20 min to block nonspecific primary antibody binding. All sections were incubated with an avidin D / biotin blocking kit (Vector Lab) for 15 min to block nonspecific binding of avidin and biotin. Sections were blocked with NSS (1 / 50 PBS) for another 5 min. Tissue sections were then incubated with primary antibodies for 1 h at RT. Anti-β-2-microglobulin (Dako Ltd; 1 / 100 in PBS) mAb and PBS alone were used as positive and negative controls, respectively. Tissue sections were washed with PBS and incubated with biotinylated goat anti-mouse / rabbit immunoglobulin (Vector Labs; 1 / 50 in NSS) for 30 min at RT. Tissue sections were washed with PBS and incubated with pre-made 1 / 50 (PBS) streptavidin-biotin / horseradish peroxidase complex (Dako Ltd) for 30 min at RT. 3,3'-Diaminobenzidine tetrahydrochloride (DAB) was used as substrate. Each section was incubated twice with 100 μl of DAB solution for 5 min each. Finally, sections were lightly counterstained with hematoxylin (Sigma-Aldrich, Poole Dorset, UK), then dehydrated in graded alcohols, cleared in xylene, and mounted on slides with stilbene, plasticizer, xylene (DPX) mounting medium (Sigma).

[0161] Identification of FG27.10 and FG27.18 heavy and light chain variable regions

[0162] Cell source and total RNA preparation: Approximately 5 × 10 from hybridomas FG27.10 and FG27.18 6Cells were taken from tissue culture, washed once in PBS, and the cell pellets were treated with 500 μl Trizol (Invitrogen). After the cells were dispersed in the reagent, they were stored at -80°C until RNA was prepared according to the manufacturer's protocol. RNA concentration and purity were determined by Nanodrop. Prior to cDNA synthesis, RNA was treated with DNase I to remove genomic DNA contamination as recommended by the manufacturer (DNase I recombinant, RNase-free, Roche Diagnostics, Burgess Hill, UK).

[0163] cDNA synthesis: First-strand cDNA was prepared from 3 μg of total RNA using a first-strand cDNA synthesis kit and AMV reverse transcriptase according to the manufacturer's protocol (Roche Diagnostics). After cDNA synthesis, reverse transcriptase activity was destroyed by incubation at 90°C for 10 minutes and cDNA was stored at -20°C.

[0164] GAPDH PCR to assess cDNA quality: PCR was used to assess cDNA quality; primers specific for the mouse GAPDH housekeeping gene (5'-TTAGCACCCCTGGCCAAGG-3' and 5'-CTTACTCCCTTGGAGGCCATG-3') were used with hot-start Taq polymerase (NEB, Hitchen, UK) for 35 cycles (95°C, 3 min, followed by 35 cycles of 94°C / 30 sec, 55°C / 30 sec, 72°C / 1 min; final correction step was 72°C for 10 min). Amplification products were assessed by agarose gel electrophoresis.

[0165] PCR primer design for cloning FG27 variable regions: Primers were designed to amplify the heavy and light chain variable regions based on the PCR product sequence data. Primers were designed to allow the cloning of the relevant chains into the unique restriction enzyme sites in the hIgG1 / κ dual expression vector pDCOrig-hIgG1. Each 5' primer targets the start codon and leader peptide of the defined variable region, with a Kozak consensus sequence immediately 5' to the start codon. Each 3' primer was designed to be complementary to the junction region of the antibody sequence to maintain the reading frame after cloning the chain, and retain the amino acid sequence commonly found at the junction region / constant region junction. All primers were purchased from Eurofins MWG.

[0166] Heavy chain variable region PCR: The immunoglobulin heavy chain variable region was determined using PCR and a previously published set of primers (Jones and Bendig 1991). Previous results using a mouse mAb typing test kit (Serotec, Oxford, UK) indicated that FG27.10 was a mouse IgG3 and FG27.18 was a mouse IgG1 antibody. Therefore, the appropriate constant region reverse primer was used to amplify from the constant region. PCR amplification was performed using 12 mouse VH region specific 5' primers and 3' primers specific for the previously determined antibody subclasses with hot start Taq polymerase for 35 cycles (94°C, 5 minutes, followed by 35 cycles of: 94°C / 1 minute, 60°C / 1 minute, 72°C / 2 minutes; final correction step was 72°C for 20 minutes). The amplification products were evaluated by agarose gel electrophoresis. The VH4 primer produced positive amplification.

[0167] Light (κ) Chain Variable Region PCR: Immunoglobulin light chain variable region usage was determined using PCR and a previously published set of primers (Jones and Bendig 1991). Previous results using a mouse mAb typing assay kit indicated that both FG27.10 and FG27.18 used κ light chains. PCR amplification was performed using mouse Vκ region-specific 5' and 3' mouse Cκ-specific primers with hot-start Taq polymerase for 35 cycles (94°C for 5 minutes, followed by 35 cycles of 94°C / 1 minute, 60°C / 1 minute, and 72°C / 2 minutes; a final correction step of 72°C for 20 minutes). Amplification products were evaluated by agarose gel electrophoresis. Vκ1 and Vκ2 primers for FG27.10 and FG27.18 resulted in positive amplification.

[0168] PCR product purification and sequencing: PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Crawley, UK). The concentration of the resulting DNA was determined by Nanodrop and the purity was assessed by agarose gel electrophoresis. PCR products were sequenced using the Nottingham University DNA sequencing tool (http: / / www.nottingham.ac.uk / life-sciences / facilities / dna-sequencing / index.aspx) using the original 5' and 3' PCR primers. The IMGT database search tool ( http: / / www.imgt.org / IMGT_vquest / vquest?livret=0&Option=mouseIg ) analysis sequence (V region identification, connection analysis). Sequencing showed that FG27.10 and FG27.18 shared the same heavy and light chain variable regions. There were enough residual constant regions in the heavy chain sequence to confirm that FG27.10 belonged to the mIgG3 subclass, while FG27.18 belonged to mIgG1, and both were generated by a single splenocyte-NSO fusion event.

[0169] Cloning strategy: Based on previous Scancell experience, it was known that direct cloning of PCR products into the pDCOrig-hIgG1 vector using restriction sites incorporated into the PCR primers was relatively inefficient. Therefore, a dual cloning strategy was adopted; PCR products generated using a proofreading polymerase were cloned simultaneously into pDCOrig-hIgG1 and a TA vector (pCR2.1; Invitrogen), with the TA vector cloned products serving as an easily amplifiable backup source of cloning material if the initial pDCOrig-hIgG1 clones failed.

[0170] FG27.18 heavy / light chain PCR for cloning: PCR amplification was performed as follows: using proofreading polymerase (Phusion; NEB) and the above cloning primers, using the previously described FG27.18 cDNA template, 35 cycles (98°C, 3 minutes, then 35 cycles: 98°C / 30 seconds, 58°C / 30 seconds, 72°C / 45 seconds; the final correction step was 72°C for 3 minutes). Successful amplification was confirmed by agarose gel electrophoresis.

[0171] Method 1-direct light chain cloning: The amplified FG27.18 light chain was digested sequentially with restriction endonucleases BsiWI and BamHI according to the manufacturer's instructions (NEB). The vector (pDCOrig-hIgG1, containing the V region of the antibody cloned previously) was digested simultaneously. The vector DNA was agarose gel purified using the QIAquick gel extraction kit (Qiagen), and the insert DNA was purified using a PCR purification kit. After quantification of the DNA by Nanodrop, the vector DNA was phosphatase treated according to the manufacturer's recommendations (Antarctic Phosphatase, NEB), and the light chain insert was ligated to the vector (T4 DNA ligase, NEB). The ligated DNA was transformed into chemically competent TOP10F' cells (Invitrogen) and plated on LB agar plates supplemented with 35 μg / ml Zeocin (Invitrogen, Toulouse, France), and then incubated overnight at 37°C.

[0172] Method 2-TOPO light chain cloning: The amplified FG27.18 light chain was treated with Taq polymerase (NEB) at 72°C for 15 minutes to add "A" overhangs suitable for TA cloning. The treated PCR product was incubated with the TOPO TA vector pCR2.1 (Invitrogen) and transformed into chemically competent TOP10F' cells according to the manufacturer's instructions. The transformed bacteria were plated on LB agar plates supplemented with ampicillin (80 μg / ml) and then incubated overnight at 37°C. The colonies were grown in liquid culture (LB supplemented with 80 μg / ml ampicillin) and plasmid DNA was prepared (spin miniprep kit, Qiagen). The presence of the insert was confirmed by sequential digestion with BsiWI and BamHI and agarose gel electrophoresis. Miniprep DNA from the colonies was sequenced using T7 and M13rev primers. The DNA insert from one such colony had the expected FG27.18 light chain sequence; 300 ml bacterial LB / ampicillin culture was grown overnight and plasmid DNA was prepared by maxiprep (plasmid maxi kit, Qiagen). The Maxiprep DNA insert was confirmed by sequencing.

[0173] TOPO heavy chain cloning: The amplified FG27.18 heavy chain was treated with Taq polymerase (NEB) at 72°C for 15 minutes to add the "A" overhang. The treated PCR product was incubated with the TOPO TA vector pCR2.1 and transformed into chemically competent TOP10F' cells as described above. The transformed bacteria were plated on LB agar plates supplemented with ampicillin and then incubated overnight at 37°C. The colonies were grown in liquid culture (LB / ampicillin) and plasmid DNA was prepared (centrifugal miniprep kit). The presence of the insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Miniprep DNA from multiple colonies was sequenced using T7 and M13rev primers. The DNA insert from one such colony had the expected FG27.18 heavy chain sequence; 300 ml of bacterial LB / ampicillin culture was grown overnight and plasmid DNA was prepared by maxiprep (plasmid large extraction kit, Qiagen). Maxiprep DNA inserts were confirmed by sequencing.

[0174] pDCOrig-hIgG1 dual expression vector light chain cloning: FG27.18 light chain was digested from the TOPO vector pCR2.1 by sequential digestion with BsiWI and BamHI, and the 400 bp insert DNA was purified from agarose gel using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's recommendations. This insert was ligated into the previously prepared pDCOrig-hIgG1 vector (see above) and transformed into chemically competent TOP10F' cells. The transformants were plated on LB agar plates supplemented with 35 μg / ml zeocin and incubated overnight at 37°C.

[0175] Colonies were grown in liquid culture (LB supplemented with 35 μg / ml bleomycin) and plasmid DNA was prepared (centrifugal miniprep kit, Qiagen). Miniprep DNA from all colonies was sequenced using the P6 sequencing primer located in the human kappa constant region. DNA inserts from colonies had the expected FG27.18 light chain sequence correctly inserted into pDCOrig-hIgG1; 300 ml bacterial LB / bleomycin cultures were grown overnight and plasmid DNA was prepared by maxiprep (plasmid maxiprep kit, Qiagen).

[0176] Heavy chain cloning of pDCOrig-hIgG1 dual expression vector: The FG27.18 heavy chain insert was digested from the TOPO vector pCR2.1 by digestion with HindIII and AfeI. The vector (pDCOrig-hIgG1-27.18k) containing the FG27.18κ light chain (prepared as above) was also digested with HindIII and AfeI. The vector DNA was then phosphatase treated according to the manufacturer's recommendations (thermosensitive phosphatase, NEB). After agarose gel electrophoresis, the 6.5kb pDCOrig-hIgG1 vector band and the 400bp FG27.18H insert band were separated using the QIAquick gel extraction kit (Qiagen) according to the manufacturer's recommendations. The insert was ligated into the pDCOrig-hIgG1 vector and transformed into chemically competent TOP10F' cells. The transformants were plated on LB agar plates supplemented with 35μg / ml bleomycin and incubated overnight at 37°C. Colonies were grown in liquid culture (LB supplemented with 35 μg / ml bleomycin) and plasmid DNA was prepared (centrifuge miniprep kit, Qiagen). The presence of the insert was confirmed by digestion with HindIII and AfeI and agarose gel electrophoresis. Miniprep DNA from multiple colonies was sequenced using the P3rev sequencing primer located in the constant region of human IgG1. The DNA insert from one colony in multiple colonies had the expected FG27.18 heavy chain sequence correctly inserted into pDCOrig-hIgG1; 300 ml bacterial LB / zeocin cultures were grown overnight and plasmid DNA was prepared by maxiprep (plasmid maxiprep kit, Qiagen). Sequencing was used to confirm the heavy and light chain loci.

[0177] Expression, purification and characterization of chimeric antibody constructs: The expression and purification methods of the chimeric antibodies described in the present invention can be achieved using methods well known in the art. In brief, antibodies can be purified by protein A or protein G affinity chromatography from supernatants collected from transient or subsequently stably transfected cells according to standard protocols, such as Sambrook et al., 2001 (Sambrook and Russell 2001).

[0178] QuikChange Rapid Site-Directed Mutagenesis Kit (Stratagene)

[0179] Prepare the reaction as follows:

[0180] 5 μl of 10× reaction buffer

[0181] 0.12 μl (25 ng) RHA or RKA template

[0182] 1.3 μl (125 ng) oligonucleotide mutagenesis primers For

[0183] 1.3μl (125ng) oligonucleotide mutagenesis primer Rev 1μl dNTPmix

[0184] 1.5 μl QuikSolution reagent ddH2O to a final volume of 50 μl

[0185] 1μl QuikChange Lightning Enzyme

[0186] 1. Cycle each reaction using the cycling parameters listed in the table below:

[0187]

[0188] 2. Add 2 μl DpnI restriction enzyme

[0189] 3. Gently and thoroughly mix each reaction, microcentrifuge briefly, and then immediately incubate at 37°C for 5 minutes to digest the parental dsDNA.

[0190] 4. Transform 2 μl of DpnI-treated DNA from each reaction into different 45 μl (+ 2 μl β-ME) aliquots of XL10-Gold supercompetent cells (see TTOP10 TM Transformation of E. coli).

[0191] 5. Screen colonies using the Phusion method, miniprep, and sequencing to check for the correct mutation.

[0192] Site-directed mutagenesis kit (NEB protocol):

[0193] 1. Assemble the following reagents in a thin-walled PCR tube:

[0194]

[0195]

[0196] cycle

[0197]

[0198] Kinase, Ligase & DpnI (KLD) Processing

[0199] 2. Add the following reagents and incubate at RT for 5 minutes: 1 μl PCR product

[0200] 5 μl 2X KLD reaction buffer 1 μl 10X KLD enzyme mix

[0201] 3 μl nuclease-free water

[0202] 3. Transform 5 μl of KDL mix from each reaction into a separate 50 μl of NEB 5-α competent E. coli. Carefully flick the tube 4-5 times to mix. Do not vortex.

[0203] 4. Place the mixture on ice for 30 minutes.

[0204] 5. Heat shock at 42℃ for 30 seconds. Place on ice for 5 minutes.

[0205] 6. Pipette 950 μl RT SOC into the mixture.

[0206] 7. Incubate at 37°C with shaking (250 rpm) for 60 minutes.

[0207] 8. Mix the cells thoroughly by flicking the tube and inverting, then spread 50 μl onto kanamycin plates and incubate overnight at 37°C.

[0208] 9. Screen the colonies using the GoTaq Green method, minipre and sequencing to check for the correct mutation.

[0209] IgG quantification:

[0210] Coat each well of a 96-well immunoplate with a 100 μl aliquot of 0.4 μg / ml goat anti-human IgG antibody (diluted in PBS) and incubate overnight at 4°C. (At this stage, the plates can be stored for 1 month). Also coat another blank plate with BSA / PBS blocking solution. Remove excess coating solution and wash the plate 3 times with 200 μl / well of wash buffer. In the blank plate, dispense 120 μl of SEC buffer into all wells except the wells in rows B to G in column 2. Prepare a 1 μg / ml human IgG1 / κ antibody solution in SEC buffer as a standard. Pipette 240 μl / well and move to the wells in rows B and C in column 2. Centrifuge the culture medium from the transfected cells (250g, 5 minutes) and save the supernatant. Pipette 240 μl of supernatant from the "no DNA" control (where cos cells were transfected without DNA) into the wells of column 2, row D. Pipette 240 μl / well of experimental supernatant into the wells of column 2, rows E, F, and G. Mix 240 μl aliquots in the wells of columns 2, rows B to G, and transfer 120 μl to the adjacent wells of column 3. Serial 2-fold dilutions of standards, control samples, and experimental samples continue until column 11. Transfer 100 μl from each well to the corresponding well of the anti-IgG coated plate. Incubate at 37°C for 1 hour. Rinse all wells 3 times with wash buffer (200 μl). After diluting goat anti-human kappa light chain peroxidase conjugate 5000-fold in SEC buffer, add 100 μl to each well. Repeat the incubation and wash steps (step 9). Add 150 μl of K-BLUE substrate to each well and incubate at room temperature for 10 minutes in the dark. Stop the reaction with STOP solution and read the optical density at 655 nm.

[0211] Lewis y Binding ELISA

[0212] Each well of a 94-well MaxiSorp plate (Nunc) was coated with 100 ng / well of Lewis Y HSA peptide in PBS and incubated overnight at 4°C. Washed 3 times with PBS-T (0.1% Tween20). Blocked a new plate with 250 μl / well of PBS / 0.2% BSA / 0.05% Tween20 and incubated for 1 hour at room temperature. Washed 3 times with PBS-T. Added 240 μl of antibody (diluted in PBS / 0.2% BSA / 0.05% Tween20 if necessary) to the wells in column 1; added 120 μl of buffer (PBS / 0.2% BSA / 0.05% Tween20) to the other wells. Transferred 120 μl from column 1 to the adjacent well in column 2. Serial 2-fold dilutions of experimental samples continued until column 12. Transferred 100 μl per well from the dilution plate to the experimental plate. Incubated for 1 hour at RT. Wash wells 3 times with PBS-T. Dilute goat anti-human Fc peroxidase conjugate 10,000-fold (or anti-mouse, 10,000-fold dilution) in PBS / 0.2% BSA / 0.05% Tween20 and add 100 μl to each well. Incubate at RT for 1 hour and repeat the wash step. Add 150 μl substrate (K-Blue) to each well and incubate at room temperature for 150 minutes. Stop the reaction by adding 50 μl RED STOP solution to each well. Read the optical density at 650 nm.

[0213] Thermal stability

[0214] Fully humanized antibodies and chimeric controls were diluted to 1 μg / ml in TBS / 0.2% Tween and dispensed into PCR tubes at appropriate volumes for EC80 concentrations. The volume was increased to 100 μl using the same buffer. Each tube was heated at 5°C intervals between 30°C and 85°C for 10 minutes and then cooled to 4°C. The 1 μg / ml stock solution was frozen for 1 hour and then diluted to the EC80 concentration. Binding assays were performed on Lewis Y HSA peptides using 100 μl of each antibody per well (measured in duplicate for each temperature) in a 96-well plate.

[0215] Thermal Drift Comparison

[0216] Prepare samples directly into a 96-well white PCR plate in a final volume of 25 μl (final concentrations of purified antibodies are 1 μM and 2 μM). Sypro Orange – Prepare stock solution at 1:100 in PBS buffer and add to final sample at 1:10 ratio (e.g., add 2.5 μl to 25 μl). Load into qPCR machine, use MxPro software, SYBR Green method (filter = FRROX, no reference dye). Thermal profile settings - 71 cycles, 1° increments. Plot results and determine Tm.

[0217] Purification of humanized mAbs

[0218] Instrument: GE Healthcare Purification system

[0219] Software: UNICORN

[0220] Column: HiTrap MabSelect SuRe, 1ml; HiLoad 16 / 600 Superdex 200pg

[0221] Mobile phase: IgG elution buffer; Dulbecco's 1xPBS

[0222] Sample preparation: Filtered through 0.22 μm

[0223] Injection volume: 200 ml Expi293 conditioned medium (1:1) in DPBS

[0224] Flow rate: Sample loading speed is 0.5 ml / min; gel filtration speed is 1.5 ml / min;

[0225] Elution at 1 ml / min

[0226] Aggregate comparison

[0227] Instrument: Agilent 1260 infinity HPLC system with thermostatted column compartment, Wyatt Technology Dawn Heleos, Wyatt Technology Optilab TRex

[0228] Analysis software: Wyatt Technology Astra version 6.1.1.17

[0229] Column: Acquity UPLC BEH200 SEC, 4.6×150mm, 1.7μm

[0230] Mobile phase: Dulbecco's 1xPBS containing 0.05% sodium azide

[0231] Sample preparation: Filtered through PES 0.22μm

[0232] Sample concentration: variable injection volume: 20μl

[0233] Analysis temperature: 30℃

[0234] Flow rate: 0.4ml / min

[0235] Cross-interaction chromatography (CIC)

[0236] Cross-interaction chromatography (CIC) analysis was performed to assess the propensity for non-specific protein-protein interactions and provide an indication of any solubility issues that could lead to downstream manufacturing problems.

[0237] Samples were analyzed by two different 20 μl injections (0.5 mg / ml); the first injection was onto 1 ml NHS activated resin (GE Healthcare) coupled to 30 mg human polyclonal IgG (Sigma 14506), and the second injection was onto a blank coupled 1 ml NHS activated resin as a control column. The mobile phase consisted of Dulbecco's PBS (Sigma D8537) containing 0.01% sodium azide (0.1 ml / min), and all experiments were performed at 25°C. The eluted samples were detected by UV absorbance (Agilent 1260 Infinity HPLC system with a thermostatted column oven), and the data were analyzed using Wyatt Technology ASTRA software (version 6.1.2.83) to determine the sample peak retention times. These were then used to calculate the retention factor k':

[0238]

[0239] Where Tr is the retention time of the sample on the poly-IgG column, and Tm is the retention time on the blank (control) column.

[0240] Surface Plasmon Resonance (SPR): Lewis Y HSA conjugate was coupled to CM5 fragment, and FG27.10 mAb and FG27.18 mAb (concentration range 0.01 μM to 1 μM) were injected at 30 μl / min. Binding data were fitted to a heterogeneous ligand model using BIA evaluation software.

[0241] Antibody internalization: Cancer cells were injected at a rate of 2×10 3Cells were plated at a density of 10 cells / well and allowed to adhere overnight. Cells were cultured in 96-well plates at 37°C for 72 hours and treated with the specified concentrations of mAbs in combination with the following secondary antibodies: saporin-conjugated goat anti-mouse (ATSbio, IT#48) or human IgG antibodies (F'ab fragments). Control wells consisted of cells incubated with culture medium alone and with the second conjugate alone. The amount of secondary antibody per well was 75ng, and the concentration of primary antibody varied from 0.01nM to 30nM. Saporin internalization was assessed by inhibiting thymidine uptake (thymidine was added in the last 24 hours of incubation).

[0242] ADCC and CDC: cells (5×10 3 ) were incubated with 100 μl PBMC, 10% autologous serum or culture medium alone, or with a range of mAb concentrations. Natural release and maximum release were assessed by incubating the labeled cells with culture medium alone or with 10% Triton X-100. After 4 hours of incubation, 50 μl of supernatant was transferred from each well to a 96-well lumaplate. The plates were dried overnight and counted on a Topcount NXT counter (PerkinElmer, Cambridge, UK). The average percentage of target cell lysis was calculated according to the following formula:

[0243]

[0244] PI uptake assay: FG27.10 and FG27.18 were incubated with AGS cells and tested for uptake of different concentrations of the small molecular weight dye propidium iodide (PI, Sigma); the number of PI-positive cells represents the number of dying / dead cells. 4 ) were incubated with 50 μl of primary antibody in 96-well round-bottom microtiter plates for 2 hours at room temperature. 1 μg of PI was added and the cells were incubated for 30 minutes at RT. Samples were analyzed on an FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot raw data. For comparison, mAbs SC104 and 791T / 36, which are known to induce membrane damage, were also included as internal controls for the experiment.

[0245] Proliferation inhibition assay: The proliferation inhibition of the constructs was evaluated by measuring the activity of cellular hydrogenases, which is proportional to the number of viable cells, using the water-soluble tetrazolium salt WST-8 (CCK8 kit, Sigma-Aldrich). Briefly, after overnight plating of cancer cells (2000 cells / 90 μl / well), different concentrations of the constructs were added in a final volume of 10 μl / well, and the plates were incubated at 37°C (5% CO2) for 72 to 96 hours. WST-8 reagent (10 μl / well) was then added, and after an additional 3 hours of incubation, the plates were read at 450 nm (Tecan Infinite F50) and the percentage of inhibition was calculated. EC was determined using nonlinear regression (curve fitting) using GraphPad Prism v 8.0 (GraphPad Inc, La Jolla, CA). 50 value.

[0246] In vivo models: These studies were conducted under a UK Home Office Licence. The NCRI Cancer Research Animal Welfare and Use Guidelines, LASA Good Practice Guidelines and FELAS Working Group Pain and Distress Guidelines were also followed. Endotoxin-free (<10 EU / ml) FG27 mAb was provided in pre-formulated aliquots ready for administration and stored at -20°C until use. Age-matched male MF-1 nude mice were obtained from Harlan Laboratories (Bichester, UK), and each group of FG27, control mAb or vehicle control consisted of n≥8 animals.

[0247] C170HM2 DLuX cells were implanted into mice and monitored by optical imaging to determine tumor formation and suitability for entry into the study. Mice were administered FG27 or positive control mAb (505 / 4, FG88.2 or FG88.7 (1 mg / ml), as appropriate): 0.1 mg, twice a week, 100 μl intravenous (iv) until termination; or PBS, mAb vehicle control: 100 μl, twice a week, iv, until termination. Bioluminescence imaging was performed on all mice weekly to obtain tumor measurements before and after dosing. In this way, each mouse provided a pre-dose control reading against which tumor growth could be compared.

[0248] All measurements and readings were converted from the original dictation / annotation into excel (tabdelineated) format for data processing in SPSS v16.0. Data integrity was checked using the explore and describe functions. Erroneous points were cross-referenced with the original data when identified and corrected accordingly. Data were screened for outliers and distribution profiles; data points (outliers) outside the 95% confidence limits were removed from the analysis but retained in the data table for reference.

[0249] During the study, mice were imaged weekly for bioluminescent tumor burden (BLI) as follows; 60 mg / kg of D-luciferin substrate was administered subcutaneously (sc), mice were anesthetized and BLI readings were obtained 15 minutes after substrate administration on an open filter block (2D) and sequential emission filters (for DLIT, 3D reconstruction). Ventral and dorsal imaging was performed; the optimal position for imaging was the uppermost abdomen. BLI was measured over the entire abdominal area, with one Region of Interest (ROI) per mouse to include all lesions present. Pre-dose or baseline images were taken for each mouse to allow calculation of the percentage of tumor growth over time; these data are averages for each group. BLI readings were also obtained after termination to identify tumors in PM tissue.

[0250] Example 1 - Generation and initial characterization of FG27 mAb:

[0251] FG27 is generated by immunization of gastric tumor cells with glycolipids

[0252] Analysis of antibody response to immunity: antibody titer was initially monitored by lipid enzyme-linked immunosorbent assay (ELISA). Subsequently, thin layer chromatography (TLC) analysis was performed using ST16 total lipids and plasma membrane lipid extracts, flow cytometry analysis (FACS) was performed using ST16 tumor cells, and western blotting was performed using ST16 whole cell extracts, total lipids, and plasma membrane lipid extracts. Mice that were considered to have the best response compared to serum controls before bleeding were strengthened intravenously (iv) with ST16 plasma membrane lipid extracts before fusion.

[0253] The binding of FG27 hybridoma supernatants to a panel of tumor cell lines was analyzed by direct immunofluorescence and FACS analysis. Both FG27.10 and FG27.18 bound to ST16, but not to human umbilical vein endothelial cells (HUVEC) or peripheral blood mononuclear cells (PBMC) ( Figure 4 ). Both FG27.10 and FG27.18 were cloned. FG27.10 is of the IgG3κ subclass and FG27.18 is of the IgG1κ subclass. To ensure that both mAbs bound to the glycolipids, glycolipids were extracted from a range of cell lines, dried onto ELISA plates, and then incubated with FG27.10 and FG27.18. Binding of glycolipids from C170, ST16, and AGS to both mAbs was observed, whereas glycolipids from cell lines that showed a lack of binding to whole cells (MKN-45, OAW28, OVCAR-3, OVCAR-4, and OAW42) did not bind to either mAb ( Figure 5 ).

[0254] Example 2—Definition of the epitope recognized by FG27 mAb

[0255] To determine the degree of specificity of FG27.10, the antibody was first evaluated by ELISA for Leuconostoc conjugated to HSA. a ,Le b ,Le x , and Le y FG27.10 failed to recognize Le a ,Le a-x or Le b , but strongly identifies Le y ( Figure 6 ). To further elucidate the fine specificity of FG27 mAb, a screening was performed against ≥600 natural and synthetic glycans by the Functional Glycomics Consortium. Binding of FG27.10 mAb and FG27.18 mAb to the glycan array showed that both mAbs bound to Le y Specific binding ( Figure 7 ). As a comparison, the binding of 692 / 29 indicated that mAb and Le b , containing Le b polysaccharides and tri-Le y In contrast, BR96 showed the strongest binding to Le y And a series of Le yVariant (Le y-x ,Le y-x-x ) combined with Le x The binding is weaker.

[0256] FG27.10 and FG27.18 were also screened for their ability to recognize glycoproteins and glycolipids by SDS / PAGE / Western blotting on a panel of tumor cell lines ( Figure 8 In addition to binding glycolipids (as evidenced by binding at the dye front), both mAbs also recognized a range of glycoproteins.

[0257] Since FG27 is Le y specific, thus binding to the cell line ( Fig. 9 ) and identification of Le y and Le x mAbBR96 and recognizes Le y and Le b All three mAbs bound to C170 cells and AGS cells. In contrast, BR96 strongly bound to Colo201, 692 / 29 moderately bound, and FG27 failed to bind ( Fig. 9 ). This indicates that this cell line does not express Le y , but may express Le x and Le b , a conclusion that could not be drawn using nonspecific mAbs. HT29 cells failed to bind any mAb, whereas FG27.10 bound best to OVCAR3 and ST16, and 692 / 29 bound best to LoVo cells.

[0258] Example 3 - Immunohistochemical evaluation of FG27

[0259] To evaluate the binding of FG27 to normal human tissues, a panel of tissue types was screened. y / b Cross-reactive mAbs (692 / 29) and Le y / x The cross-reactive mAb (BR96), FG27, only bound to a subset of cells in the normal stomach, lung, tonsil, pancreas, and duodenum (Table 1). y Compared to the cross-reactive mAb, FG27 failed to stain the colon, jejunum, breast, kidney, or ileum. Representative IHC images of normal staining of the esophagus, stomach, duodenum, ileum, colon, pancreas, lung, and kidney are shown. Fig.10 shown.

[0260] Table 1: the combination of FG27 and normal human tissues assessed by immunohistochemistry (IHC). The staining of these tissue microarrays (Tissue microarray, TMA) was analyzed by new viewer software 2010, and semi-quantitative scoring was given according to the staining intensity of tumor tissue. Strong staining was recorded as 3 points, moderate staining was recorded as 2 points, weak staining was recorded as 1 point, and negative was recorded as 0 points. The results also demonstrated the differential staining of specific cell types in these tissues.

[0261] Organization Type <![CDATA[692 / 29(Le y / b )]]> <![CDATA[BR96(Le y / x )]]> <![CDATA[FG27(Le y )]]> placenta 0 2 0 esophagus 0 3 0 rectum 0 1 0 gallbladder 1 2 0 skin 0 0 0 Fat 0 0 0 heart 0 0 0 skeleton 0 0 0 bladder 0 0 0 Ileum 2 3 0 spleen 0 0 0 brain 0 0 0 jejunum 3 3 0 Stomach 2 3 0 / 2 Breast 0 3 0 kidney 0 1 0 testis 0 0 0 Cerebellum 0 0 0 liver 0 0 0 Thymus 1 0 0 cervix 0 0 0 lung 1 3 0 / 1 smooth muscle 0 0 0 colon 1 1 0 Ovaries 0 0 0 tonsil 2 3 0 / 1 Diaphragm 0 0 0 pancreas 1 3 0 / 2 Uterus 0 0 0 Duodenum 1 3 0 / 2 thyroid 0 0 0

[0262] To determine the therapeutic value of FG27, human gastric, colorectal, ovarian, and breast TMAs were screened by IHC. It stained 80% of gastric (69 / 86), 100% of colorectal (55 / 55), 95% of ovarian (36 / 38), and 85% of breast (39 / 46) tumor tissues with varying intensities (Table 2).

[0263] Table 2: Binding of FG27 mAb to human gastric, colorectal, ovarian and breast tumor tissues assessed by immunohistochemistry. The staining of these tissue microarrays was analyzed by the Novel Viewer software 2010 and a semi-quantitative score was given based on the intensity of staining of the tumor tissues. Strong staining was scored 3 points, moderate staining was scored 2 points, weak staining was scored 1 point, and negative was scored 0 points.

[0264]

[0265] The scores showed that FG27 was widely associated with gastric tumors, with 59% of tumors positively stained and 18% of tumors strongly stained. 19% of tumors were moderately stained, and 22% of tumors were weakly stained. The results showed that FG27 has the potential to target a variety of gastric tumors and is tightly associated with 18% of them. Kaplan Meier analysis showed no association between the expression of the FG27 antigen and survival (data not shown). However, there was a correlation between FG27 antigen expression and M30 (apoptosis marker) expression (Pearson chi-square p = 0.049). There was also a correlation between FG27 antigen expression and tumor site (Pearson chi-square p = 0.008), of which 65% of gastric tumors were specifically stained with FG27, of which 26% were strongly stained. In contrast, only 53% of tumors in the gastroesophageal junction area were stained, and 37% of them were strongly stained. 46% of tumors in the lower third of the esophagus were stained with FG27, of which 53% were strongly stained.

[0266] Due to the cross-reactivity of FG27 with ovarian cancer cell lines, an ovarian cancer TMA with 360 tumors was screened by immunohistochemistry. Scoring showed that FG27.18 bound poorly to ovarian tumors, with 85% of tumors showing negative staining. Only 15% of tumors showed positive staining, and only 3% showed strong staining. 3% of tumors showed moderate staining, and 9% of tumors showed weak staining.

[0267] Kaplan Meier survival analysis showed that patients whose tumors expressed the FG27 antigen had significantly (log-rank p = 0.049) better survival than those who did not express it. There was a correlation between FG27 antigen expression and FIGO stage (Pearson chi-square p = 0.024). There was also a correlation between FG27 antigen expression and ovarian cancer type (Pearson chi-square p = 0.024), with more than one-third of mucinous and endometrioid tumors expressing FG27, while less than 14% of serous clear cells and undifferentiated tumors were stained by this mAb. Cox multivariate analysis showed that FG27 did not provide any further prognostic value for FIGO stage, residual disease, and chemotherapy response.

[0268] Table 3. Comparison of tumor staining by Lewis y-specific mab FG27 and other Lewis y-cross-reactive mabs BR96 (Lewisy / x) and 692 / 29 (Lewisy / b).

[0269]

[0270] The monospecific Lewis y mab FG27.10 stained 3% to 39% more tumors than Lewis y / b 692 / 29mab and 6% to 48% more tumors than Lewis y / x Br96mab, reflecting its unique specificity. The greatest changes were seen in ovarian and breast tumors.

[0271] Unlike FG27, which showed only moderate binding to normal stomach, pancreas and duodenum in one of two samples, and weak binding to lung and tonsil in one of two samples, BR96 showed strong binding to many tissues, including esophagus, ileum, jejunum, stomach, breast, lung, tonsil, pancreas and duodenum. BR96 also showed moderate binding to placenta and gallbladder, and weak binding to rectum, kidney and colon. In contrast, 692 / 29 strongly bound to jejunum, moderately bound to ileum, stomach and tonsil, and weakly bound to gallbladder, thymus, lung, colon, pancreas and duodenum (Table 1). This limited normal binding spectrum of FG27 (especially with respect to gastrointestinal tissue) did not reduce the binding of the antibody to tumor tissue, with 80% of gastric tumor tissue and 100% of colorectal tumor tissue showing antigen expression. In contrast, 692 / 29 and BR96 showed good distribution on colorectal tumors, binding to 82% and 90% respectively (Noble, Spendlove et al. 2013), and for the latter, similar numbers, i.e. 15 / 18 samples (i.e. 83%), were also reported in US 5491088. 39% and 49% of these tumors were moderately or strongly stained, compared to 85% for FG27. 2-25LE showed a poorer distribution, binding to only 52% of colorectal tumors, of which 19% were moderately stained and no tumors were strongly stained (Noble, Spendlove et al. 2013).

[0272] Thus, it was demonstrated that FG27 had a significantly better normal binding profile (especially with respect to gastrointestinal tissues), and each Le y The differential binding between the mAbs demonstrates the unique properties of the present invention.

[0273] Example 4 - In vivo antitumor activity of FG27 in an established colorectal metastatic xenograft model

[0274] Comparison of therapeutic efficacy of mAb FG27 in the C170HM2 DLuX human liver metastasis model: The mouse C170HM2DLuX human liver metastasis model was used to study the antitumor activity of murine FG27 mAb. The C170HM2 DLuX cell line is a bioluminescent variant of a liver metastatic colon tumor cell line that subcultures into the liver parenchyma when implanted into the peritoneal cavity and metastasizes to the living body 10 days before the start of mAb treatment. The growth and distribution / location of such labeled cells and tissues can be assessed non-invasively in real time in a suitable optical imaging system, as well as in resected tissues post mortem (PM).

[0275] Antitumor data: As assessed by bioluminescence intensity, twice weekly administration of FG27 (100 μg, iv) reduced peritoneal cavity and associated tumor growth compared to vehicle control ( Fig.11a The study was terminated on day 32. Treatment with FG27 resulted in a significant reduction in bioluminescent tumor burden by the last day of the study compared to vehicle controls (p = 0.014). Treatment with FG27 significantly reduced the growth of tumors at the injection site (p = 0.05; Fig.11b ).

[0276] In a second in vivo study with extended treatment duration, FG27 demonstrated a significant reduction in the percentage of tumor growth by day 51 compared to the treatment control group (p=0.019) ( Fig.12a ). Analysis by log-rank Mantel-Cox test showed that the survival rate of the FG27 (p = 0.0251) treated group was significantly improved compared with the vehicle only control ( Figure 12b ).

[0277] Example 5—Chimeric mAbs

[0278] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. The chimeric (or humanized) antibody of the present invention can be prepared based on the sequence of the mouse mAb prepared as described above. When the gene and amino acid sequence of the antibody were analyzed, it was shown that FG27.10 (Fig. 1) and FG27.18 (Fig. 2) had the same variable region, but had different constant regions IgG3 and IgG1, respectively. This indicates that subclass switching and then amplification occurred in a single B cell during the immunization protocol, and both clones were successfully fused.

[0279] Compared to the closest germline sequence, FG27 contains 7 mutations in its heavy chain (all non-silent) and 3 mutations in its light chain (all non-silent). Four of the amino acid differences from germline are in the heavy chain CDRs and three are in FR3. In CDR1, the amino acid at position 36 is changed from D to H. In CDR2, the amino acids at positions 59 and 63 are changed from G to D, and the amino acid at position 64 is changed from S to N ( Figure 2a ). For the light chain, there are 2 amino acid differences in the CDR1 germline, S to I at position 28 and S to T at position 32, and 1 amino acid difference in FR2, namely the only K51N.

[0280] There are several other mAbs that bind Lewis y but cross-react with other related Lewis antigens. FG27.10 / 18 is the only antibody with monospecificity for Lewis y. This is reflected in the similar but distinct sequences of the FG27 variable heavy and light chain regions ( Fig.13a , Fig.13b Compared to other cross-reactive Lewis Y mAbs, FG27 showed 16%-68% differences in amino acid sequence CDR within the heavy chain, 4.3%-16% differences in amino acid sequence CDR in the light chain, 8.6%-49.5% differences in amino acid sequence of the heavy chain variable region, and 2%-24% differences in amino acid sequence of the light chain variable region (Table 4). The greatest variation was within CDRH2, where 3 to 5 of the 8 amino acids varied in different mAbs.

[0281] Table 4. Amino acid sequence differences between FG27 and anti-Lewis Y cross-reactive mAbs in the heavy chain variable region and the light chain variable region.

[0282]

[0283] The FG27.18 heavy and light chain variable regions were cloned into human IgG1 and IgG2 expression vectors. The expression vectors were transfected into CHO-S cells and the human antibodies were purified on protein G. The chimeric mAbs were expressed in Leuconostoc cells, including the gastric cell line AGS. y Combination of tumor cell lines ( Fig.14 ).

[0284] Example 6—Direct Cell Killing

[0285] Many anti-glycan mAbs have been shown to directly induce cell death in antigen-positive cell lines without the need for effector cells or complement. This could potentially enhance their efficacy in vivo, as tumors may develop mechanisms to avoid immune-mediated cell death. This ability is primarily associated with glycan-binding mAbs of the mIgG3 isotype, and this property is therefore lost upon chimerization or humanization.

[0286] To determine whether FG27.10 and FG27.18 have the ability to directly cause cell death, ST16 cells were incubated with FG27.10, FG27.18, and anti-sialic acid Le a mAb SC104 was incubated for 2 h at room temperature, and cell death was then measured by uptake of propidium iodide, a DNA intercalator that is only taken up by dying cells ( Fig.15a Interestingly, despite sharing the same variable region, only FG27.10 was able to directly induce cell death, whereas no PI uptake was observed for FG27.18. Fig.15b Direct killing of a range of antigen positive (AGS, Colo201, C170, C170HM, MCF-7, OVCAR4) cell lines was shown, with both mAbs showing varying amounts of killing. Neither mAb showed killing of antigen negative cell lines (LoVo, MKN45, 791T, SKOV3).

[0287] Fig.15c PI uptake in the colorectal cell line C170 by a range of mAbs is shown. FG27.10 and the positive control antibody SC101 showed strong titratable killing. FG27.18 showed weak non-titratable killing. The human IgG1 chimera showed moderate titratable killing, while the IgG2 variant did not.

[0288] Example 6 - Internalization

[0289] The internalization of FG27.10, FG27.18, and CH27 was evaluated using various tumor cell lines ( Fig.16 FG27.18 exhibited dose-dependent internalization in most cell lines tested, with cell viability reductions ranging from 60% (C170) to as high as 75% (MCF7) and 85% (AGS) at the highest concentration tested. FG27.10 exhibited the best internalization against AGS (up to 50% reduction in viability) and MCF7 (up to 70% reduction in viability). Internalization was more variable in the H322 cell line for both murine mAbs. The CH27 mAb was efficiently internalized by MCF7, with up to 75% reduction in cell viability observed, and by H322 with approximately 40% reduction in cell viability.

[0290] Example 7—In vitro antitumor activity of FG27

[0291] ADCC and CDC: Murine and chimeric FG27 mAbs were screened for their ability to induce tumor cell death via ADCC. Human PBMCs were used as a source of effector cells, while a panel of tumor cells were used as target cells. ADCC analysis was initially performed using FG27.10 IgG3 supernatant ( Fig.17a The extent of ADCC observed corresponded to the expression of Lewis Y antigen, such that ADCC was observed in ST16 and OVCAR3, only low levels were observed with C170, and only background was observed with LoVo, HT29, or Colo201 ( Fig.17a After incubation at 37°C for 18 hours, the ADCC killing degree of mouse FG27.10 (mIgG3), FG27.18 (mIgG1), CH27 IgG1 and IgG2 on MCF7 and OVCAR3 target cells was measured and compared; CH27 IgG1 had the highest killing level on cell lines MCF7 and OVCAR3 ( Fig.17b ).

[0292] CDC is known to be an important mechanism involved in the elimination of tumor cells in vivo. The ability of CDC induced by mAbs FG27.10, FG27.18, CH27 IgG1, and CH27 IgG2 to kill ST16 cells was determined in the presence or absence of 37°C human serum as a complement source for 18 hours. Fig.17c As shown, among the FG27 mAbs, FG27.10 (mIgG3) produced the highest level of killing at 3 μg / ml, while CH27 IgG2 did not produce significant killing, which is expected given the properties of IgG2 antibodies. Similarly, when using AGS cells, FG27.10 produced the highest level of killing, followed by CH27 IgG1. Again, no killing was seen with CH27 IgG2 ( Fig.17d ).

[0293] In conclusion, FG27 mAb strongly induced ADCC using human PBMCs as effector cells and significantly induced CDC using human serum as a complement source.

[0294] Example 9— Generation of a humanized version of the FG27 antibody

[0295] Human VH and VK cDNA database

[0296] The human immunoglobulin sequence database in the form of Kabat alignment was compiled using protein sequences of human and mouse immunoglobulins from the International Immunogenetics Database 2009 (Lefranc 307-10) and the Kabat Database Release 5 of Sequences of Proteins of Immunological interest (last updated November 17, 1999) (Kabat et al. 1-3242). Our database contains 10906 VH sequences and 2912 VK sequences.

[0297] FG27 frame selection

[0298] Humanization requires identification of appropriate human V regions. The sequence analysis program Gibbs was used to query the human VH and VK databases with FG27 VH and VK protein sequences using various selection criteria. Using the program Discovery Studio (Accelrys), the CDR residues in the mouse FG27 antibody structure were identified. The FW residues in Neighboring residues". Fig.19 Shows The human VH sequence with the highest identity to FG27 VH in the neighboring residues, as well as a list of these envelope residues and VCI, and FW, VCI or The number of residues in the neighborhood that have identity with the mouse equivalent position. Fig.18 Shows Fig.19 Alignment and residue identity of all VH sequences in . The number of significant somatic mutations in each FW identified by comparison with the most identical human germline V gene is shown in Fig. 20 middle.

[0299] Humanized sequences, murine sequences, and incomplete sequences were removed from the analysis. Sequence AJ579110 was selected as a human donor candidate due to its higher sequence identity and similarity. It has no somatic mutations different from germline ABO19439 or HM855436.

[0300] Design of FG27 RHA and FG27 RHB

[0301] Since a suitable human framework had been identified, synthetic protein and DNA sequences could be designed. The initial design of the humanized version of FG27 was to graft CDR 1, CDR 2, and CDR 3 from FG27 VH into the acceptor FW of AJ579110. A The adjacent residues were not conserved in FG27 RHA and were backmutated to the mouse equivalent residues to generate the humanized version, FG27RHB. Fig.21 The murine and humanized versions of the FG27 VH protein sequence were compared.

[0302] FG27 RKA Frame Selection

[0303] To humanize the light chain, a human kappa chain was identified in a similar process to that of the heavy chain. Fig. 22 , Fig.23 shows the FW or The number of residues with identity to the mouse equivalent position in the neighboring residues, the alignment of the VK sequence, and the comparison to the human germline. Initial analysis identified several potential donor candidates, but all of them turned out to be poorly expressed human VK4. Extending the analysis to include CDR1 with one less residue resulted in a single candidate, X72449, which had higher sequence homology to the murine antibody than the VK4 candidate. This candidate had a single somatic mutation that differed from the human VK2 germline, but no somatic mutations that differed from the human VK2D germline ( Fig.24 ).

[0304] Design of FG27 RKA and FG27 RKB

[0305] The framework from X72449 was used to design DNA and proteins for humanized constructs. CDR 1, CDR 2, and CDR 3 from FG27VK were grafted into the acceptor FW of X72449 to generate the initial version of humanized FG27. There is a single mismatch in FG27RKA Adjacent residue 5, which was backmutated to the equivalent mouse residue in variant FG27 RKB ( Fig.25 ).

[0306] Generation and characterization of a humanized version of FG27

[0307] Generation of FG27 humanized antibody

[0308] The genes of FG27 RHA and FG27 RKC were synthesized by Genewiz and codon-optimized for human sequences ( Figure 3a , Figure 3b ). The native human framework sequences M65092 and X72449 for the heavy and light chains, respectively, and the native mouse CDR sequences were assembled in silico and named FG27 RHA to FG27 RHB and FG27 RKA to FG27 RKD. The sequences were optimized by silent mutagenesis to use codons preferentially used by human cells and synthesized using Genewiz proprietary software algorithms. The RHA / B and RKA / B constructs were PCR amplified with specific primers for the expression vector + insert (as described above for the chimeric version) and inserted into pHuG1 and pHuK in a ligase-independent cloning reaction and used to transform TOP10 bacteria. RKA and RHA were then modified by PCR mutagenesis to obtain Fig.21 and Fig.25 All the variants of the annotations in .

[0309] The clones were sequenced and expression plasmid DNA was prepared using the QIAGEN Plasmid Miniprep Kit or the Qiagen Plasmid Maxiprep kit. The expression construct sequences (RHA, RHB, RKA and RKB) are shown in Fig.21 and Fig.25 Expi293 cells were transfected with expression plasmid preparations encoding (humanized or chimeric) VH and VK, cultured in serum-free medium for 10 days, and the conditioned medium containing the secreted antibodies was harvested.

[0310] Antibody expression

[0311] The concentration of IgG1κ antibodies in the conditioned medium of Expi293 cells was measured by ELISA and is shown in Table 5. Most antibodies were produced at satisfactory levels, except for those highlighted in red.

[0312] Table 5. IgG levels in conditioned medium of transfected Expi293 cells

[0313]

[0314] FG27 antibody binds to Lewis Y-HSA

[0315] Binding activity to Lewis Y-HSA antigen was measured by binding ELISA. Initial experiments showed that there was essentially no difference in binding potency between the mouse antibody or the chimeric antibody. This result gave us some confidence that the humanization process was successful.

[0316] Fig.26 The data shown in show the binding potency of the initial versions of humanized FG27. Although all versions bind to Lewis Y-HSA. Antibody versions consisting of RHA / RKB, RHA / RKC, RHA / RKD, and RHB / RKC were selected as possible lead candidates.

[0317] Thermal stability of humanized antibody candidates to high temperatures

[0318] The purpose of this experiment was to compare the thermal stability of humanized antibodies. When subjected to higher temperatures, ranging from 30°C to 85°C for 10 minutes, cooled to 4°C, and subjected to ELISA at the EC80 concentration of each candidate. All antibodies tested appeared to be equally stable ( Fig. 27 ), become inactive at >72°C.

[0319] Determination of Tm of humanized candidate antibodies

[0320] To determine the melting temperatures of chimeric and humanized lead antibodies RHA / RKB, RHA / RKC, RHA / RKD, and RHB / RKC, all antibodies were purified in a 2-step affinity chromatography and gel filtration system and tested in a thermal shift assay. The samples were incubated with a fluorescent dye (Sypro Orange) in a qPCR thermal cycler for 71 cycles with an increase of 1°C per cycle. Fig.28 The Tm of the chimeric antibody and four humanized antibodies are shown. All candidate antibodies and the chimeric antibody had the same Tm (72°C).

[0321] Affinity of humanized antibody candidates

[0322] Antibody affinity determination using SPR analysis was performed using Biacore T3000. Lewis Y-HSA was covalently fixed on a CM5 chip (Catalog No.: BR-1005-30) according to the manufacturer's instructions. Chimeric FG27, RHA / RKB, RHA / RKC, RHA / RKD, and RHB / RKC antibodies were diluted in buffer to a concentration range of 200 nM to 0.274 nM (serial 3-fold dilutions) and then injected at a flow rate of 50 μl / min. The association time was set to 120 seconds and the disassociation time was set to 600 seconds.

[0323] The binding / dissociation kinetics were analyzed based on the best fit to the heterogeneous ligand model using the BIAcore 3000 evaluation software package. The sensorgram shows an overlay of the 200 nM binding curve ( Fig.29 RHA / RKB, RHA / RKC, and RHB / RKC showed similar affinities to chimeric FG27 (RHA / RKB>RHA / RKC>RHB / RKC>>RHA / RKD). These results indicate that humanization successfully retained binding activity within the desired parameters, with RHA / RKB or RHA / RKC having the highest affinity.

[0324] Aggregation of humanized antibody candidates

[0325] The samples were injected into a size exclusion column in the HPLC system at 0.4 ml / min and analyzed by multi-angle light scattering to determine the absolute molar mass and check for aggregation. All variants showed no signs of aggregation, with an average molecular weight range of 140.19 kDa, which is the expected range for IgG monomers in this analytical setting. All samples were monodisperse (Mw / Mn < 1.05). The mass recovery (ratio of calculated mass to injected mass) was 100%, indicating good protein recovery and that the samples did not appear to stick to the column or contain insoluble aggregates that would be retained by the guard column. Overall, the data indicate that there were no aggregation issues in any of the anti-FG27 antibody samples analyzed.

[0326] Nonspecific protein-protein interactions (CIC)

[0327] Cross-interaction chromatography using large amounts of purified human polyclonal IgG is a technique that monitors nonspecific protein-protein interactions and can be used to distinguish soluble from insoluble antibodies. An increased retention index (k') indicates a tendency for self-interaction and low solubility. The retention index for RHA / RKB, RHA / RKC, and RHB / RKC antibodies was less than 0.044, indicating a low tendency for nonspecific interactions and good solubility ( Fig.30 ).

[0328] The antibody was engineered and expressed as a fully humanized antibody without losing Lewis Y HSA binding potency ( Fig.26 and Fig.29 ). Experiments with chimeric antibodies consisting of mouse variable regions attached to human constant regions showed similar potency to mouse antibodies in the Lewis Y HSA binding ELISA. This result gave some confidence in the positive outcome of the humanization process.

[0329] Initial experiments showed that fully humanized heavy and light chains (i.e., no framework mutations to introduce mouse 4A-adjacent residues) did not bind to the Lewis YHSA peptide as well as the chimeric positive control antibody, but versions with a single back mutation bound in an equivalent manner to the chimeric positive control. Assays measuring antibody solubility, thermal stability, and aggregation showed that all humanized candidates had acceptable biophysical properties.

[0330] In our opinion, excellent binding, expression, thermal stability, affinity, and cell binding make the RHA heavy chain version, together with the RKC light chain version, the best candidate antibodies for further development.

[0331] Example 10—FG27 Binding Studies

[0332] The humanized mAb (humanized 27) and chimeric mAb (CH27) showed similar cell binding patterns in titrations against cancer cell lines ( Fig.31 ). The equilibrium affinity constants were generally of the same order of magnitude, but slightly higher than the parental murine antibody, while the maximum binding capacity of humanized 27 and CH27 was greater than that of the murine mAb. This result indicates that the outcome of the humanization process was successful.

[0333] Example 11—Direct killing by humanized mAbs

[0334] To enhance direct cell killing of human IgG1 27 chimera (27hIgG1), we transferred selected mIgG3 constant region residues into the hIgG1 Fc domain, generating an improved 'i27G1' LewisY glycan-binding mAb with improved in vitro direct cell killing ability. Fig.33 The amino acid and nucleic acid sequences of each of the light and heavy chains of the 'i27G1' antibody are shown. This antibody forms a further aspect of the invention.

[0335] Fig.32 Improved direct cell killing, functional affinity, and effector function of i27G1 were shown. y Our i27G1, which contains selected mIgG3 constant region residues, showed significantly improved direct cell killing on AGS(i) and MCF7(ii) cells compared to 27hIgG1 and was equivalent to 27mG3 mAb. In addition, i27G1 exhibited significantly improved functional Lewis antigen binding activity compared to 27hIgG1(iii). y Affinity. Importantly, in MCF7, the effector functions ADCC and CDC of i27G1 were equivalent to those of 27hIgG1 (iv and v). Overall, the results suggest that the transfer of selected regions from the mIgG3 constant region into the 27hIgG1 backbone generated a hybrid mAb with direct cell killing ability, increased functional affinity, and potent immune effector functions.

[0336] References

[0337] Altschul, SF, W. Gish, W. Miller, EW Myers and DJ Lipman (1990). "Basiclocal alignment search tool." JMol Biol 215(3):403-410.

[0338] Altschul, SF, TL Madden, AASchaffer, J. Zhang, Z. Zhang, W. Miller and D. J. Lipman (1997). "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs." Nucleic Acids Res 25(17):3389-3402.

[0339] Alvarez-Rueda,N.,S.Leprieur,B.Clemenceau,S.Supiot,V.Sebille-Rivain,A.Faivre-Chauvet,F.Davodeau,F.Paris,J.Barbet,J.Aubry and S.Birkle(2007)."Binding activities and antitumor properties of a new mouse / human chimericantibody specific for GD2 ganglioside antigen." Clin Cancer Res 13(18Pt 2):5613s-5620s.

[0340] Ausubel,F.(1992). Short protocols in molecular biology ,John Wiley&Sons.

[0341] Barbas,C.F.,3rd,D.Hu,N.Dunlop,L.Sawyer,D.Cababa,R.M.Hendry,P.L.Naraand D.R.

[0342] Azinovic,I.,G.L.DeNardo,K.R.Lamborn,G.Mirick,D.Goldstein,B.M.Bradt,and S.J.

[0343] DeNardo.2006.'Survival benefit associated with human anti-mouseantibody(HAMA)in patients with B-cell malignancies',Cancer ImmunolImmunother,55:1451-8.

[0344] Burton(1994)."In vitro evolution of a neutralizing human antibody tohuman immunodeficiency virus type 1to enhance affinity and broaden straincross-reactivity." Proc Natl Acad Sci USA 91(9):3809-3813.

[0345] Bird,R.E.,K.D.Hardman,J.W.Jacobson,S.Johnson,B.M.Kaufman,S.M.Lee,T.Lee,S.H.Pope,G.S.

[0346] Riordan and M.Whitlow(1988)."Single-chain antigen-binding proteins." Science 242(4877):423-426.

[0347] Bodanzsky,M.and A.Bodanzsky(1984). The practice of peptide synthesis .New York,Springer Verlag.

[0348] Chou,H.H.,H.Takematsu,S.Diaz,J.Iber,E.Nickerson,K.L.Wright,E.A.Muchmore,D.L.Nelson,S.T.Warren and A.Varki(1998)."A mutation in humanCMP-sialic acid hydroxylase occurred after the Homo-Pan divergence." Proc Natl Acad Sci USA 95(20):11751-11756.

[0349] D'Arcy,C.A.,and M.Mannik.2001.'Serum sickness secondary to treatmentwith the murine-human chimeric antibody IDEC-C2B8(rituximab)',ArthritisRheum,44:1717-8.

[0350] Durrant,L.G.,P.Noble and I.Spendlove(2012)."Immunology in the clinicreview series;focus on cancer:glycolipids as targets for tumourimmunotherapy." Clin Exp Immunol 167(2):206-215.

[0351] Eppstein,D.A.,Y.V.Marsh,M.van der Pas,P.L.Felgner and A.B.Schreiber(1985)."Biological activity of liposome-encapsulated murine interferon gammais mediated by a cell membrane receptor." Proc Natl Acad Sci USA 82(11):3688-3692.

[0352] Furukawa,K.,S.Welt,B.W.Yin,H.J.Feickert,T.Takahashi,R.Ueda andK.O.Lloyd(1990)."Analysis of the fine specificities of 11mouse monoclonalantibodies reactive with type 2blood group determinants." Mol Immunol 27(8):723-732.

[0353] Gram,H.,L.A.Marconi,C.F.Barbas,3rd,T.A.Collet,R.A.Lerner and A.S.Kang(1992)."In vitro selection and affinity maturation of antibodies from a naivecombinatorial immunoglobulin library." Proc Natl Acad Sci USA 89(8):3576-3580.

[0354] Hellstrom,I.,H.J.Garrigues,U.Garrigues and K.E.Hellstrom(1990)."Highly tumor-reactive,internalizing,mouse monoclonal antibodies to Le(y)-related cell surface antigens." Cancer Res 50(7):2183-2190.

[0355] Holliger,P.,T.Prospero and G.Winter(1993).""Diabodies":small bivalentand bispecific antibody fragments." Proc Natl Acad Sci USA 90(14):6444-6448.

[0356] Holliger,P.and G.Winter(1993)."Engineering bispecific antibodies." Curr Opin Biotechnol 4(4):446-449.

[0357] Huston,J.S.,D.Levinson,M.Mudgett-Hunter,M.S.Tai,J.Novotny,M.N.Margolies,R.J.Ridge,R.E.Bruccoleri,E.Haber,R.Crea and et al.(1988)."Protein engineering of antibody binding sites:recovery of specific activityin an anti-digoxin single-chain Fv analogue produced in Escherichia coli." Proc Natl Acad Sci USA 85(16):5879-5883.

[0358] Hwang,K.J.,K.F.Luk and P.L.Beaumier(1980)."Hepatic uptake anddegradation of unilamellar sphingomyelin / cholesterol liposomes:a kineticstudy." Proc Natl Acad Sci USA 77(7):4030-4034.

[0359] Jespers,L.S.,A.Roberts,S.M.Mahler,G.Winter and H.R.Hoogenboom(1994)."Guiding the selection of human antibodies from phage display repertoires to asingle epitope of an antigen." Biotechnology (NY)12(9):899-903.Jones,S.T.andM.M.Bendig(1991)."Rapid PCR-cloning of full-length mouse immunoglobulinvariable regions." Biotechnology(NY) 9(1):88-89.

[0360] Karlin,S.and S.F.Altschul(1990)."Methods for assessing thestatistical significance of molecular sequence features by using generalscoring schemes." Proc Natl Acad Sci USA 87(6):2264-2268.

[0361] Karlin,S.and S.F.Altschul(1993)."Applications and statistics formultiple high-scoring segments in molecular sequences." Proc Natl Acad Sci US A 90(12):5873-5877.

[0362] Kitamura,K.,E.Stockert,P.Garin-Chesa,S.Welt,K.O.Lloyd,K.L.Armour,T.P.Wallace,W.J.Harris,F.J.Carr and L.J.Old(1994)."Specificity analysis ofblood group Lewis-y(Le(y))antibodies generatedagainst synthetic and naturalLe(y)determinants." Proc Natl Acad Sci U S A 91(26):12957-12961.

[0363] Lefranc,M.P.,V.Giudicelli,C.Ginestoux,J.Jabado-Michaloud,G.Folch,F.Bellahcene,Y.Wu,E.Gemrot,X.Brochet,J.Lane,L.Regnier,F.Ehrenmann,G.Lefrancand P.Duroux(2009)."IMGT,the international ImMunoGeneTics informationsystem." Nucleic Acids Res 37(Database issue):D1006-1012.

[0364] Loo,D.,N.Pryer,P.Young,T.Liang,S.Coberly,K.L.King,K.Kang,P.Roberts,M.Tsao,X.Xu,B.Potts and J.P.Mather(2007)."The glycotope-specific RAV12monoclonal antibody induces oncosis in vitro and has antitumor activityagainst gastrointestinal adenocarcinoma tumor xenografts in vivo." Mol Cancer Ther 6(3):856-865.

[0365] Marks,J.D.,A.D.Griffiths,M.Malmqvist,T.P.Clackson,J.M.Bye andG.Winter(1992)."By-passing immunization:building high affinity humanantibodies by chain shuffling." Biotechnology (N Y) 10(7):779-783.

[0366] Miotti,S.,D.R.Negri,O.Valota,M.Calabrese,R.L.Bolhuis,J.W.Gratama,M.I.Colnaghi,and S.Canevari.1999.'Level of anti-mouse-antibody responseinduced by bi-specific monoclonal antibody OC / TR in ovarian-carcinomapatients is associated with longer survival',Int J Cancer,84:62-8.

[0367] Myers,E.W.and W.Miller(1989)."Approximate matching of regularexpressions." Bull Math Biol 51(1):5-37.Noble,P.,I.Spendlove,S.Harding,T.Parsons and L.G.Durrant(2013)."Therapeutic targeting of Lewis(y)and Lewis(b)with a novel monoclonal antibody 692 / 29." PLoS One 8(2):e54892.

[0368] Pai,L.H.,R.Wittes,A.Setser,M.C.Willingham and I.Pastan(1996)."Treatment of advanced solid tumors with immunotoxin LMB-1:an antibody linkedto Pseudomonas exotoxin." Nat Med 2(3):350-353.

[0369] Pearson,W.R.and D.J.Lipman(1988)."Improved tools for biologicalsequence comparison." Proc Natl Acad Sci U S A 85(8):2444-2448.

[0370] Pluckthun,A.(1991)."Antibody engineering:advances from the use ofEscherichia coli expression systems." Biotechnology(NY) 9(6):545-551.

[0371] Rabu,C.,R.McIntosh,Z.Jurasova and L.Durrant(2012)."Glycans as targetsfor therapeutic antitumor antibodies." Future Oncol 8(8):943-960.

[0372] Reff,M.E.(1993)."High-level production of recombinant immunoglobulinsin mammalian cells." Curr Opin Biotechnol 4(5):573-576.

[0373] Remington,R.(1980). Remington's pharmaceutical sciences ,Mack Pub.Co.

[0374] Sambrook,J.(1989). Molecular cloning:A laboratory manual ,Cold SpringHarbor Laboratory Press.

[0375] Sambrook,J.and D.Russell(2001). Molecular cloning:A laboratory manual .Cold Spring Harbor,N.Y.,Cold Spring Harbor Laboratory Press.

[0376] Schier,R.,A.McCall,G.P.Adams,K.W.Marshall,H.Merritt,M.Yim,R.S.Crawford,L.M.Weiner,C.Marks and J.D.Marks(1996)."Isolation of picomolaraffinity anti-c-erbB-2 single-chain Fv by molecular evolution of thecomplementarity determining regions in the center of the antibody bindingsite." J Mol Biol 263(4):551-567.

[0377] Schuster,M.,P.Umana,C.Ferrara,P.Brunker,C.Gerdes,G.Waxenecker,S.Wiederkum,C.Schwager,H.Loibner,G.Himmler and G.C.Mudde(2005)."Improvedeffector functions of a therapeutic monoclonal Lewis Y-specific antibody byglycoform engineering." Cancer Res 65(17):7934-7941.

[0378] Scott,A.M.,D.Geleick,M.Rubira,K.Clarke,E.C.Nice,F.E.Smyth,E.Stockert,E.C.Richards,F.J.Carr,W.J.Harris,K.L.Armour,J.Rood,A.Kypridis,V.Kronina,R.Murphy,F.T.Lee,Z.Liu,K.Kitamura,G.Ritter,K.Laughton,E.Hoffman,A.W.Burgessand L.J.Old(2000)."Construction,production,and characterization of humanizedanti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solidtumors." Cancer Res 60(12):3254-3261.

[0379] Sidman,K.R.,W.D.Steber,A.D.Schwope and G.R.Schnaper(1983)."Controlledrelease of macromolecules and pharmaceuticals from synthetic polypeptidesbased on glutamic acid." Biopolymers 22(1):547-556.

[0380] Schroff,R.W.,K.A.Foon,S.M.Beatty,R.K.Oldham,and A.C.Morgan,Jr.1985.'Human anti murine immunoglobulin responses in patients receiving monoclonalantibody therapy',Cancer Res,45:879-85.

[0381] Soejima,M.and Y.Koda(2005)."Molecular mechanisms of Lewis antigenexpression." Leg Med (Tokyo) 7(4):266-269.

[0382] Stemmer,W.P.(1994)."Rapid evolution of a protein in vitro by DNAshuffling." Nature 370(6488):389-391.Stewart,J.and J.Young(1984). Solid phase peptide synthesis .Rockford,Illinois,Pierce Chemical Company.

[0383] Tolcher,A.W.,S.Sugarman,K.A.Gelmon,R.Cohen,M.Saleh,C.Isaacs,L.Young,D.Healey,N.Onetto and W.Slichenmyer(1999)."Randomized phase II study of BR96-doxorubicin conjugate in patients with metastatic breast cancer." J Clin Oncol 17(2):478-484.

[0384] Torelli,A.and C.A.Robotti(1994)."ADVANCE and ADAM:two algorithms forthe analysis of global similarity between homologous informationalsequences." Comput Appl Biosci 10(1):3-5.

[0385] Traunecker,A.,A.Lanzavecchia and K.Karjalainen(1991)."Bispecificsingle chain molecules(Janusins)target cytotoxic lymphocytes on HIV infectedcells." EMBO J 10(12):3655-3659.

[0386] Trill,J.J.,A.R.Shatzman and S.Ganguly(1995)."Production of monoclonalantibodies in COS and CHO cells." Curr Opin Biotechnol 6(5):553-560.

[0387] Ward,E.S.,D.Gussow,A.D.Griffiths,P.T.Jones and G.Winter(1989)."Binding activities of a repertoire of single immunoglobulin variable domainssecreted from Escherichia coli." Nature 341(6242):544-546.

[0388] Yin,B.W.,C.L.Finstad,K.Kitamura,M.G.Federici,M.Welshinger,V.Kudryashov,W.J.Hoskins,S.Welt and K.O.Lloyd(1996)."Serological andimmunochemical analysis of Lewis y(Ley)blood group antigen expression inepithelial ovarian cancer." Int J Cancer 65(4):406-412.

[0389] Yuriev,E.,W.Farrugia,A.M.Scott and P.A.Ramsland(2005)."Three-dimensional structures of carbohydrate determinants of Lewis system antigens:implications for effective antibody targeting of cancer." Immunol Cell Biol 83(6):709-717.

[0390] Zhang,G.,H.Zhang,Q.Wang,P.Lal,A.M.Carroll,M.de la Llera-Moya,X.Xu andM.I.Greene(2010)."Suppression of human prostate tumor growth by a uniqueprostate-specific monoclonal antibody F77 targeting a glycolipid marker." Proc Natl Acad Sci U S A 107(2):732-737.

[0391] Zhong, L.T., A. Manzi, E. Skowronski, L. Notterpek, A.L. Fluharty, K.F. Faull, I. Masada, S. Rabizadeh, M. Varsanyi-Nagy, Y. Ruan, J.D. Oh, L.L. Butcher and D.E. Bredesen (2001). "A monoclonal antibody that induces neuronal apoptosis binds a metastasis marker." Cancer Res 61(15):5741-5748. Sequence Listing <110> Scancell Limited <120> Binding Member <130> P125851WO <150> GB1910899.2 <151> 2019-07-31 <160> 22 <170> PatentIn version 3.5 <210> 1 <211> 1401 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of 27.10 <400> 1 atgaacttct ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60 gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120 tgtgcaacct ctggattcac tttcagtcac tattacatgt attgggttcg ccagactcca 180 gagaagaggc tggagtgggt cgcatacatt agtaatgatg gtgataacac ctattatcca 240 gacactataa ggggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300 caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag agggaagtac 360 gacggggcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagctaca 420 acaacagccc catctgtcta tcccttggtc cctggctgca gtgacacatc tggatcctcg 480 gtgacactgg gatgccttgt caaaggctac ttccctgagc cggtaactgt aaaatggaac 540 tatggagccc tgtccagcgg tgtgcgcaca gtctcatctg tcctgcagtc tgggttctat 600 tccctcagca gcttggtgac tgtaccctcc agcacctggc ccagccagac tgtcatctgc 660 aacgtagccc acccagccag caagactgag ttgatcaaga gaatcgagcc tagaataccc 720 aagcccagta cccccccagg ttcttcatgc ccacctggta acatcttggg tggaccatcc 780 gtcttcatct tccccccaaa gcccaaggat gcactcatga tctccctaac ccccaaggtt 840 acgtgtgtgg tggtggatgt gagcgaggat gacccagatg tccatgtcag ctggtttgtg 900 haacaag aagtacacac agcctggaca cagccccgtg aagctcagta cacactacc 960 ttccgagtgg tcagtgccct cccatccag caccaggact ggatgagggg caggttc 1020 aaatgcaagg tcaacaa agccctcccca gccccatcg agagaccact ctcaaaccc 1080 aaaggaagg cccagacacc tcaagtatac accataccc cacctcgtga acaaatgtcc 1140 aagagaagg ttagtctgac ctgcctgtc accaacttct tctctgaagc catcagtgtg 1200 gagtgggaaa ggaacggaga actggagcag gattacaga acactccacc catcctggac 1260 tcagatggga cctacttcct ctcagcaag ctcactgtgg atacagacag ttggttgcaa 1320 ggagaaattt ttacctgctc cgtggtgcat gaggctctcc ataccacca cacacagaag 1380 ctgtctcgct cccctgtaa a 1401 <210> 2 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain protein sequence of 27.10 <400> 2 Met Asn Phe Trp Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp 50 55 60 Val Ala Tyr Ile Ser Asn Asp Asp Asn Thr Tyr Tyr Pro Asp Thr Ile 65 70 75 80 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Thr Leu Tyr 85 90 95 Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 100 105 110 Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala Ala Thr Thr Thr Ala Pro Ser Val Tyr 130 135 140 Pro Leu Val Pro Gly Cys Ser Asp Thr Ser Gly Ser Ser Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Lys Trp 165 170 175 Asn Tyr Gly Ala Leu Ser Ser Gly Val Arg Thr Val Ser Ser Val Leu 180 185 190 Gln Ser Gly Phe Tyr Ser Leu Ser Ser Leu Val Thr Val Pro Ser Ser 195 200 205 Thr Trp Pro Ser Gln Thr Val Ile Cys Asn Val Ala His Pro Ala Ser 210 215 220 Lys Thr Glu Leu Ile Lys Arg Ile Glu Pro Arg Ile Pro Lys Pro Ser 225 230 235 240 Thr Pro Pro Gly Ser Ser Cys Pro Pro Gly Asn Ile Leu Gly Gly Pro 245 250 255 Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Ala Leu Met Ile Ser 260 265 270 Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 275 280 285 Pro Asp Val His Val Ser Trp Phe Val Asp Asn Lys Glu Val His Thr 290 295 300 Ala Trp Thr Gln Pro Arg Glu Ala Gln Tyr Asn Ser Thr Phe Arg Val 305 310 315 320 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Arg Gly Lys Glu 325 330 335 Phe Lys Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg 340 345 350 Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln Val Tyr Thr 355 360 365 Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val Ser Leu Thr 370 375 380 Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val Glu Trp Glu 385 390 395 400 Arg Asn Gly Glu Leu Glu Gln Asp Tyr Lys Asn Thr Pro Pro Ile Leu 405 410 415 Asp Ser Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Thr 420 425 430 Asp Ser Trp Leu Gln Gly Glu Ile Phe Thr Cys Ser Val Val His Glu 435 440 445 Ala Leu His Asn His His Thr Gln Lys Asn Leu Ser Arg Ser Pro Gly 450 455 460 Lys 465 <210> 3 <211> 1386 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of 27.18 <400> 3 atgaacttct ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60 gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120 tgtgcaacct ctggattcac tttcagtcac tattacatgt attgggttcg ccagactcca 180 gagaagaggc tggagtgggt cgcatacatt agtaatgatg gtgataacac ctattatcca 240 gacactataa ggggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300 caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag agggaagtac 360 gacggggcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa 420 acgacacccc catctgtcta tccactggcc cctggatctg ctgcccaaac taactccatg 480 gtgaccctgg gatgcctggt caagggctat ttccctgagc cagtgacagt gacctggaac 540 tctggatccc tgtccagcgg tgtgcacacc ttcccagctg tcctggagtc tgacctctac 600 actctgagca gctcagtgac tgtcccctcc agccctcggc ccagcgagac cgtcacctgc 660 aacgttgccc acccggccag cagcaccaag gtggacaaga aaattgtgcc cagggattgt 720 ggttgtaagc cttgcatatg tacagtccca gaagtatcat ctgtcttcat cttcccccca 780 aagcccaagg atgtgctcac cattactctg actcctaagg tcacgtgtgt tgtggtagac 840 atcagcaagg atgatcccga ggtccagttc agctggtttg tagatgatgt ggaggtgcac 900 acagctcaga cgcaaccccg ggaggagcag ttcaacagca ctttccgctc agtcagtgaa 960 cttcccatca tgcaccagga ctggctcaat ggcaaggagt tcaaatgcag ggtcaacagt 1020 gcagctttcc ctgcccccat cgagaaaacc atctccaaaa ccaaaggcag accgaaggct 1080 ccacaggtgt acaccattcc acctcccaag gagcagatgg ccaaggataa agtcagtctg 1140 acctgcatga taacagactt cttccctgaa gacattactg tggagtggca gtggaatggg 1200 cagccagcgg agaactacaa gaacactcag cccatcatga acacgaatgg ctcttacttc 1260 gtctacagca agctcaatgt gcagaagagc aactgggagg caggaaatac tttcacctgc 1320 tctgtgttac atgagggcct gcacaaccac catactgaga agagcctctc ccactctcct 1380 ggtaaa 1386 <210> 4 <211> 462 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence of 27.18 <400> 4 Met Asn Phe Trp Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 Ser His Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Asn Asp Gly Asp Asn Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Ile Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro 130 135 140 Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met 145 150 155 160 Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Glu Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val 195 200 205 Pro Ser Ser Pro Arg Pro Ser Glu Thr Val Thr Cys Asn Val Ala His 210 215 220 Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys 225 230 235 240 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 245 250 255 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro 260 265 270 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 275 280 285 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 290 295 300 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 305 310 315 320 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 325 330 335 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 355 360 365 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 370 375 380 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 385 390 395 400 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asn Thr Asn 405 410 415 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 420 425 430 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 435 440 445 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 450 455 460 <210> 5 <211> 714 <212> DNA <213> Artificial sequence <220> <223> Light chain DNA sequence FG27.10 <400> 5 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc catcagtgat 60 gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120 tcttgcagat ctagtcagat cattgtacat actaatggaa acacctattt agaatggtac 180 ctgcagaaac caggccagtc tccaaacctc ctgatctaca aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300 agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 360 acgttcggct cggggacaaa gttggaaata aaacgggctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct acccaaaga catcaatgtc aagtgaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgacagttg gactgatcag gagcaag acagcaccta height 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acacctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt 714 <210> 6 <211> 238 <212> PRT <213> Artificial sequence <220> <223> Light chain protein sequence FG27.10 <400> 6 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ile Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Gln Ile Ile 35 40 45 Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro 130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp 195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr 210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 7 <211> 1404 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence CH27.10 <400> 7 atgaacttct ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60 gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120 tgtgcaacct ctggattcac tttcagtcac tattacatgt attgggttcg ccagactcca 180 gagaagaggc tggagtgggt cgcatacatt agtaatgatg gtgataacac ctattatcca 240 gacactataa ggggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300 caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag agggaagtac 360 gacggggcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tagcgcttcc 420 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 720 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 960 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1080 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1140 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttctctcct ctacagcaag ctcaccgtgg acagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaccacta cacgcagaag 1380 agccctctccc tgtctccgggg taaa 1404 <210> 8 <211> 468 <212> PRT <213> Artificial sequence <220> <223> Heavy chain protein sequence CH27.10 <400> 8 Met Asn Phe Trp Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 His Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Asn Asp Gly Asp Asn Thr Tyr Tyr Pro 65 70 75 80 Asp Thr With Arg Gly Arg Phe Thr With Arg Asn Ala Arg Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met Identification of Leu Glu Ala Leu Identification of Asn and Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465 <210> 9 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> CH27.10 light chain DNA sequence <400> 9 atgaagttgc ctgttaggct gttggtgctg atgttctgga tcctgctc catcagtgat 60 gttttgatga cccaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120 tcttgcagat ctagtcagat cattgtacat actaatgga acacctattt agaatgtac 180 ctgcagaaac caggccagtc tccaaacctc ctgatctaca aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgga tcaggcacag atttcacact acaggcagc 300 <h2 style=";text-align:left;direction:ltr">agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acgttcggct cggggacaaa gttggaaata aaacgtacgg tagcggcccc atctgtcttc 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 714<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 238<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Artificial sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> CH27.10 light chain protein sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Ile Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile 35 40 45 Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 11 <211> 357 <212> DNA <213> Artificial sequence <220> <223> FG27 RHA <400> 11 caggtccagc tggtcgaatc agggggaggc gtcgtgcagc ccggaaggtc actgagactg 60 tcatgtgccg catcagggtt tactttctca cactactata tgtactgggt gcgacaggct 120 ccaggcaagg gactggagtg ggtcgcatat atcagcaacg acggcgataa tacttactat 180 cctgacacca tccgggggag attcacaatc tctagata acagtaagaa cactctgtac 240 ctgcagatga actctctgag ggcagaagac accgccgtgt actattgcgc ccgcggggaaa 300 tacgatggag cctggttgc ttattgggga cagggcaccc tggtgacagt cagctcc 357 <210> 12 <211> 119 <212> PRT <213> Artificial sequence <220> <223> FG27 RHA <400> 12 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Asp Gly Asp Asn Thr Tyr Tyr Pro Asp Thr Ile 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 13 <211> 990 <212> DNA <213> Artificial sequence <220> <223> HuG1 DNA sequence <400> 13 gcctccacca agggcccttc cgtgttccct ctggcccctt cctccaagtc cacctccggc 60 ggcaccgccg ctctgggctg cctggtgaag gactacttcc ctgagcctgt gaccgtgtcc 120 tggaactctg gcgccctgac cagcggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180 ggcctgtact ccctgtcctc cgtggtgacc gtgccttcct cctccctggg cacccagacc 240 tacatctgca acgtgaacca caagccttcc aacaccaagg tggacaagaa ggtggagcct 300 aagtcctgcg acaagaccca cacgtgccct ccatgcccag ctcccgagct gctgggcgga 360 ccaagcgtgt ttctgttccc tcctaagcct aaggacaccc tgatgatctc ccggacccct 420 gaggtgacgt gcgtggtggt ggacgtgtcc cacgaggacc cagaggtgaa gttcaattgg 480 tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ctcgggagga acagtacaac 540 tccacctacc gggtggtgtc tgtgctgacc gtgctgcacc aggactggct gaacggcaag 600 gaatacaagt gcaaagtctc caacaaggcc ctgcctgccc ccatcgaaaa gaccatctcc 660 aaggccaagg gccagcctcg cgagcctcag gtgtacaccc tgcctccaag cagggaggaa 720 atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttaccc ttccgatatc 780 gccgtggagt gggagtccaa cggccagcct gagacaacact acagaccac ccctcctgtg 840 ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtcccggtgg 900 cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 960 cagaagagcc tctccctgtc cccgggtaaa 990 <210> 14 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> HuG1 protein sequence <400> 14 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 15 <211> 396 <212> DNA <213> Artificial sequence <220> <223> FG27 RKC DNA sequence <400> 15 atggaagccc cagcgcagct tctcttcctc ctgctactct ggctccctga taccaccgga 60 gacgtcgtga tgacccagag cccactgagc ctgcctgtga cacccggcga gcccgcaagt 120 atttcctgta gaagttccca gattatcgtg cacactaacg ggaataccta cctggagtgg 180 tatctgcaga agcccggcca gagtcctcag ctgctgatct acaaagtgag taaccggttc 240 tcaggagtcc cagaccggtt cagcggctcc gggtctggaa ccgatttcac actgaagatt 300 agcagggtgg aggccgaaga cgtgggagtc tactattgct ttcagggctc ccatgtcccc 360 ttcacatttg gccaggggac caagctggaa atcaaa 396 <210> 16 <211> 132 <212> PRT <213> Artificial sequence <220> <223> FG27 RKC protein sequence <400> 16 Put Glu Wing Pro Wing Gln Slow Slow Phe Slow Slow Slow Slow Trp Slow Pro 1 5 10 15 Asp Thr Thr Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile 35 40 45 Ile Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Three Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys 115 120 125 Leu Glu Ile Lys 130 <210> 17 <211> 321 <212> DNA <213> Artificial sequence <220> <223> HuK DNA sequence <400> 17 agaactgtgg cagcaccaag cgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agxaggaca gcacctacag cctcagcagc accctgacgc tgagcaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agctcaca ggggagagtg t 321 <210> 18 <211> 107 <212> PRT <213> Artificial sequence <220> <223> HuK protein sequence <400> 18 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Ala Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Serve Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Server Lys Ala Asp Tyr Glu 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 19 <211> 714 <212> DNA <213> Artificial sequence <220> <223> 27 Light chain DNA sequence (variable region + Kappa constant) <400> 19 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc catcagtgat 60 gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120 tcttgcagat ctagtcagat cattgtacat actaatggaa acacctattt agaatggtac 180 ctgcagaaac caggccagtc tccaaacctc ctgatctaca aagtttccaa ccgattttct 240 ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300 agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tgttccattc 360 acgttcggct cggggacaaa gttggaaata aaacgtacgg tagcggcccc atctgtcttc 420 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 480 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 540 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 600 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 660 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 714 <210> 20 <211> 238 <212> PRT <213> Artificial sequence <220> <223> 27 Light chain protein sequence (variable region + Kappa constant) <400> 20 Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ile Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ile Ile 35 40 45 Val His Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Asn Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 100 105 110 Phe Gln Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu 115 120 125 Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro 130 135 140 Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 145 150 155 160 Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170 175 Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser 180 185 190 Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala 195 200 205 Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly 210 215 220 Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 21 <211> 1404 <212> DNA <213> Artificial sequence <220> <223> Heavy chain DNA sequence of i27v1 (variable + ‘improved’ v1 constant region) <400> 21 atgaacttct ggctcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60 gtgaagctgg tggagtctgg gggaggctta gtgcagcctg gagggtccct gaaactctcc 120 tgtgcaacct ctggattcac tttcagtcac tattacatgt attgggttcg ccagactcca 180 gagaagaggc tggagtgggt cgcatacatt agtaatgatg gtgataacac ctattatcca 240 gacactataa ggggccgatt caccatctcc agagacaatg ccaggaacac cctgtacctg 300 caaatgagcc gtctgaagtc tgaggacaca gccatgtatt actgtgcaag agggaagtac 360 gacggggcct ggtttgctta ctggggccaa gggactctgg tcactgtctc tagcgcttcc 420 accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca 480 gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 540 tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 600 tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc 660 tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct 720 tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca 780 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 840 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 900 960 taccgagtgg tcagtgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1020 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaaccc 1080 aaaagagag cccagacacc tcaagtatac accatacccc cacctcgtga acaaatgtcc 1140 aagaagaagg ttagtctgac ctgcctggtc accaacttct tctctgaagc catcagtgtg 1200 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1260 tccgacggct ccttctctcct ctacagcaag ctcaccgtgg acagagcag gtggcagcag 1320 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaccacta cacgcagaag 1380 agccctctccc tgtctccgggg taaa 1404 <210> 22 <211> 468 <212> PRT <213> Artificial sequence <220> <223> Heavy chain Protein sequence of i27v1 (variable + 'improved' v1 constant region) <400> 22 Met Asn Phe Trp Leu Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly 1 5 10 15 Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 His Tyr Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu 50 55 60 Glu Trp Val Ala Tyr Ile Ser Asn Asp Gly Asp Asn Thr Tyr Tyr Pro 65 70 75 80 Asp Thr Ile Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn 85 90 95 Thr Leu Tyr Leu Gln Met Ser Arg Leu Lys Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Ala Arg Gly Lys Tyr Asp Gly Ala Trp Phe Ala Tyr Trp 115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200 205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 290 295 300 Val His Thr Ala Trp Thr Gln Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 Ile Glu Lys Thr Ile Ser Lys Pro Lys Gly Arg Ala Gln Thr Pro Gln 355 360 365 Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met Ser Lys Lys Lys Val 370 375 380 Ser Leu Thr Cys Leu Val Thr Asn Phe Phe Ser Glu Ala Ile Ser Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 450 455 460 Ser Pro Gly Lys 465

Claims

1. An isolated antibody or antibody fragment capable of being associated with Fuca1-2Galb1-4 (Fuca1-3)GlcNAc (Le y ) binds specifically and contains CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 as shown in the following amino acid sequences: CDRH1:GFTFSHYY; CDRH2:ISNDGDNT; CDRH3:ARGKYDGAWFAY; CDRL1:QIIVHTNGNTY; CDRL2:KVS; and CDRL3:FQGSHVPFT.

2. The antibody or antibody fragment according to claim 1, in, CDRs are carried by human antibody frameworks.

3. The antibody or antibody fragment according to claim 1, in, The antibody or antibody fragment comprises: VH with the following amino acid sequence: EVKLVESGGGLVQPGGSLKLSCATSGFTFSHYYMYWVRQTPEKRLEWVAYISNDGDNTYYPDTIRG RFTISRDNARNTLYLQMSRLKSEDTAMYYCARGKYDGAWFAYWGQGTLVTVSA, and VL with the following amino acid sequence: DVLMTQTPLSLPVSLGDQASISCRSSQIIVHTNGNTYLEWYLQKPGQSPNLLIYKVSNRFSGVPDRFS GSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIKR.

4. The antibody or antibody fragment according to any of the preceding claims, in, The antibody or antibody fragment also comprises a human constant region.

5. The antibody or antibody fragment according to claim 1, in, The antibody or antibody fragment comprises: Heavy chains with the following amino acid sequences: EVKLVESGGGLVQPGGSLKLSCATSGFTFSHYYMYWVRQTPEKRLEWVAYISNDGDNTYYPDTIRGRFTISRDNARNTLYLQMSRLKSEDTAMYYCARGKYDGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Light chains with the following amino acid sequences: DVLMTQTPLSLPVSLGDQASISCRSSQIIVHTNGNTYLEWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

6. The antibody or antibody fragment according to claim 1, in, The antibody or antibody fragment comprises: Heavy chains with the following amino acid sequences: QVQLVESGGGVVQPGRSLRLSCAASGFTFSHYYMYWVRQAPGKGLEWVAYISNDGDNTYYPDTIRGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKYDGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Light chains with the following amino acid sequences: MEAPAQLLFLLLLWLPDTTGDVVMTQSPLSLPVTPGEPASISCRSQIIVHTNGNTYLEWYLQKPGQSPQLLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

7. The antibody or antibody fragment according to claim 1, in, The antibody or antibody fragment is linked or associated with a chemotherapeutic agent or a cytotoxin agent.

8. A pharmaceutical composition comprising an antibody or antibody fragment as claimed in any of the preceding claims and a pharmaceutically acceptable excipient, diluent, carrier, buffer or stabilizer.

9. Use of an antibody or antibody fragment according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment or prevention of tumors, in, The tumor is a colorectal tumor, a gastric tumor, an ovarian tumor or a breast tumor.

10. A product containing the antibody or antibody fragment and an active agent according to any one of claims 1 to 6, as a combination preparation for use simultaneously, separately or sequentially in tumor treatment, in, The tumor is a colorectal tumor, a gastric tumor, an ovarian tumor or a breast tumor.

11. A nucleic acid encoding an antibody or antibody fragment according to any one of claims 1 to 6.

12. A nucleic acid, the nucleic acid comprising: Nucleotide sequence encoding heavy chain: atgaacttctggctcagcttgattttccttgtccttgtttttaaaaggtgtccagtgtgaagtgaagtggtgggtggagtctggggcttagtgcagcctgggggtccctgaaactctcctcctgaaactctcctgtgcaacctggattcactttcagtcactattcagtcactatttgaagttgggttcgccaga ctcccagagagaaggctgggtggtcgcatcattagtaatgatggtgataacctattatcccagacactattaagggccgattcaccatctcccagagacaatgccaggaacccctgtacctgcaaatgagccgtctgaagttactgtgcaagaatgagccgtctgaagtctgaaggacacagccatgttactgtgcaaga gggaagtacgacggcctggtttgcttactggggccagccagggccctggctggctggccaggactctggtcactgtctctagcgcttccaccaagggccatchggtcttcccccctggcccctggccaggccctggtccctgtcaaggactacttccccgaacccggtgtgac ggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga aagttgagcccaaatcttgtgacaaaactcacacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccccaaaaccccaaggacacctcatgatctccccccggccctgaggtgagccaccctgagtgagccaccctgaggtgagccacgaagaccctgagtgagccacgaagaccctgagtgagccacgaagaccctgagtgagccacgaagaccctgagtgagtgagccacgaagaccctgag gtcaagttcaactggtacgtggacggcgtgggtgcatacagcctggcaagcctggcaagccccgtgaaggaagcagtacaagtacagtacagtacagtacagtacagtcagtggtcagtcagtcctcaccgtcctgcaccaggactggctgaatggcaaggagtgcaagtcccccccccccc catcgagaaaaccatctcccaaacccaaaggaagcccagacacctcaagtacaccaccccccacctcgtgaacaaatgtcccaagaaggttagtgaaccttagtgacctggcctggtcaccacacttcttctctgaagccatcatcagtgtggagtgggagagaagcaatgggcagccggccgggagaacaca actacaagaccacgcctcccgtgctggactccgacggctccttcttcctcttcctctctctctcagcaagctcaccgtggacaaggagcaggtggcaggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacgcagaagcctctccctgtctccggatgcacaaccactacgcagaagcctctccctgtctccgggtaaa, and Nucleotide sequence encoding light chain: atgaagttgcctgttaggctgttggtgctgatgttctggatgttctggattcctgctgcttccatcagtgatgttttgatgacccaaactccactctccctgcctgtcagtcttggagatcaagcctccatctcttgcagatctagtcagatcatcatcattgtacatactataggaaacacctatttagaatggttttagaatggtt acctgcagaaaccaggccagtctccaaacctcctgatctacaaagtttccaaccgatttctggggtcccagacaggttcaggacagatttcagatcagatttcacaagatcagcagagtgggaggctgaggctggagtctggaggatctggaggagtttactgcttcaaggttcacaagtcagcagagtggagctgaggagtctggagtttactgcttcaaggttcacatgttcca ttcacgttcggctcgggggacaaagttggaaataaaacgtacggttagcggccccatctgtcttcatcttcccgccatctgatgaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaagtggaagtggataacgcccctccacaaagtacagtggaagtggataacgcccctcca atcgggtaactcccaggagagtgtcacagagcaggcaaggacaagcaagcacctcacctcagcacctcagcacctcagcacctgagctgagcaaagcagctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcaccatcaggcctgagctcgccgtcacaaagctcaaggggagagtgt.

13. An antibody or antibody fragment, comprising: Heavy chains with the following amino acid sequences: MNFWLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCATSGFTFSHYYMYWVRQTPEKRLEWVAYISNDGDNTYYPDTIRGRFTISRDNARNTLYLQMSRLKSEDTAMYYCARGKYDGAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHTAWTQPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Light chains with the following amino acid sequences: MKLPVRLLVLMFWIPASISDVLMTQTPLSLPVSLGDQASISCRSSQIIVHTNGNTYLEWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

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