Anti-Fcosil-GM1 Antibody

Abstract

The present invention relates to specific binding members, such as antibodies and fragments thereof, capable of specifically binding to fucosyl-GM1 (Fuc-GM1). The invention also relates to the use of such binding members in medicine and nucleic acids encoding such binding members, methods for detecting Fuc-GM1, and methods for treating various diseases, including cancer, using anti-Fuc-GM1 antibodies.

Description

[Technical field]

[0001] The present invention relates to specific binding members, such as antibodies and fragments thereof, capable of specifically binding to Fucosyl-GM1 (Fuc-GM1). The invention also relates to the use of such binding members in medicine and nucleic acids encoding such binding members, methods for detecting Fuc-GM1, and methods for treating various diseases, including cancer, using anti-Fuc-GM1 antibodies. [Background technology]

[0002] Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc-ceramide (hereafter referred to as Fuc-GM1 glycolipid) is a sphingolipid monosialoganglioside composed of a ceramide lipid moiety that anchors the molecule in the cell membrane and a carbohydrate moiety that is exposed at the cell surface. It is biosynthesized by the sequential addition of sugar and sialic acid to ceramide (sphingosine and fatty acid) through different glucosyltransferases (type I transmembrane protein glycosylceramide synthetase, β-galactosyltransferase, GM1 synthetase, α1,2-fucosyltransferase) (Figure 1) (Kartal Yandim, Apohan, and Baran 2013; Tokuda et al. 2006). Overexpression of some of these enzymes has been associated with small cell lung cancer (SCLC) (Martin-Satue et al. 1998), further suggesting that Fuc-GM1 is involved in tumor progression. Carbohydrate antigens are the most abundantly expressed antigens on the cell surface of cancer (Feizi 1985). In some tumor types, such as SCLC, the initial response to chemotherapy is impressive, but chemotherapy-resistant relapse soon follows. Novel immunotherapeutic interventions can be successful in overcoming drug-resistant relapse (Johnson 1995). Some carbohydrate antigens, such as gangliosides GD3 and GD2, have been shown to serve as effective targets for passive immunotherapy with monoclonal antibodies (mAbs) (Irie and Morton 1986; Houghton et al. 1985). Ganglioside antigens have also been shown to be effective targets for active immunotherapy with vaccines in clinical trials (Krug et al. 2004; Dickler et al. 1999; Livingston et al. 1994). Indeed, sera from SCLC patients who developed antibody titers against Fuc-GM1 after vaccination with KLH-conjugated antigens showed specific binding to tumor cells and tumor-specific complement-dependent cytotoxicity (CDC).Anti-Fuc-GM1 titer-related toxicity was mild and transient, and three patients with limited-stage SCLC were relapse-free at 18, 24, and 30 months (Krug et al. 2004; Dickler et al. 1999).

[0003] Fuc-GM1 expression has been demonstrated in a high percentage of SCLC cases, 75-90% (Drivsholm et al. 1994), and unlike other ganglioside antigens, Fuc-GM1 glycolipid is little or not expressed in normal tissues (Nilsson et al. 1984; Krug et al. 2004; Brezicka et al. 1989; Zhang et al. 1997; Brezicka et al. 2000; Fredman et al. 1986; Brezicka et al. 1991; Nilsson et al. 1986). Fuc-GM1 has been demonstrated to be present in culture medium from SCLC cell lines, in tumor extracts and in serum of nude mouse xenografts, and in serum of SCLC patients with advanced stage disease (Vangsted et al. 1991; Vangsted et al. 1994). These reports provide solid evidence for Fuc-GM1 as a highly specific tumor antigen that can be targeted by immunotherapeutic agents.

[0004] Therefore, effective agents that recognize lung cancer antigens that mainly target sphingoglycolipids, such as Fuc-GM1 glycolipids, and methods of using such agents are desired. mAb that binds to Fuc-GM1 ("F12") is known in the art (Brezicka et al. 1989; Brezicka et al. 2000). Furthermore, WO 2007 / 067992 and WO 2016 / 049256A1 disclose anti-Fc-GM1 antibodies. Summary of the Invention

[0005] In a first aspect, the present invention provides an isolated specific binding member capable of specifically binding to Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc-ceramide (Fuc-GM1 glycolipid) but not Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc (free sugar).

[0006] The isolated binding members of the present invention bind to Fuc-GM1 glycolipids but not to free sugars. As detailed in the Examples herein, mAbs were generated against Fuc-GM1 glycolipids first formulated in liposomes with immune adjuvants to ensure that the antibodies recognize Fuc-GM1 in the cell membrane. However, this failed to induce a high affinity antibody response. To improve the immunogenicity of Fuc-GM1 glycolipid, it was conjugated to human serum albumin (HSA), a T cell carrier. This was achieved by removing one of the lipid chains by ozonolysis and chemical conjugation to HSA to provide T cell support for affinity maturation. Further immunization was performed with Fuc-GM1-HSA. Unexpectedly, this generated IgG antibodies that bind to Fuc-GM1 glycolipids but not to free sugars. It is very surprising that lipid chains must be present for antibody binding, since antibodies do not generally bind to hydrophobic moieties such as lipids. Interestingly, despite the fact that they were raised against Fuc-GM1-HSA, these antibodies bound to Fuc-GM1 glycolipids and tumour cells with 1,000-10,000-fold higher affinity. The anti-FucGM1 mab presented herein, F12, also binds to GM1, whereas the binding members of the invention do not cross-react with GM1.

[0007] The inventors have provided specific binding members that exhibit potent in vivo anti-tumor activity. The specific binding members of the invention exhibit potent immune-mediated cytotoxic activity against human SCLC cells in vitro via antibody-dependent cellular cytotoxicity (ADCC) and CDC. The inventors have generated anti-Fuc-GM1 glycolipid mAbs by immunization with cell / glycan conjugates. Examples of these mAbs are the IgG3 murine mAbs referred to herein as "FL133.63" (Figures 2a and d) and "FL133.67" (Figures 2b and e), the IgG1 mAb referred to as "FL134.33" (Figures 2c and f) and the hIgG1 chimeric Ab referred to as "CH134" (Figure 3).

[0008] As normal tissues do not express 2-hydroxy fatty acid containing lipids, the specific binding members of the invention have a very limited normal distribution. However, they bind strongly to SCLC. The specificity is reflected in the distinct sequences of the FL133.63 / FL133.67 / FL134.33 / CH134.33 variable heavy and light chain regions. The specific binding members of the invention may bind to SCLC-specific gangliosides expressed on 2-hydroxy fatty acid containing lipids.

[0009] Specific binding members of the invention preferably comprise one or more binding domains selected from binding domains having an amino acid sequence substantially as set out as residues 27-38 (CDRH1), 54-65 (CDRH2) or 105-116 (CDRH3) of Figure 2a, b, c or 3a. A specific binding member may comprise a binding domain comprising an amino acid sequence substantially as set out as residues 105-116 (CDRH3) of the amino acid sequence of Figure 2a, b, c or 3a. Such specific binding members may further comprise one or both, preferably both, of binding domains having an amino acid sequence substantially as set out as residues 27-38 (CDRH1) and residues 56-65 (CDRH2) of the amino acid sequence shown in Figures 2a, b, c and 3a. Preferred specific binding members are (a) CDRH1, CDRH2 and CDRH3 in Figure 2a; (b) CDRH1, CDRH2 and CDRH3 in Figure 2b; (c) CDRH1, CDRH2 and CDRH3 of Figure 2c; or (d) CDRH1, CDRH2, and CDRH3 in Figure 3a Includes.

[0010] The binding member may comprise an amino acid sequence substantially as set out as 1-127 (VH) of Figure 2a, b, c or 3a.

[0011] The specific binding member may comprise one or more binding domains selected from binding domains having the amino acid sequence of residues 27-38 (CDRL1), 56-65 (CDRL2) or 105-113 (CDRL3) of Figure 2d, e, f or 3b. The binding member may comprise a binding domain having an amino acid sequence substantially as set out as residues 105-113 (CDRL3) of the amino acid sequence of Figure 2d, e, f or 3b. Such a specific binding member may further comprise one or both, preferably both, of binding domains having an amino acid sequence substantially as set out as residues 27-38 (CDRL1) and residues 56-65 (CDRL2) of the amino acid sequence shown in Figure 2d, e, f or 3b. Preferred specific binding members are (a) CDRH1, CDRH2 and CDRH3 in Figure 2d; (b) CDRH1, CDRH2, and CDRH3 in Figure 2e; (c) CDRH1, CDRH2 and CDRH3 of FIG. 2f; or (d) CDRH1, CDRH2 and CDRH3 in Figure 3b Includes.

[0012] Specific binding members comprising multiple binding domains of the same or different sequence or combinations thereof are included within the present invention. Thus, each binding domain may be carried by a human antibody framework. For example, one or more binding domains may be substituted into the complementarity determining regions (CDRs) of a fully human antibody or its variable regions.

[0013] An isolated specific binding member of the present invention comprises an amino acid sequence substantially as set out as residues 1 to 124 (VL) of the amino acid sequence shown in Figure 2d, e, f or 3b.

[0014] An isolated specific binding member of the invention may comprise one or more, preferably all, of a binding domain having an amino acid sequence substantially as set out as residues 27-38 (CDRH1), 56-65 (CDRH2) or 105-116 (CDRH3) of Figure 2a, b, c or 3a in combination with one or more, preferably all, of a binding domain having an amino acid sequence substantially as set out as residues 27-38 (CDRL1), 56-65 (CDRH2) or 105-113 (CDRL3) of Figure 2d, e, f or 3b. Preferred isolated specific binding members of the invention include (a) one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRH1), 56-65 (CDRH2) or 105-116 (CDRH3) of Figure 2a in combination with one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRL1), 56-65 (CDRH2) or 105-113 (CDRH3) of Figure 2d; (b) one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRH1), 56-65 (CDRH2) or 105-116 (CDRH3) of Figure 2b in combination with one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRL1), 56-65 (CDRH2) or 105-113 (CDRH3) of Figure 2e; (c) one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRH1), 56-65 (CDRH2) or 105-116 (CDRH3) of Figure 2c in combination with one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRL1), 56-65 (CDRL2) or 105-113 (CDRL3) of Figure 2f; or (d) one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRH1), 56-65 (CDRH2) or 105-116 (CDRH3) of Figure 3a in combination with one or more, preferably all, of a binding domain having the amino acid sequence substantially as set out as residues 27-38 (CDRL1), 56-65 (CDRL2) or 105-113 (CDRL3) of Figure 3b. Includes.

[0015] The binding member may comprise an amino acid sequence substantially as set out as residues 1 to 127 (VH) of the amino acid sequence of Figure 2a, b, c or 3a and an amino acid sequence substantially as set out as residues 1 to 124 (VL) of the amino acid sequence of Figure 2d, e, f or 3b. Preferably the binding member comprises: (a) an amino acid sequence substantially as set forth as residues 1 to 127 (VH) of the amino acid sequence of Figure 2a and an amino acid sequence substantially as set forth as residues 1 to 124 (VL) of the amino acid sequence of Figure 2d; (b) an amino acid sequence substantially as set forth as residues 1 to 127 (VH) of the amino acid sequence of Figure 2b and an amino acid sequence substantially as set forth as residues 1 to 124 (VL) of the amino acid sequence of Figure 2e; (c) an amino acid sequence substantially as set forth as residues 1 to 127 (VH) of the amino acid sequence of Figure 2c and residues 1 to 124 (VL) of the amino acid sequence of Figure 2f; or (d) an amino acid sequence substantially as set forth as residues 1 to 127 (VH) of the amino acid sequence of Figure 3a and an amino acid sequence substantially as set forth as residues 1 to 124 (VL) of the amino acid sequence of Figure 3b. Includes.

[0016] Once a single original mAb, e.g., Fuc-GM1 glycolipid mAb, with the desired properties described herein is isolated, it is easy to generate other mAbs with similar properties by using methods known in the art. For example, the method of Jespers et al., 1994 (Jespers et al. 1994) may be used to guide the selection of mAbs with the same epitope, and thus similar properties, as the original mAb. Using phage display, the heavy chain of the original antibody is first paired with a repertoire of (preferably human) light chains to select for ganglioside-binding mAbs, and then the new light chains are paired with a repertoire of (preferably human) heavy chains to select for (preferably human) ganglioside-binding mAbs with the same epitope as the original mAb.

[0017] The specific binding member may be an antibody or antibody fragment, Fab, (Fab')2, scFv, Fv, dAb, Fd or diabody. The antibody may be a polyclonal antibody. The antibody may be a monoclonal antibody (mAb). The antibody of the invention may be a humanized, chimeric or veneered antibody or any species of non-human antibody.

[0018] Murine 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 either humanized or fully human IgG antibodies.

[0019] A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as set out in Figure 2a and a light chain having an amino acid sequence substantially as set out in Figure 2d.

[0020] A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as set out in Figure 2b, and a light chain having an amino acid sequence substantially as set out in Figure 2e.

[0021] A specific binding member of the present invention may comprise a heavy chain having an amino acid sequence substantially as set out in Figure 2c and a light chain having an amino acid sequence substantially as set out in Figure 2f.

[0022] A specific binding member of the invention may comprise a heavy chain having an amino acid sequence substantially as set out in Figure 3a, and a light chain having an amino acid sequence substantially as set out in Figure 3b.

[0023] The present invention further provides a binding member which competes for binding to Fuc-GM1 containing gangliosides with an antibody comprising a VH chain having the amino acid sequence of residues 1 to 127 of Figure 2a, b, c or 3a and a VL chain having the amino acid sequence of residues 1 to 124 of Figure 2d, e, f or 3b, preferably the VH chain of Figure 2a combined with the VL chain of Figure 2d, the VH chain of Figure 2b combined with the VL chain of Figure 2e, the VH chain of Figure 2c combined with the VL chain of Figure 2f, or the VH chain of Figure 3a combined with the VL chain of Figure 3b.

[0024] Included in the invention are specific binding members which are capable of specifically binding to Fuc-GM1 glycolipid but not to free saccharide (Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc) and which have a VH and / or VL domain that is at least 90%, at least 95% or at least 99% identical to the VH or VL domain of Figure 2 or 3. Included in the invention are specific binding members which are capable of specifically binding to Fuc-GM1 and which are at least 90%, at least 95% or at least 99% identical to the heavy and / or light chain of Figure 2 or 3. Preferably, such antibodies differ from the sequences of Figure 2 or 3 by a small number of functionally insignificant amino acid substitutions (e.g. conservative substitutions), deletions or insertions.

[0025] A specific binding member of the present invention may carry a detectable or functional label.

[0026] In a further aspect, the invention provides isolated nucleic acids encoding specific binding members of the invention and methods for preparing specific binding members of the invention comprising expressing said nucleic acid under conditions which result in expression of said binding member and recovering the binding member.Included in the invention are isolated nucleic acids which encode specific binding members capable of specifically binding to Fuc-GM1 glycolipid but not to free sugars (Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc) and which are at least 90%, at least 95% or at least 99% identical to the sequences provided herein.

[0027] The specific binding members of the invention may be used in methods of treatment or diagnosis of the human or animal body, for example a method of treating a tumour in a patient (preferably a human) comprising administering to the patient (preferably a human) an effective amount of a specific binding member of the invention. The invention also provides specific binding members of the invention for use in medicine, preferably for use in the treatment of tumours, as well as the use of a specific binding member of the invention in the manufacture of a medicament for the diagnosis or treatment of a tumour. The tumour may be small cell lung carcinoma (SCLC).

[0028] The antigens that the specific binding members of the present invention bind to are disclosed herein.The Fuc-GM1 glycolipids that the specific binding members of the present invention can more preferably specifically bind to can be provided.The Fuc-GM1 glycolipids can be provided in isolated form and used in screening to develop further specific binding members thereto.For example, a library of compounds can be screened for library members that specifically bind to Fuc-GM1.The Fuc-GM1 can be on a lipid backbone.

[0029] In a further aspect, the present invention provides an isolated specific binding member of the first aspect of the invention for use in the diagnosis or prognosis of SCLC.

[0030] The present invention further provides a method for the diagnosis of cancer comprising using a specific binding member of the invention to detect Fuc-GM1 containing GSLs in a sample from an individual, in which the pattern of gangliosides detected by the binding member may be used to stratify treatment options for the individual.

[0031] These and other aspects of the invention are described in further detail below. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] As used herein, a "specific binding member" is a member of a pair of molecules that have binding specificity for each other. The members of a specific binding pair may be naturally occurring or wholly or partially synthetically produced. One member of the molecular pair has an area, which may be a protrusion or a cavity on its surface, which specifically binds to, and is therefore complementary to, a particular spatial and diametric configuration of the other member of the molecular pair. In this way, the members of the pair have the property of specifically binding to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention generally relates to antigen-antibody type reactions, but also to small molecules that bind to antigens as defined herein.

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

[0034] As used herein, a "tumor" is an abnormal growth of tissue. It may be localized (benign) or may invade nearby (malignant) or distant tissues (metastatic). Tumors include neoplastic growths that give rise to cancer, including SCLC as well as cancerous tissues or cell lines.

[0035] The term "antibody" as used herein refers to immunoglobulin molecules, whether natural or partially or wholly synthetically produced, and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The term also encompasses any polypeptide or protein having a binding domain that is or is homologous to an antibody binding domain. They may be from natural sources or they may be partially or wholly synthetically produced. Examples of antibodies of the invention are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments that contain the antigen binding domain, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Preferred isotypes are IgG1 and IgG3. Antibodies can be polyclonal or monoclonal. Monoclonal antibodies can be referred to as "mAbs".

[0036] Using monoclonal and other antibodies and recombinant DNA techniques, it is possible to generate other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region or CDRs of an antibody into the constant region, or constant region plus framework regions, of a different immunoglobulin. See, for example, EP-A-184187, GB-A-2188638A or EP-A-239400. Hybridomas or other cells that produce the antibodies may be subjected to genetic mutations or other changes that may or may not alter the binding specificity of the antibodies produced.

[0037] Since antibodies can be modified in many ways, the term "antibody" should be interpreted as including any specific binding member or substance having a binding domain with the required specificity. The term therefore includes antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanized antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetically produced. Thus, chimeric molecules or equivalents comprising an immunoglobulin binding domain fused to another polypeptide are included. Cloning and expression of chimeric antibodies are described in European Patent Applications A-0120694 and A-0125023. Humanized antibodies can be modified antibodies having variable regions of a non-human, e.g. murine, antibody and constant regions of a human antibody. Methods for producing humanized antibodies are described, for example, in US Pat. No. 5,225,539.

[0038] It has been shown that fragments of a whole antibody can perform the function of binding antigen. Examples of binding fragments are: (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain (Ward et al. 1989); (v) an isolated CDR region; (vi) an F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al. 1988; Huston et al. al. 1988); (viii) bispecific single chain Fv dimers (PCT / US92 / 09965); and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; (Holliger, Prospero, and Winter 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 linked (e.g. by a peptide linker) but unable to associate with each other to form an antigen-binding site: the antigen-binding site is formed by association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO 94 / 13804).

[0040] If bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger and Winter 1993), e.g., prepared chemically or from hybrid hybridomas, or can be any of the bispecific antibody fragments mentioned above. It may be preferable to use scFv dimers or diabodies rather than whole antibodies. Diabodies and scFvs can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti-idiotypic reactions. Other forms of bispecific antibodies include the single chain "Janusins" described in (Traunecker, Lanzavecchia, and Karjalainen 1991).

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

[0042] A "binding domain" is a part of a specific binding member that comprises the area that specifically binds to and is complementary to part or all of an antigen. If the binding member is an antibody or an antigen-binding fragment thereof, the binding domain may be a CDR. If the antigen is large, the antibody may only bind to a specific part of the antigen, called the epitope. An antigen-binding domain may be provided by one or more antibody variable domains. The antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

[0043] "Specific" is generally used to refer to the situation where one member of a specific binding pair does not exhibit any significant binding to molecules other than its specific binding partner, e.g., has less than about 30%, preferably 20%, 10% or 1% cross-reactivity with any other molecule. The term is also applicable, for example, when an antigen-binding domain is specific for a particular epitope carried by multiple antigens, in which case a specific binding member carrying the antigen-binding domain is capable of binding to a variety of antigens carrying the epitope.

[0044] "Isolated" refers to the state in which specific binding members of the invention or nucleic acids encoding such binding members should preferably be in accordance with the present invention. Members and nucleic acids are generally free or substantially free of materials with which they are naturally associated, such as other polypeptides or nucleic acids found therewith in their natural environment or in the environment in which they are prepared (e.g. cell culture) if such preparation is by recombinant DNA techniques carried out in vitro or in vivo. Specific binding members and nucleic acids may be formulated with a diluent or adjuvant and are nevertheless isolated for practical purposes - for example members will typically be mixed with gelatin or other carrier when used to coat microtiter plates for use in immunoassays, or mixed with a pharma- ceutically acceptable carrier or diluent when used diagnostically or therapeutically. Specific binding members may be glycosylated, either naturally or by heterologous eukaryotic systems, or they may be unglycosylated (e.g. when made by expression in prokaryotic cells).

[0045] By "substantially as described" it is meant that the amino acid sequence of the present invention is either identical or highly homologous to the referenced amino acid sequence. By "highly homologous" it is contemplated that there may be 1-5, 1-4, 1-3, 2 or 1 substitutions that may be made in the sequence.

[0046] The present invention also includes within its scope a polypeptide having an amino acid sequence as depicted in Figure 2 or 3, a polynucleotide having a nucleic acid sequence as depicted in Figure 2 or 3, and sequences having substantial identity thereto, for example at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity thereto. 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 best alignment with the second sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is the alignment of the two sequences that results in the greatest percent identity. The 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 a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990 (Karlin and Altschul 1990), modified as in Karlin and Altschul, 1993 (Karlin and Altschul 1993). The NBLAST and XBLAST programs of Altschul et al., 1990 (Altschul et al. 1990) incorporate such an algorithm. To obtain nucleotide sequences that are homologous to the nucleic acid molecules of the present invention, BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12. To obtain amino acid sequences that are homologous to the protein molecules of the present invention, BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST, described in Altschul et al., 1997 (Altschul et al. 1997), can be used. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules (Id.). When using BLAST, Gapped BLAST and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http: / / www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, 1989 (Myers and Miller 1989). The ALIGN program (version 2.0), which is part of the GCG sequence alignment software package, incorporates such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCE and ADAM, described in Torellis and Robotti, 1994 (Torelli and Robotti 1994); and FASTA, described in Pearson and Lipman, 1988 (Pearson and Lipman 1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

[0048] The isolated specific binding members of the invention are capable of binding to specific glycosphingolipids (GSLs) but are unable to bind to the free sugars of glycosphingolipids. Quite unusually, the binding members of the invention require the presence of lipids in order to bind. GSLs are a highly diverse group of molecules consisting of glycans structurally linked to a ceramide lipid moiety (sphingosine linked to a fatty acid by an amide bond). The ceramide structures vary in length and saturation, which increases the diversity of GSL species, but the main source of diversity comes from the glycan head group. Compared to amino acids or nucleic acids, which can only be linked linearly, carbohydrates can be linked to each other at multiple points. Class-switched, affinity-matured antibody targeting domains comprising amino acid sequences substantially as set forth as residues 105-116 (CDRH3) of Figure 2a, b, c or 3a and 105-113 (CDRL3) of Figure 2d, e, f or 3b may be carried in a conformation that allows binding of these regions to Fuc-GM1 ganglioside.

[0049] The structures for carrying the binding domains of the invention are generally antibody heavy or light chain sequences or substantial portions thereof, in which the binding domains are located at 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 may be determined by reference to http: / / www.imgt.org / . The amino acid sequence substantially as set forth as residues 105-116 of Figure 2a, b, c or 3a may be carried as CDR3 in a human heavy chain variable domain or substantial portion thereof, and the amino acid sequence substantially as set forth as residues 105-113 of Figure 2d, e, f or 3b may be carried as CDR3 in a human light chain variable domain or substantial portion thereof.

[0050] The variable domains can be derived from any germline or rearranged human variable domain, or can be synthetic variable domains based on consensus sequences of known human variable domains. The CDR3-derived sequences of the present invention can be introduced into a repertoire of variable domains lacking CDR3 regions using recombinant DNA technology. For example, Marks et al., 1992 (Marks et al. 1992) describes a method for generating a repertoire of antibody variable domains, using a consensus primer directed to or adjacent to the 5' end of the variable domain region, together with a consensus primer for the third framework region of the human VH gene, to provide a repertoire of VH variable domains lacking CDR3. Marks et al., 1992 (Marks et al. 1992) further describes how this repertoire can be combined with the CDR3 of a particular antibody. Similar techniques may be used to shuffle the CDR3 derived sequences of the invention with a repertoire of VH or VL domains lacking CDR3, and the shuffled complete VH or VL domains may be combined with cognate VL or VH domains to provide a specific binding member of the invention. The repertoire may then be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, so that suitable specific binding members may be selected. The repertoire may be a phage display system of at least 10 4 individual members, e.g. 10 6 ~10 8 or 10 10 It may consist of any one of the members.

[0051] Similar shuffling or combinatorial techniques have also been disclosed by Stemmer, 1994 (Stemmer 1994), who describe techniques involving beta-lactamase genes, but state that this approach can be used to generate antibodies. A further alternative is to use random mutagenesis of, for example, Fuc-GM1 VH or VL genes to generate mutations within the entire variable domain, to generate novel VH or VL regions carrying the CDR3-derived sequences of the invention. Such techniques have been described by Gram et al., 1992 (Gram et al. 1992), using error-prone PCR.

[0052] Another method that can be used is to direct mutagenesis to the CDR regions of the VH or VL genes. Such techniques are disclosed by Barbas et al., 1994 (Barbas et al. 1994) and Schier et al., 1996 (Schier et al. 1996). A substantial portion of an immunoglobulin variable domain generally comprises at least three CDR regions together with their intervening framework regions. This portion may also comprise at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N- or C-terminus of the substantial portion of the variable domain may be residues not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the invention produced by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other engineering steps, including the introduction of linkers to link a variable domain of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (e.g. in the production of diabodies) or protein tags as discussed in 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 set out in Figures 2 and 3, namely amino acids 1 to 127 (VH) of Figure 2a, b, c or 3a and amino acids 1 to 124 (VL) of Figure 2d, e, f or 3b. Single binding domains based on any of these sequences form further aspects of the invention. In the case of binding domains based on the amino acid sequence of the VH region substantially as set out in Figure 2a, b, c or 3a, such binding domains may be used as targeting agents, as it is known that immunoglobulin VH domains are capable of specifically binding to target antigens. In the case of any of the single chain specific binding domains, these domains may be used to screen for complementary domains capable of forming a two domain specific binding member with in vivo properties as good or comparable to the FL133 / 4 antibody disclosed herein.

[0054] This can be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO 92 / 01047, in which individual colonies containing either H or L chain clones are used to infect the complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding members are selected according to phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al., 1992 (Marks et al. 1992).

[0055] Specific binding members of the invention may further comprise an antibody constant region or part thereof. For example, specific binding members based on the VL region shown in Figures 2d, e, f or 3b may be linked at their C-terminus to an antibody light chain constant domain. Similarly, specific binding members based on the VH region shown in Figures 2a, b, c or 3a may be linked at their C-terminus to all or part of an immunoglobulin heavy chain from any antibody isotype, such as IgG, IgA, IgE and IgM, and any of the isotype subclasses, in particular IgG1, IgG2 and IgG4.

[0056] Specific binding members of the present invention may be used in methods for the diagnosis and treatment of tumours in human or animal subjects.

[0057] When used in diagnostics, the specific binding members of the invention may be coupled to a detectable label, such as a radiolabel, which may be linked to the specific binding member of the invention using conventional chemistry known in the art of antibody imaging. 131 I or 99 The label may be labeled with Tc. Labels also include enzyme labels, such as horseradish peroxidase. Labels further include chemical moieties, such as biotin, that can be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

[0058] The specific binding members of the present invention may be labelled with a functional label. Functional labels include substances which are designed to be targeted to the site of cancer and cause its destruction. Such functional labels include toxins such as ricin and enzymes such as bacterial carboxypeptidases or nitroreductases which are capable of converting a prodrug into an active drug. Furthermore, the specific binding members may be coupled to chemotherapeutic or cytotoxic agents such as maytansine (DM1 and DM4), onides, auristatins, calicheamicin, duocamicin, doxorubicin or radiolabels such as 90 Y or 131 It may be bound to or otherwise associated with I.

[0059] Furthermore, specific binding members of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. Thus, the invention further provides a product containing a specific binding member of the invention and an active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of tumours. Active agents may include chemotherapeutic or cytotoxic agents, including 5-fluorouracil, cisplatin, mitomycin C, oxaliplatin and tamoxifen, which may act synergistically with the binding members of the invention. Other active agents may include analgesics, such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates, e.g. morphine, or an antiemetic drug in an appropriate dose.

[0060] Without wishing to be bound by theory, the ability of the binding members of the invention to synergize with active agents to promote tumour killing may be a direct result of the binding of the binding members to cell surface-bound Fuc-GM1 ganglioside, rather than through an immune effector mechanism.Cancer immunotherapy involving antibodies against immune checkpoint molecules has shown efficacy against a variety of malignancies and in combination with different immune tumour treatment modalities.

[0061] The specific binding members of the present invention are usually administered in the form of a pharmaceutical composition which may contain at least one component in addition to the specific binding member. The pharmaceutical composition may contain, in addition to the active ingredient, pharma- ceutically acceptable excipients, diluents, carriers, buffers, stabilizers or other substances well known to those skilled in the art. Such substances must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substance will depend on the route of administration, which may be oral or by injection, for example intravenous. Injection is expected to be the primary route for therapeutic administration of the composition, although delivery through catheters or other surgical tubing may also be used. Some suitable routes of administration include intravenous, subcutaneous, intraperitoneal and intramuscular administration. Liquid formulations may be utilized following reconstitution from a powder formulation.

[0062] For intravenous injection or injection at the affected site, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has appropriate pH, isotonicity and stability.Those skilled in the art are well able to prepare appropriate solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as necessary.

[0063] Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other sugar solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. When the formulation is liquid, it may be, for example, a saline solution containing a non-phosphate buffer solution of pH 6.8-7.6 or a lyophilized powder.

[0064] The composition may also be delivered via microspheres, liposomes, other microparticulate delivery systems, or sustained release formulations that are placed in certain tissues, including blood.Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, such as suppositories, or microcapsules.Implantable or microencapsulated sustained release matrices include polylactic acid (U.S. Pat. No. 3,773,919; EP-A-0058481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al. 1983), poly(2-hydroxyethyl-methacrylate). Liposomes containing polypeptides are prepared by known methods: DE-A-3,218,121A; (Eppstein et al. 1985); (Hwang, Luk, and Beaumier 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. Usually, liposomes are of the small (about 200-800 angstroms) unilamellar type with a lipid content of more than about 30 mol.% cholesterol, the selected proportion being adjusted to provide an optimal rate of polypeptide leakage. The composition can be administered locally to the tumor site or other desired site, or it can be delivered so that it targets tumor or other cells.

[0065] 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 and the rate and time course of administration will depend on the nature and severity of what is being treated. Prescription of treatment, such as the choice of dosage, is within the responsibility of general practitioners and other physicians, and will generally take into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to physicians. The compositions of the present invention are particularly suitable for the treatment of existing tumors, particularly cancers, and for the prevention of recurrence of such conditions after initial treatment or surgery. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980 (Remington 1980).

[0066] The optimal dose can be determined by a 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, a serum concentration of polypeptide and antibody that allows receptor saturation is desired. A concentration of more than approximately 0.1 nM is usually sufficient. For example, a serum concentration of 100 mg / m 2 of antibody is generally sufficient. 2 provides a serum concentration of approximately 20 nM for approximately 8 days.

[0067] As a rough guideline, antibody doses range from 10 to 300 mg / m 2 The antibody fragment may be given weekly in an amount of 1000 μg / patient / dose. Equivalent doses of antibody fragment should be used at more frequent intervals to maintain serum levels in excess of concentrations that allow saturation of the Fuc-GM1 carbohydrate. The dose of the composition will depend on the properties of the binding member, such as its binding activity and in vivo plasma half-life, the concentration of the polypeptide in the formulation, the route, site and rate of administration, the clinical tolerability of the patient involved, the medical condition the patient is suffering from, etc., as is well within the skill of the physician. For example, a dose of 300 μg antibody / patient / dose is preferred, although doses may range from about 10 μg to 6 mg / dose. Different doses are utilized during a series of successive inoculations; the physician may administer an initial inoculation followed by a booster inoculation with a relatively smaller antibody dose.

[0068] The present invention is also directed to optimized immunization schedules for promoting a protective immune response against cancer.

[0069] The binding members of the present invention may be made wholly or partly by chemical synthesis. They may be readily prepared according to well-established standard solution-phase, or preferably solid-phase peptide synthesis methods, general descriptions of which are widely available (see, for example, JM Stewart and JD Young, 1984 (Stewart and Young 1984), M. Bodanzsky and A. Bodanzsky, 1984 (Bodanzsky and Bodanzsky 1984)), or they may be prepared in solution by solution-phase methods, or by any combination of solid-phase, solution-phase and solution chemistry, for example by first completing the respective peptide moiety and then, if desired and appropriate, after removing any protecting groups present, introducing residue X by reaction of the respective carboxylic or sulfonic acid or reactive derivative thereof.

[0070] Another convenient way of producing a binding member according to the invention is to express nucleic acid encoding it, by use of nucleic acid in an expression system.

[0071] The present invention further provides isolated nucleic acids encoding specific binding members of the invention. Nucleic acids include DNA and RNA. In a preferred aspect, the present invention provides nucleic acids encoding specific binding members of the invention as defined above. Examples of such nucleic acids are shown in Figures 2 and 3. The skilled artisan will be able to determine substitutions, deletions and / or additions to such nucleic acids which still provide a specific binding member of the invention.

[0072] The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes comprising at least one nucleic acid as above. The present invention also provides recombinant host cells comprising one or more constructs as above. As mentioned, nucleic acids encoding the specific binding members of the present invention form an aspect of the invention, as do methods of producing specific binding members comprising expression from encoding nucleic acid. Expression may conveniently be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the specific binding member may be isolated and / or purified using any suitable technique and then used as appropriate.

[0073] Systems for cloning and expression of 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 available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common and preferred bacterial host is E. coli. Expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see e.g. Pluckthun, 1991 (Pluckthun 1991). Expression in eukaryotic cells in culture is also available to the skilled artisan as an option for producing specific binding members, see e.g. Reff, 1993 (Reff 1993); Trill et al., 1995 (Trill, Shatzman and Ganguly 1995) for the most recent reviews.

[0074] A suitable vector can be selected or constructed, containing appropriate control sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences, as needed. The vector can be a plasmid, a virus, such as a phage or a phagemid, as needed. For further details, see, for example, Sambrook et al., 1989 (Sambrook 1989). Many known techniques and protocols for the manipulation of nucleic acids, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and analysis of gene expression and proteins, are detailed in Ausubel et al., 1992 (Ausubel 1992).

[0075] Thus, a further aspect of the invention provides a host cell containing a nucleic acid as disclosed herein. Yet a further aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction may use any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses, such as vaccinia, or in the case of insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. After introduction, expression from the nucleic acid may be caused or allowed, for example by culturing the host cells under conditions for expression of the gene. The nucleic acids of the invention can be integrated into the genome (e.g., chromosome) of a host cell. Integration can be facilitated by the inclusion of sequences that facilitate recombination with the genome, according to standard techniques.

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

[0077] Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis.Prior art documents referred to herein are incorporated to the fullest extent permitted by law.

[0078] The FL133 / 4 mAb disclosed herein exhibited potent in vitro cytotoxic activity through ADCC and CDC.

[0079] The invention will now be further described in the following non-limiting examples and the accompanying drawings. [Brief description of the drawings]

[0080] [Figure 1] Biosynthetic pathway of Fuc-GM1 glycolipid. Cer, ceramide; Fuc, fucose; FucGm1, fucosyl-GM1; Gal, galactose; GalNac, N-acetylgalactosamine; Glc, glucose; LacCer, lactosylceramide; SA, sialic acid. [Figure 2a] Amino acid and nucleotide sequence of the murine FL133.63 IgG3 heavy chain variable domain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 2b] Amino acid and nucleotide sequence of the murine FL133.67 IgG3 heavy chain variable domain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 2c] Amino acid and nucleotide sequence of mouse FL134.33 IgG1 heavy chain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 2d] Amino acid and nucleotide sequences of mouse FL133.63 kappa chain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 2e]Amino acid and nucleotide sequences of mouse FL133.67 kappa chain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 2f] Amino acid and nucleotide sequences of mouse FL134.33 kappa chain. Numbers refer to the standardized IMGT system for numbering antibody sequences (Lefranc et al. 2009). [Figure 3a] Amino acid and nucleotide sequences of the mouse FL134.33 heavy chain variable region chimerized to a human IgG1 heavy chain constant region. [Figure 3b] Amino acid and nucleotide sequences of the mouse FL134.33 kappa chain variable region chimerized to the human kappa chain constant region. [Figure 4] Schematic representation of the ozonolysis of the GSL Fucosyl GM1 and subsequent conjugation to a protein carrier via reductive amination. Reactive ozone attacks the double bond of the sphingosine moiety of the GSL, generating a stable free aldehyde group that can then be used to conjugate the GSL to a protein by a step of reductive amination. Here, the aldehyde reacts with a primary amine to first form an unstable Schiff base, which must be further reduced to form a stable secondary amine. [Diagram 5] Fourier transform mass spectrometry of ozonolyzed and original Fucosyl GM1. A) Ozonolyzed and B) original Fucosyl GM1 were examined by FTMS and analyzed using a high resolution mass spectrometry system, Exactive. The analysis shows various Fucosyl GM1 species depending on the length of the fatty acyl chain. The Fucosyl GM1 ion was singly charged. The legend indicates the molecular weight of the species present. [Figure 6]Detection of HSA-FucosylGM1 conjugates by ELISA and Western blot analysis. A) Three samples of HSA-FucosylGM1 conjugates detected by ELISA reading absorbance at 450 nm against a background of 620 nm. Plates were coated with either HSA or HSA-FucosylGM1 conjugates. Primary antibodies were either anti-HSA IgG antibodies (MOD6 6L) or mouse serum containing anti-FucosylGM1 mAb F12. Primary antibodies were detected using anti-mouse IgG-biotin and streptavidin-HRP. Wells to which no primary antibody was added served as negative controls. B) Western blot analysis of three samples of HSA-FucosylGM1 conjugates detected by either anti-HSA IgG antibodies (MOD6 6L) or mouse serum containing anti-FucosylGM1 mAb F12. Lanes 1-4 were used as controls, lane 1 = original HSA, lane 2 = HSA obtained through the reactive amination procedure but without gangliosides added, lane 3 = HSA-Lewis Y conjugate, lane 4 = HSA-GD3 conjugate, lane 5 = HSA-Fucosyl GM1 conjugate sample 1, lane 6 = HSA-Fucosyl GM1 conjugate sample 2 and lane 7 = HSA-Fucosyl GM1 conjugate sample 3. Primary antibodies were detected using anti-mouse IgG-HRP. An identical membrane blotted with only the secondary antibody was used as a negative control. Positive bands were developed by ECL. [Figure 7] Binding of mouse serum IgG to HSA-Fuc-GM1 conjugates and purified Fuc-GM1. Mouse serum IgG was screened for binding to either HSA-Fuc-GM1 conjugates (A, C, E, G) or purified Fuc-GM1 (B, D, F, H). Mouse serum was analyzed by ELISA reading absorbance at 450 nm against a 620 nm background. Secondary antibody alone was used as a control. Mouse serum containing anti-HSA IgG antibody and anti-Fuc-GM1 mAb F12 was used as a positive control. * denotes p<0.05 compared to no primary antibody, only 1 / 100 dilution was analyzed. [Figure 8]Binding of mouse serum IgG to the cell surface of DMS79 cells. Binding of mouse serum IgG to the cell surface of the Fuc-GM1 positive cell line DMS79. Cells were first incubated with mouse serum at 1 / 100 dilution. Binding of the primary antibody was detected using anti-mouse IgG FITC at 10 μg / ml. Cells were analyzed by flow cytometry. Isotype IgG1 was used as a negative control (data not shown) and mAb F12 was used as a positive control, both at 10 μg / ml. The intensity of antibody binding is expressed by the geometric mean. [Figure 9] Binding of Fuc-GM1-specific mAbs to the cell surface of Fuc-GM1 expressing cells. Binding of purified mAbs, FL133.63, FL133.67 and FL134.33, to the cell surface of Fuc-GM1 positive cell lines DMS79, DMS53, H128 and H69 was assessed by indirect immunofluorescence and flow cytometry analysis. Surface binding was detected using anti-mouse IgG FITC. Isotype IgG1 was used as a negative control (data not shown) and mAb F12 was used as a positive control. The intensity of antibody binding is expressed as the geometric mean. [Figure 10] Binding of Fuc-GM1-specific mAbs to whole blood of healthy human volunteers. Binding of A) isotype IgG1, B) isotype IgG3, C) anti-MHC class I mAbs, D) mAb F12, E) mAb FL133.63, F) mAb FL133.67 and G) mAb FL134.33 to whole blood of healthy human volunteers assessed by indirect immunofluorescence and flow cytometry analysis. Whole blood was first incubated with 10 μg / ml of primary antibody and then detected with 10 μg / ml of anti-mouse IgG FITC. Red blood cells were lysed before analysis. Mouse IgG1 isotype, mouse IgG3 isotype and mAb F12 were used as negative controls. Anti-MHC class I mAbs were used as positive controls. Negative controls were used to set up dot plot quadrants. [Figure 11]Binding of Fuc-GM1 mAb to various purified glycolipids. Binding of A) FL133.63, B) FL133.67, C) FL134.33 to various purified gangliosides analyzed by ELISA reading absorbance at 450 nm against a background of 620 nm. Primary antibody binding was detected by biotinylated anti-mouse IgG and streptavidin-HRP. D) F12, E) anti-GD3 mAb R24 and F) anti-GM1 toxin CTxB-HRP were used as positive controls. MAbs against the remaining gangliosides have not yet been generated. * denotes p<0.05 compared to lactosylceramide, only 1 / 100 dilution was analyzed. [Figure 12] Competitive binding of Fuc-GM1 mAb preincubated with purified ganglioside Fuc-GM1 to the DMS79 cell line. 2 μg of mAb was first incubated with 5 μg of purified Fuc-GM1 for 1 h at room temperature. Binding to the cell surface of DMS79 cells was assessed by indirect immunofluorescence and flow cytometry analysis. Isotype IgG1 (data not shown) and MHC I were used as negative controls. F12 was used as a positive control. A primary antibody that was not preincubated with purified Fuc-GM1 was also used as a positive control. The intensity of antibody binding is expressed as the geometric mean. [Figure 13] FL133.63 and FL134.33 were screened for binding against a glycan array from The Consortium for Functional Glycomics consisting of 610 mammalian glycan targets. Comparing fine specificity between a) FL133.63, b) FL134.33. [Figure 14]Binding of Fuc-GM1 mAb to purified Fuc-GM1 and DMS79 PM TGL. Binding of A) FL133.63, B) FL133.67 and C) FL134.33 to purified ganglioside Fuc-GM1 (5 μg / lane) and GM1 (5 μg / lane) to DMS79 PM TGL (1×107 cells / lane). Binding was analyzed by TLC analysis with immunodetection. Primary antibodies were used at 1 μg / ml. Primary antibody binding was detected by IRDye 680CW anti-mouse IgG used at 1 / 10000. D) TLC plate immunoblotted with anti-Fuc-GM1 mAb F12 was used as positive control. E) TLC plate immunoblotted with secondary antibody only was used as negative control. [Figure 15] Binding of anti-Fuc-GM1 mAbs to HSA-Fuc-GM1 conjugates. Binding of A) mAb FL133.63, B) mAb FL133.67, C) mAb FL134.33 D) mAb F12 to HSA-Fuc-GM1 conjugates was analyzed by ELISA reading absorbance at 450 nm against a 620 nm background. Primary antibodies were detected using anti-mouse IgG biotin and streptavidin HRP conjugates. Original HSA was used as a negative control. MAb F12 was used as a positive control. [Figure 16] Binding of anti-Fuc-GM1 mAbs to purified Fuc-GM1 and DMS79 cells. Anti-Fuc-GM1 mAbs (FL133.63, FL133.67 and FL134.33) were evaluated by ELISA for binding to purified Fuc-GM1 in the concentration range of 100-0.001 nM. Isotype IgG1 was used as a negative control. EC50 values ​​were established using nonlinear dose-response curve fitting (GraphPad Prism). The mAbs were also evaluated for binding to the SCLC cell line DMS79 by flow cytometry using the same concentration range. Isotype IgG1 was used as a negative control. Kd values ​​were established using nonlinear binding curve fitting (single-site specific binding) in Graphpad Prism. [Figure 17]Fuc-GM1 mAb-mediated ADCC and CDC of DMS79 cells. MAbs FL133.63, FL133.67 and FL134.33 mediated A) ADCC and B) CDC of the Fuc-GM1 positive cell line DMS79. Anti-Lewisy / bmAb SC101 was used as a positive control. * denotes p<0.05. [Figure 18] Sequences of the VH regions of mAbs FL134.33, FL133.63 and FL133.67. Germline antibody sequences are shown as amino acid codons. Mutations in each Fuc-GM1 specific antibody are shown below. [Figure 19] Sequences of the VL regions of mAbs FL134.33, FL133.63 and FL133.67. Germline antibody sequences are shown as amino acid codons. Mutations in each Fuc-GM1 specific antibody are shown below. [Figure 20] Specificity of antibody binding to purified antigen by ELISA. Fuc-GM1 coated plates were incubated with antibodies and detected using anti-mouse Fc specific or anti-human gamma chain specific IgG biotin and streptavidin HRP. MAbF12 was used as a positive control. [Figure 21] Assessment of antibody cell surface binding by indirect immunofluorescence. mAbs FL134.33, FL133.63, FL133.67 and CH134.33 were incubated with cells and binding was detected using anti-mouse Fc specific or anti-human gamma chain specific IgG FITC. A) Histograms generated by flow cytometry with the cell lines. B) Bar graph of Gm values ​​for all cell lines. MAb F12 was used as a positive control. EXAMPLES

[0081] method All experiments were performed in accordance with applicable safety regulations. Experimental methods were performed according to standard operating procedures with modifications for specific conditions.

[0082] immunity Animal work was performed under a Home Office project license. All reagents used were purchased from Sigma-Aldrich (Poole, UK) unless otherwise stated. All immunization experiments were performed using either Balb / c or CD1 mice aged 6–8 weeks. Mice were bred and maintained under specified pathogen-free conditions by staff at the Biomedical Services Unit, University of Nottingham. Mice were immunized with antigen diluted in up to 100 μl PBS either subcutaneously (sc) or intraperitoneally (ip) using a 0.5 ml insulin syringe. The frequency of injections varied for each immunization and is outlined in the appropriate results section. Mice were bled via the tail vein 7 days after each immunization to assess mouse antibody responses to immunization. Blood was centrifuged twice for 2 min at maximum speed (17968×g) (SIGMA, 1-15PK Microfuge, Osterode am Harz, Germany) to remove red blood cells and serum was stored at −20° C. until further use.

[0083] cell culture All tissue cultures were performed aseptically in a class II safety cabinet. Human-derived cancer cell lines were maintained in Roswell Park Memorial Institute medium (RPMI1640) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), except for H128, which was maintained in RPMI1640 supplemented with 20% HI-FBS, and DMS53, which was maintained in Weymouth medium supplemented with 10% HI-FBS. Freshly purchased cell lines were delivered on dry ice and were kept frozen at -80°C. To remove the cell culture freezing medium, cells were thawed in a 37°C water bath and transferred to a 30 ml universal container (Sterilin, Newport, UK) with 10 ml of the appropriate culture medium. Cells were centrifuged at 1000g for 5 min. The supernatant was then removed and the cell pellet was resuspended in 7 ml of the appropriate culture medium. Cells were then transferred to a T25 Cellstar cell culture flask (VWR, Lutterworth, UK) and incubated at 37°C in 5% CO2. Once the cells reached approximately 80% confluency, they were split or transferred to a larger cell culture flask. For adherent cell lines, the spent culture medium was aspirated from the flask and 5ml of 1x trypsin / EDTA was added. Trypsinized cells were incubated at 37°C until the cells detached (usually around 10 min). The detached cells were then collected in a 30mL universal vessel and 5ml of culture medium was added to reduce the effect of trypsin on the cells. The cells were then centrifuged at 1000g for 5 min. The supernatant was removed and the cell pellet was resuspended in fresh culture medium. The cells were then transferred to a T75 Cellstar culture flask. Cells growing as floating aggregates were split after their culture medium was consumed (discolored from orange to yellow) or when the cells appeared unhealthy / started to die. The cells with culture medium were transferred directly to a T75 culture flask and 10ml of fresh culture medium was added. To expand and maintain stocks of newly purchased cell lines, cells were harvested from a T75 culture flask at 80% confluency by the method described above. Cells were counted using a hemocytometer and trypan blue viability stain applied at a 1:1 ratio. 5 x 10 cells were cultured in 5% dimethylsulfide (DMSO) / appropriate culture medium.6 The cells were resuspended at 500 / ml and 1 ml was added per 2 ml microtube (Sarstedt, Leicester, UK). The vials were first placed in a -80°C freezer for a minimum of 24 hours, then transferred to liquid nitrogen and kept at -170°C.

[0084] When larger stocks of live cells were required for various experiments, the cells were gradually expanded into T175 culture flasks and kept healthy by replacing spent culture medium with fresh and splitting to ensure log-phase growth of the cells. When working with non-adherent cell lines, the cells were transferred to the universal vessel and left to stand for 5 minutes. This allowed the floating aggregates containing live cells to sink to the bottom of the universal vessel, and the supernatant containing dead cells was then removed. The cell pellet was then resuspended in the appropriate culture medium and the cells were returned to the culture flask.

[0085] antibody Anti-HLA-ABC (clone W6 / 32) and anti-CD44 were purchased from eBiosciences (Hatfield, UK). Anti-CD59 (BRIC 229) was purchased from IBGRL (Bristol, UK). Anti-sialyl Lewis a (CA19.9), anti-Lewis b (2-25 Le), anti-Lewis X (P12), anti-GD2 (clone 2Q549), anti-GM2, anti-Gb3, and anti-prohibitin (clone II-14-10) were purchased from Abcam (Cambridge, UK). Anti-NeuGc GM3 (clone M2590) was purchased from COSMO BIO CO., LTD (Tokyo, Japan). Anti-CD46, anti-CD14 (clone M5E2), anti-CD11c (clone B-ly6), and IgG1 isotype were purchased from BD Biosciences (Crawley, UK). Anti-Lewis Y (BR96) was purchased from ATCC (Middlesex, UK). Anti-sialyl Lewis X (KM93) and anti-CA125 (OC125) were purchased from Calbiochem (Darmstadt, Germany). Anti-Globo H (clone MBr1) was purchased from ENZO Life Sciences (Exeter, UK). Anti-Fuc-GM1 (F12) was purchased from Fujirebio (Tokyo, Japan). Anti-transferrin (UNCONJ) was purchased from Invitrogen (Paisley, UK). Anti-EGFR (IF4) and anti-calnexin were purchased from Cell Signalling Technology (Danvers, MA, USA). Anti-EpCAM (BerEP4) was purchased from Dako (Cambridgeshire, UK). Anti-EGFR (Erbitux) and anti-Her2 (Herceptin) were kind gifts from the NHS. Anti-GD3(R24) was a gift from Philip O. Livingston from Memorial Sloan Kettering Cancer Center. Streptavidin-HRPO conjugate was purchased from Invitrogen (Paisley, UK). Rabbit anti-mouse immunoglobulin FITC and swine anti-rabbit IgG1 FITC were purchased from Dako (Cambridgeshire, UK).Streptavidin PE was purchased from eBiosciences (Hatfield, UK). Cholera toxin, B subunit biotin was purchased from Stratech Scientific Ltd., (Newmarket, UK). IRDye 680RD donkey anti-mouse and IRDye 800CW streptavidin were purchased from LI-COR, Nebraska, USA.

[0086] Antigen preparation The dried cell pellet (washed once with phosphate-buffered saline (PBS)) was stored in a -80 °C freezer until use. To extract the plasma membrane (PM), 2 × 10 cells were 8 Cell pellets from each were pooled together and manually homogenized in 2 ml of 50 mM mannitol / 5 mM Hepes (pH 7.4) and 10 mM calcium chloride solution using successively smaller needles (23G, 25G and finally 29G). Samples were then placed on ice for 20 min and then centrifuged at 3000×g for 15 min to remove intracellular membranes and nuclei. The supernatant was transferred to a polyallomer ultracentrifuge tube (Beckmann, High Wycombe, UK) and centrifuged at 48,000×g for 30 min at 4° C. to separate the PM-containing pellet from the supernatant containing the cytosol. The pellet was then resuspended in 200 μl of PBS and stored at −20° C. until use.

[0087] PM TGL was extracted by adding 2 ml of methanol and 2 ml of chloroform either directly to the PM pellet in a polyallomer ultracentrifuge tube or to PM suspended in 200 μl of PBS. The material was then transferred to a 15 ml polypropylene tube, vortexed thoroughly, and incubated on a roller for 30 min at room temperature (RT). After centrifugation at 2000×g for 10 min, the TGL-containing supernatant was collected and incubated overnight at −20° C. The next day, the samples were centrifuged again with the same settings and the supernatant was collected and stored at 4° C.

[0088] Adjuvants Various adjuvants were used in the course of this thesis to promote immune responses against antigens. During the formulation of liposomes, α-GalCer (alpha-galactosylceramide), α-GalCer analog 7 or α-GalCer analog 8, (ENZO Life Sciences, Exeter, UK) were dried together with the rest of the lipids (10 μg-25 μg / mouse). Anti-mouse CD40 (R&D Systems, Abingdon, UK), C-type CpG oligonucleotides and monophosphoryl lipid A (MPLA) from S. mennesota (InvivoGen, San Diego, CA, USA) were used at 10 μg / mouse and mixed into the already formed liposomes. Freund's complete or incomplete adjuvants were used to promote immune responses against protein antigens. These were mixed with the antigens in a 1:1 ratio (v:v).

[0089] Conjugation of purified gangliosides to proteins Purified gangliosides were covalently conjugated to HSA by indirect reductive amination, in which aldehyde-containing gangliosides react with amine (lysine)-containing proteins to form stable amine bonds. To introduce reactive aldehyde groups into gangliosides, a previously described ozonolysis method (Song et al., 2011) was used. Reactive ozone was freshly generated from dry air (Air drier MAG-600, Ozone Solutions, Hull, Iowa, US) by an ozone generator OZV-8 (Ozone Solutions). Ozone was passed through glycolipid-containing samples resuspended for 1 min in a minimum volume of 500 μl of 2:1 chloroform:methanol, and the blue color persisted. 50 μl of dimethyl sulfide was then added to destroy residual ozone, and after 1 h of incubation at room temperature, the solution was dried under a stream of nitrogen. Ozonolyzed Fuc-GM1 (Matreya, PA, USA) resuspended in DMSO was added in 10-fold molar excess to human serum albumin (HSA) resuspended in carbonate-bicarbonate buffer, pH>9. 10 μl of 5 M sodium borohydride (HBNa) in 1 M NaOH was then added per ml of reaction. Each sample was then incubated for 2 hours at room temperature. Unreacted aldehyde sites were blocked by adding 20 μl of 3 M ethanolamine per ml of reaction for 15 minutes. The conjugate was purified by dialysis against PBS.

[0090] Protein-Fuc-GM1 conjugates were analyzed by total protein MALDI-TOF analysis. To prepare samples, ziptip C4 pipette tips (Millipore, MA, US) were first activated with 50% (v / v) acetonitrile:H2O solution and then equilibrated with 0.1% TFA in H2O. Samples were then bound to the resin of the ziptip by pipetting up and down 15 times. The resin-bound samples were then washed 15 times with 0.1% TFA in H2O and dispensed onto the MALDI plate with elution solution (saturated sinapinic acid in 50% (v / v) acetonitrile:H2O with 0.1% TFA).

[0091] Generation of mAbs Several flasks with NS0 bone marrow cells were grown in RPMI medium supplemented with 10% HI-FCS at least one week prior to fusion. The flask containing the healthiest looking cells was selected and refed with culture medium one day prior to fusion. The removed spent NS0 medium was stored at -4°C. On the day of fusion, all reagents were first heated to 37°C. NS0 cells were harvested and counted four times. They were then washed using serum-free RPMI by centrifugation twice at 1000 x g for 5 min. The immunized mice were sacrificed and the spleens were removed using 70% (v / v) ethanol to keep the incision site sterile. A single cell suspension was prepared by pushing RPMI medium through a needle into the spleen and simultaneously applying pressure with forceps. The splenocytes were then centrifuged in RPMI medium at 1000 x g for 10 min and counted four times. The splenocytes were then fused with NS0 bone marrow cells at a 10:1 cell ratio. More specifically, 1 x 10 8 1 x 10 splenocytes and 1 x 10 7 NS0 bone marrow cells were combined and centrifuged at 1000×g for 5 min. The supernatant was removed and 800 μl of polyethylene glycol 1500, which serves to permeabilize the cell plasma membrane, was gently mixed into the cell pellet for 1 min. Next, 1 ml of RPMI medium was slowly added to the cells over 1 min with gentle mixing. An additional 5 ml of RPMI was layered slowly over the cells for 5 min. Finally, an additional 20 ml of RPMI was layered on top. To aid in the fusion process, the cells were then centrifuged at 1200×g for 7 min. The supernatant was removed and the cells were carefully resuspended in RPMI medium supplemented with 10% HI-FBS, 5% hypoxanthine methotrexate thymidine (HMT selection reagent; Invitrogen, Paisley, UK), 5% hybridoma cloning factor (HCF; PAA, Piscataway, US) and 10% filtered spent NS0 supernatant. The cells were then seeded into 96-well culture plates (Thermo Fisher Scientific, Rockford, UK) and incubated at 37°C and 5% CO2.

[0092] Approximately two weeks after fusion, when the established hybridomas occupied approximately 1 / 3 of the wells, they were screened for antigen-specific antibody production by enzyme-linked immunosorbent assay (ELISA). Hybridoma colonies producing antibodies with the desired specificity were selected for further cloning, the aim of which was to generate single-cell colonies, a critical step in monoclonal antibody production. To do this, the selected hybridomas were seeded into 96-well culture plates at a cell density of 0.3 cells / well. The hybridomas were incubated at 37°C, 5% CO2 until the colonies were large enough to be screened again. Cloning was repeated a minimum of two times until all clones showed positive binding to the antigen.

[0093] MAb purification The double-cloned hybridomas were expanded in GIBCO Hybridoma Serum-Free Medium (Life Technologies, Paisley, UK) supplemented with 10% low Ig newborn calf serum (Life Technologies). Spent medium was collected and centrifuged at 2000×g for 15 min to remove hybridoma cells along with cell debris. The antibody-containing supernatant was then filtered through a 0.2 μm Minisart disposable filter (Sartorius Stedim, Surrey, UK). Antibodies were purified by FPLC using a 1 ml recombinant protein G column (GE Healthcare, Buckinghamshire, UK), eluted with 100 mM glycine pH 12, neutralized and dialyzed overnight against 2 L PBS. The final concentration of purified antibodies was determined by spectrophotometer, reading absorbance at 280 nm. A mouse monoclonal antibody isotyping test kit (ABD Serotec, Kidlington, UK) was used according to the manufacturer's guidelines.

[0094] Enzyme-linked immunosorbent assay (ELISA) For glycolipid ELISA, 96-well flexible PVC flat-bottom plates (BD biosciences, Oxford, UK) were loaded with 100 ng purified ganglioside / well or 1 × 10 cells. 4 5 x 10 cells / well equivalent to WC TGL extract or resuspended in 100% ethanol 4 Each well was coated with either PM TGL or equivalent and allowed to dry overnight at room temperature.

[0095] Purified gangliosides were resuspended at 1 mg / ml in 2:1 (v / v) chloroform:methanol. Extraction of PM TGL was described above in antigen preparation. WC TGL was prepared by adding 1 ml methanol and 1 ml chloroform to the dried cell pellet. Cells were vortexed vigorously and incubated on a roller for 30 min at RT. After centrifugation at 2000×g for 10 min, the TGL-containing supernatant was collected and stored at −20° C. overnight. The next day, samples were centrifuged again and the supernatant was collected and stored at 4° C.

[0096] For protein ELISA, 96-well flexible PVC flat-bottom plates were coated with 100 ng protein / well resuspended in carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. The next day, plates were blocked with 2% bovine serum albumin (BSA) in PBS for 1 h at RT. The blocking buffer was then removed and primary antibodies or mouse serum prepared in 1% BSA in PBS was then added to the wells. After 1 h of incubation at RT, plates were washed three times in PBS. Primary antibody binding was detected by appropriate secondary and tertiary antibodies. Plates were then washed and developed by adding 90 μl per well of 3,3'5,5'-tetramethylbenzidine (TMB) in phosphate / citrate / perborate buffer. The reaction was stopped by adding 30 μl of 2M H2SO4. The results were analyzed by reading the absorbance at 450 nm against a 620 nm background in a spectrophotometer. The mean and standard deviation of the data were calculated using Microsoft Excel. Significant differences between groups were calculated using ordinary one-way ANOVA with multiple comparisons against the negative control, and p<0.05 was considered statistically significant.

[0097] Glycome analysis To clarify the fine specificity of FL133 and 134 mAbs, the antibodies were FITC-labeled and sent to the Consortium for Functional Glycomics (http: / / www.functionalglycomics.org / static / consortium / resources / resourcecoreh8.shtml) where they were screened against >600 natural and synthetic glycans. Briefly, synthetic and mammalian glycans were printed on N-hydroxysuccinimide (NHS)-activated glass microscope slides with amino linkers to form amide bonds. This work was performed by Core H of CFG at Emory University. FG27 was tested at 1 μg / ml in PBS at room temperature. Briefly, antibody samples were applied to the printed surface of the microarray and incubated for 1 h in a humidified chamber. The slides were then rinsed 4 times with PBS, followed by the addition of fluorescently labeled (Alexa Fluor 488) anti-rabbit IgG and incubation for 1 h. The slides were then rinsed four times in PBS and fluorescence was measured on a Perkin-Elmer microarray XL4000 scanner and analyzed using Imagene software (BioDiscovery).

[0098] Further details of the protocol used are given below: Glycan-binding assay with unlabeled monoclonal antibodies 1. Introduction: The primary objective of 1.1.Core H is to determine the binding specificities of researcher-submitted glycan-binding proteins (GBPs) and various organisms using printed glycan microarrays.

[0099] 2. References: www.functionalglycomics.org

[0100] 3. Materials needed: 3.1. Glycan printed slide (Core D) with a barcode imprinted in white and the words -DO NOT TOUCH THIS AREA- printed in black on the side of the slide. 3.2. Coverslip (Fisher Scientific, 12-545F) 3.3. A humidified slide processing chamber (Fisher Scientific, NC9091416) or a homemade system using a Petri dish with a damp paper towel placed at the bottom of the chamber. 3.4. 100ml Coplin Jar for Washing Slides 3.5.Tris-HCl (Fisher scientific, BP152-1) 3.6.NaCl(Fisher scientific,S271-3) 3.7.CaCl2(Fisher scientific,C79-500) 3.8.MgCl2(Fisher scientific,BP214-500) 3.9. Potassium phosphate monobasic (Fisher scientific, P285-3) 3.10.dH20 3.11. Cyanine 5-streptavidin (ZYMED 43-4316) 3.12. Appropriate secondary antibodies, fluorescently labeled if necessary 3.13.BSA(Fisher scientific,Bp1600-100) 3.14.Tween-20(EMD Biosciences,655205) 3.15. Sodium azide (Fisher Scientific, S227-500) 3.16.ProScanArray Scanner(Perkin Elmer) 4.Buffer: 4.1.TSM=20mM Tris-HCl, pH7.4 150mM NaCl, 2mM CaCl2, 2mM MgCl2 4.2. TSM Wash Buffer (TSMW) = TSM buffer + 0.05% Tween-20 4.3.TSM Binding Buffer (TSMBB)=TSM Buffer+0.05%Tween20+1%BSA 5. Protocol: 5.1. Make a working stock of wash buffer (TSM, TSM wash buffer and HO) or collect reagents and bring to room temperature if they are in the refrigerator. 5.1.1. Buffer (A) TSM-20mM Tris-HCl, pH7.4 150mM NaCl, 2mM CaCl2, 2mM MgCl2 5.1.2. Buffer (B) TSM wash buffer (TSMW) - TSM buffer + 0.05% Tween-20 5.1.3. Buffer (C) TSM binding buffer (TSMBB) - TSM buffer + 0.05% Tween20 + 1% BSA 5.1.4.dH2O 5.2. Prepare 100 μl samples by diluting the antibody in TSMBB or appropriate binding buffer based on the properties of the antibody to a final concentration of 50 μg / ml or the appropriate concentration required for the analysis. 5.3. Remove the slide from the desiccator and write the sample name on the slide outside the black markings, near the barcode. 5.4. Hydrate the slides by placing them in a glass Coplin staining jar containing 100 ml TSMW for 5 minutes. 5.5. Remove excess liquid from the slide by holding the slide upright and allowing the liquid to drain off. 5.6. Carefully apply 70 μl of sample near the left-most slide between the black marks (see 5.2). 5.7. Gently place the coverslip onto the slide to avoid forming air bubbles in the sample under the coverslip. Remove any air bubbles if necessary by gently tapping the coverslip with a pipette tip or by slowly lifting one side of the coverslip. Ensure that the coverslip is between the black marks.

[0101] Thin Layer Chromatography (TLC) 1 μg / lane of purified lipid or WC TGL (1 × 10 cells 6 cells / lane) or PM TGL extracted from cell lines as described above (1 × 10 cells 7 1000 pieces / well) were loaded onto a TLC plate (nanosilica gel on a TLC plate). To aid in loading of large amounts of cell-derived TGL, the TGL was first dried using a heating block set at 60°C and then resuspended in up to 20 μL of 2:1 chloroform / methanol. After loading, the plate was developed twice in solvent 1 (chloroform:methanol:dH2O, 60:30:5) to transfer lipids to a distance of 5 cm and once in solvent 2 (hexane:diethyl ether:glacial acetic acid, 80:20:1.5) to transfer lipids to a distance of 8 cm. Lipids were then visualized either by orcinol (20 mg orcinol in 20 ml of 5% H2SO4) developed at 120°C for 20 min or by immunoblotting.

[0102] For immunoblotting, after transfer of glycolipids, TLC plates were first blocked with a thin spray coat of polyisobutyl methacrylate 50 mg / 50 ml in acetone. Plates were then further blocked by incubation in 1% (w / v) BSA in PBS for 1 h at RT. Plates were then incubated in primary antibody (10 μg in 10 ml per plate) or mouse serum (1 / 100 dilution in 10 ml per plate) and detected with anti-mouse IgG biotin (1 / 1000) and donkey anti-mouse IRD 800 CW (LICOR Biosciences Ltd, Cambridge, UK). Each antibody incubation was performed with gentle rocking and lasted for 1 h. Tertiary antibody incubation was performed in the dark. All antibodies were prepared in 1% (w / v) BSA in PBS. After each antibody incubation, plates were washed by pouring PBS directly onto the plate. Finally, the plates were washed, dried overnight in the dark and analyzed by an Odyssey SA Infrared Imaging System (LICOR).

[0103] Antibody and Complement-Dependent Cytotoxicity Radiolabeled sodium chromate (Cr) released by dying cells due to increased permeability of the plasma membrane of dying cells. 51 ) was used to assess antibody and complement-dependent cytotoxicity in normal saline. 6 Target cells were inoculated with 40 μl (1 mBq) of Cr 51 (Perkin Elmer, Cambridge, UK) and incubated at 37°C for a minimum of 1 hour. After incubation, the labeled target cells were washed twice in 25 ml of SF RPMI medium at 1400 x g for 5 minutes. They were then resuspended in 25 ml of SF RPMI medium and left at 37°C for 20 minutes. They were then centrifuged again to remove the SF RPMI medium, resuspended in 1 ml of culture medium (RPMI supplemented with 10% FCS and 1% penicillin-streptomycin) and counted. Finally, the labeled target cells were washed at 1 x 10 cells. 5 Resuspend the cells at 5 × 10 cells / 1 ml and add 50 μl (5 × 10 cells) 3 100 µl) were added to the appropriate wells of a 96-well round bottom plate. Dilutions of antibody were prepared in culture medium to ensure that the working dilution was 4x the final concentration. A volume of 50 µl of diluted antibody was added to the appropriate wells.

[0104] Peripheral mononuclear cells (PBMCs) were isolated from healthy volunteers on the day of the experiment using heparin-coated vacutainer tubes with green caps (BD, Plymouth, UK). First, whole blood was diluted 1:1 in SF RPMI. To isolate PBMCs, 25 ml of diluted blood was gently layered on top of 15 ml of Histopaque-1077 in a 50 ml Falcon tube. Blood was centrifuged at 2100 × g for 20 min with acceleration set to 1 and deceleration set to 0. The buffy coat containing PBMCs was then collected using a 10 ml pipette and washed twice in 20 ml of SF RPMI at 2000 × g for 5 min. PBMCs were then counted and 5 × 10 cells were collected. 6 The cells were resuspended in 1 ml culture medium and 100 μl of cells were added to the appropriate wells.

[0105] To prepare serum, blood from healthy volunteers was collected in red-capped clotting activator-containing vacutainer tubes (BD, Plymouth, UK). The clotted blood was transferred to a 30 ml universal container and centrifuged twice for 5 min at 2000 × g to ensure removal of all red blood cells. Finally, 20% serum in culture medium was prepared and 100 μl was added to the appropriate wells.

[0106] Maximal cell death was induced by applying one drop (25 μl) of Triton X-100 to 50 μl of labeled target cells and 125 μl of medium. Spontaneous cell death was assessed by incubating 50 μl of labeled target cells in 150 μl of culture medium. To assess the level of direct PBMC-induced killing, 50 μl of target cells were incubated with 100 μl of PBMC and 50 μl of culture medium.

[0107] Plates were incubated at 37°C for 24 hours, after which 50 μl of supernatant was transferred to a Lumaplate and allowed to dry for 24 hours. Lumaplates were then analysed using a TopCount Scintillation Counter (Perkin Elmer, Cambridge, UK).

[0108] Affinity research Affinity constants were determined using the principle of surface plasmon resonance (SPR) with Biacore X (GE Healthcare, Buckinghamshire, UK). Multivalent HSA-Fuc-GM1 conjugates (5 μg / ml in-house conjugate, 686 response units / chip) were coupled to the flow cells of a CM5 biosensor chip via amine coupling according to the manufacturer's instructions. A reference flow cell treated similarly but without conjugate was used as the reference cell. Binding kinetic parameters were determined from several known concentrations of antibody diluted and dialyzed in HBS-P buffer (10 mmol / L HEPES, pH 7.4, 150 mmol / L NaCl, 0.005% surfactant P20) using a Fuc-GM1-coated flow cell at a flow rate of 50 μl / min. Curve fitting software (BiaEvaluation) provided by the Biacore instrument was used to generate estimates of association and dissociation rates from which affinities are calculated using a bivalent analyte model.

[0109] Data analysis One-way ANOVA Dunnett's multiple comparison test was used to determine the statistical significance of the ELISA results. Differences were considered statistically significant at a level of P<0.05. * The symbol indicates P ≤ 0.05. ** The symbol indicates P ≤ 0.01. *** The symbol indicates P ≤ 0.001. **** The symbol indicates P≦0.0001.

[0110] Binding to blood: 50 μl healthy donor blood was incubated with 50 μl primary antibody for 1 h at 4° C. The blood was washed with 150 μl RPMI 10% NBCS and centrifuged at 1,000 rpm for 5 min. The supernatant was discarded and 50 μl FITC-conjugated anti-mouse IgG Fc-specific mAb (1 / 100 in RPMI 10% NBCS) was used as secondary antibody. The cells were incubated in the dark at 4° C. for 1 h, then washed with 150 μl RPMI 10% NBCS and centrifuged at 1,000 rpm for 5 min. After discarding the supernatant, red blood cells were lysed using 50 μl / well Cal-Lyse (Invitrogen, Paisley, UK) followed by 500 μl / well distilled water. The blood was subsequently centrifuged at 1,000 rpm for 5 min. The supernatant was discarded and the cells were fixed using 0.4% formaldehyde. Samples were analyzed on an FC-500 flow cytometer (Beckman Coulter). WinMDI 2.9 software was used to analyze and plot the raw data.

[0111] RNA extraction and cDNA synthesis Approximately 1×10 cells from hybridomas FL134.33, FL133.63, and FL133.67 6 Cells were harvested from tissue culture, washed once in PBS, and treated with 500 μL Trizol (life technologies). Homogenized samples were treated with 0.1 ml chloroform and centrifuged to separate RNA from contaminating DNA and protein. RNA was precipitated using 0.25 ml propan-2-ol and centrifuged to form a small pellet. The pellet was then washed with ethanol 75% and resuspended in RNAse-free water. RNA was treated with DNAase (DNAse I recombinant, RNase-free, Roche) to remove genomic DNA, following the manufacturer's recommendations. Oligo(dT) was purified using 0.1 ml chloroform and centrifuged to form a small pellet. 15First-strand cDNA was prepared from 1 μg of total RNA using the AMV reverse transcriptase kit (Roche Diagnostics. Basel, Switzerland) with primers. After cDNA synthesis, the enzyme was denatured by incubation at 95°C for 5 min. The cDNA was then stored at -20°C.

[0112] Variable region PCR The antibodies were previously evaluated by isotyping test kit (Serotec, Kidlington, UK) and FL134.33 was determined to be of subtype IgG1, and FL133.63 and 133.67 were determined to be IgG3 (data not shown). Variable regions were determined by PCR using previously published primer sets (Jones & Bendig, 1991). There were 13 V for the light chain. K Region-specific primers and one C K PCR amplification was performed using specific primers and 12 VH region specific primers for the heavy chain and 1 constant region subclass specific primer. A 50 μl PCR reaction was set up using 1 U of polymerase (AmpliTaq Gold 360, Applied Biosystems. California, USA), a mixture of 2'-deoxynucleoside 5'-triphosphates (dNTPs) at a final concentration of 0.2 mM each, magnesium chloride at a final concentration of 1.5 mM, and both forward and reverse primers at 1 μM. The hot start step of the reaction was performed at 95°C for 5 min. Then, the amplification was performed for 35 cycles: 94°C for 1 min, followed by 60°C for 1 min and 72°C for 2 min. Finally, a polishing step was performed at 72°C for 20 min. Amplification products were assessed using agarose gel electrophoresis prepared with 1% UltraPure agarose (Invitrogen. Carlsbad, USA) in TAE buffer with ethidium bromide and run at 90 V. Gels were visualized using UV transillumination.

[0113] Purification of PCR products (heavy and light chains) To avoid carryover of residual nucleotides or primers and to obtain a clear sequence graph, the PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen. Venlo, Netherlands). 150 μl of heavy and light chain PCR products were loaded onto a 1% agarose gel and run at 85 volts. DNA fragments were excised from the gel, dissolved and purified on a column according to the manufacturer's protocol. The final concentration of the resulting DNA was determined by UV spectroscopy (Nanodrop, Thermoscientific. Waltham, USA).

[0114] Proofreading PCR Primers for proofreading PCR were designed based on sequence data, maintaining the reading frame and conserving the amino acid sequence. Start codons, Kozak consensus sequences for the initiation of the translation process, and restriction enzyme sites were incorporated to allow integration into dual expression vectors. PCR amplification was performed using cDNA as template with cloning primers and a proofreading polymerase (PHUsion, NEB. Ipswich, UK). The reactions were performed in triplicate to improve product yield. 50 μL reactions were set up as described before. A hot start step was performed at 98°C for 3 min. Then, 35 cycles of amplification were performed: 98°C for 30 s, followed by 58°C for 30 s and 68°C for 60 s. A polishing step was performed at 72°C for 10 min.

[0115] TOPO cloning of PCR products The PCR products generated by the proofreading polymerase were treated with Taq polymerase (NEB) for 15 min at 72°C to add adenine overhangs according to the manufacturer's instructions, cloned into the TA (TOPO) vector (pCR2.1, Invitrogen), and transformed into chemically competent TOP10F cells for subsequent enzymatic digestion and ligation into the pDCOrig-hIgG1 vector. Transformed bacteria were grown on LB agar plates or in liquid medium supplemented with 80 μg / ml ampicillin.

[0116] Nucleic acid purification Plasmid DNA was prepared from overnight cultures of transformed bacteria in liquid culture supplemented with the appropriate antibodies at 37°C and 120 rpm. Small amounts were prepared using a spini miniprep kit (Qiagen) and large amounts were prepared with a plasmid maxi kit (Qiagen), both according to the manufacturer's instructions. DNA purification was performed by agarose gel electrophoresis as previously described and use of a gel extraction kit to recover DNA, according to the manufacturer's procedures.

[0117] Restriction enzyme digestion and cloning of dual expression vectors Enzymatic digestion of DNA was performed by adding 8 units of each restriction enzyme to 10 μL of DNA in the presence of bovine serum albumin (BSA) and each enzyme's own optimal buffer. Incubation was performed for 2 hours at the activity temperature of each enzyme. The dual expression vector PDCOrig-hIgG1 and the light chain insert were digested, purified on agarose gel, and ligated overnight at 16°C using T4 DNA ligase (NEB) according to the manufacturer's instructions. A second digestion of the vector and the heavy chain was followed by another purification via gel extraction and a second ligation overnight at 16°C. After each digestion, the vector was transformed into chemically competent TOP10F cells (Invitrogen). Transformed bacteria were grown on LB agar plates or in liquid medium supplemented with 35 μg / ml Zeocin (Invivogen).

[0118] Sequencing and transfection of chimeric antibody vectors PCR products and plasmids were sequenced using appropriate 5' and 3' primers at the University of Nottingham DNA sequencing facility and analyzed using the GeneTool software package and the IMGT database for mouse immunoglobulin nucleotide sequences. CHO cells were transfected using Lipofectamine (Invitrogen) and Opti-mem serum-reduced medium (Gibco, Life technologies. Waltham, USA). Cells were cultured in suspension in CHO-S-SFMII medium (Gibco) until the medium was consumed. The supernatant was collected and the antibody was purified by preparative chromatography (Akta FPLC, GE Healthcare, Little Chalfont, UK) using a Protein-G Sepharose pre-packed column (HiTrap Protein G HP, GE Healthcare). After purification, the antibody was dialyzed and the final yield was determined by UV spectroscopy.

[0119] Example 1 - Generation and initial characterization of FUC-GM1 mAb: Mice were immunized with classical liposomes containing 10 μg Fucosyl-GM1 per immunization of mice administered ip. Alpha-GalCer was used as the adjuvant in the first and third immunizations, while anti-CD40 mAb was used as the adjuvant in the second and fourth immunizations. All mice received four immunizations. The first three immunizations were given at two-week intervals, and the last immunization was given four weeks after the third immunization. From the second immunization onwards, mice were bled one week after immunization and sera were screened by ELISA for IgG and IgM binding to purified Fucosyl-GM1. After immunization, mice developed a significant IgG immune response to Fucosyl-GM1 with an endpoint titer of 1 / 100. Mouse serum IgG was also assessed for binding to the cell surface of the Fucosyl-GM1 expressing DMS79 cell line. Unfortunately, only weak binding was observed to the cell line. It is possible to presume that the mouse sera recognized an epitope on the Fucosyl-GM1 antigen that is inaccessible once the lipid is incorporated into the plasma membrane of the cell.

[0120] As purified glycolipids incorporated into liposomes were unable to generate high titer IgG responses, other immunization methods were evaluated. Traditionally, carbohydrates and glycolipids are classified as T cell independent antigens that elicit mainly IgM antibody responses. High affinity IgG antibody responses arise mainly in response to protein antigens. It was therefore hypothesized that associated T cell help would generate larger and more consistent high affinity IgG antibody responses against carbohydrate and glycolipid antigens. Fucosyl GM1 was therefore conjugated to human serum albumin (HSA).

[0121] The conjugation of fucosyl GM1 to proteins followed a two-step process. First, purified ganglioside fucosyl GM1 was subjected to ozonolysis, where reactive ozone oxidizes the carbon-carbon double bond present in the sphingosine moiety of glycosphingolipids to generate free aldehyde groups. In the next step, the ozonolyzed fucosyl GM1 was conjugated to HSA by a step of reductive amination, which allows the newly generated aldehydes of ozonolyzed fucosyl GM1 to react with the primary amine groups of lysines in HSA, first forming a labile Schiff base, which, when reduced by sodium borohydride, forms a highly stable secondary amine bond. A schematic representation of the preparation of the conjugate is illustrated in (Figure 4).

[0122] After ozonolysis, samples from both original and ozonolyzed fucosyl GM1 were analyzed by Fourier transform mass spectrometry (FTMS). The original sample contained several species of fucosyl GM1 with varying fatty acid chain lengths. The molecular weight of the most abundant species was 1,746 Da. The ozonolysis sample also contained species with fatty acid chains of various sizes. In the ozonolysis fucosyl GM1 sample, the molecular weight of the main species was 1,566 Da, showing a mass shift of exactly 180 Da, which corresponds to the loss of the sphingosine moiety. Furthermore, the original fucosyl GM1 species was not detectable in the ozonolysis sample (Figure 5). Ozonolysis fucosyl GM1 resuspended in DMSO was added to HSA resuspended in carbonate-bicarbonate buffer. To this, a reducing agent, sodium borohydride (HBNa), was added and the reaction was incubated at room temperature for 8 hours and then resuspended in 100 μl of PBS / EDTA buffer. Sulfo-MBS crosslinker was then added and incubated for 1 hour at room temperature.

[0123] The success of the production of HSA-fucosyl GM1 conjugates was evaluated by ELISA, Western blotting and mass spectrometry. In ELISA, mouse serum containing IgG antibodies against HSA (1 / 10,000 endpoint antibody titer) detected the original HSA as well as all three HSA-fucosyl GM1 conjugate samples, while anti-fucosyl GM1 mAb F12 (1 / 10,000 endpoint antibody titer) detected only the HSA-fucosyl GM1 conjugates (Figure 6A). Western blot analysis also confirmed the production of HSA-fucosyl GM1 conjugates in all three samples. Anti-HSA mouse serum bound to the original HSA, to HSA that had been subjected to reductive amination but without the addition of fucosyl GM1, to the HSA-LewisY conjugate, to the HSA-GD3 conjugate, and to all three samples of HSA-fucosyl GM1 conjugates. On the other hand, anti-Fucosyl-GM1 mAb F12 detected only HSA-Fucosyl-GM1 conjugates, which were of the expected size of 67 kDa. This analysis also showed a slight increase in the molecular weight of the HSA-Fucosyl-GM1 conjugates compared to the original HSA, consistent with the addition of a Fucosyl-GM1 molecule to HSA (Figure 6B).

[0124] Fuc-GM1-containing liposomes (10 μg / immunoglobulin) and Fuc-GM1-positive SCLC cell line DMS79 (cells 1 × 10 6 Mice were immunized with HSA-Fuc-GM1 conjugate (10 μg / immunization) together with 15 mice (100 μg / immunization). Fifteen mice were divided into five groups of three mice each, and different types and orders of immune adjuvants were used.

[0125] Group 1 was first immunized three times with Fuc-GM1 liposomes, followed by two immunizations with HSA-Fuc-GM1 conjugates. The first immunization was adjuvanted with α-GalCer, the next two immunizations with anti-CD40, and for the HSA-Fuc-GM1 conjugates, incomplete Freund's adjuvant (IFA) was used. To improve the chance of generating mAbs capable of recognizing Fuc-GM1 on the cell surface, group 2 was first immunized with 1 × 10 6 Group 1 was immunized with 100 DMS79 cells, followed by two immunizations with Fuc-GM1-containing liposomes and two immunizations with HSA-Fuc-GM1 conjugates. The second and third immunizations used α-GalCer, while incomplete Freund's adjuvant was used for HSA-Fuc-GM1 conjugates. Group 2 was immunized first with HSA-Fuc-GM1 conjugates and then with Fuc-GM1 liposomes. Complete Freund's adjuvant was used for HSA-Fuc-GM1 conjugates, anti-CD40 was used as an adjuvant for the second immunization, and α-GalCer was used for the third immunization. Group 4 reversed the order of these antigens; mice were first immunized twice with Fuc-GM1 liposomes, followed by one immunization with HSA-Fuc-GM1 conjugates. The first immunization used α-GalCer as adjuvant, the second anti-CD40, and the third IFA. Group 5 was immunized three times with HSA-Fuc-GM1 conjugate. The first immunization used CFA as adjuvant, and the following two immunizations used IFA. The groups and their immunogens are summarized in (Table 1). [Table 1]

[0126] Mice were immunized via the ip route. The first three immunizations were given at two-week intervals. Groups 1 and 2 received the fourth immunization 6 weeks after the third and the fifth immunization 3 weeks after the fourth.

[0127] After each immunization except the first, mice were tail bled and mouse serum IgG was screened for binding to the HSA-Fuc-GM1 conjugate by ELISA. In addition, because we intended to generate mAbs that only recognize Fuc-GM1 (and not Fuc-GM1 linked to lysine residues), mouse serum was also screened for binding to purified Fuc-GM1. These analyses showed that all mice developed IgG immune responses to the HSA-Fuc-GM1 conjugate (endpoint antibody titer 1 / 10,000), but only 6 of 15 mice developed the desired purified Fuc-GM1 response. In two mice from group 1 (3x liposome + 2x conjugate), significant IgG responses to purified Fuc-GM1 were detected, both with endpoint antibody titers of 1 / 1,000. Additionally, two mice from group 2 (DMS79 cells + 2x liposomes + 2x conjugate) had detectable antibody titers, with mouse 2P having an endpoint titer of 1 / 1000 and mouse 2R having an endpoint titer of 1 / 100, and two mice from G5 (3x conjugate) with mouse 5P having an endpoint titer of 1 / 100 and mouse 5R having an endpoint titer of 1 / 1000. Only data from mice showing a positive anti-Fuc-GM1 response are shown in (Figure 7).

[0128] Serum analysis showed that six mice developed IgG immune responses against purified Fuc-GM1. To assess whether these antibodies could also recognize Fuc-GM1 in the context of intact cell plasma membranes, they were screened for binding to DMS79 cells by flow cytometry. This analysis showed that only two mice from group 2 (immunized with DMS79 cells, then immunized twice with liposomes and twice with conjugates) produced antibodies capable of binding to Fuc-GM1 on the cell surface (GM≈500). None of the other groups that developed Fuc-GM1-specific IgG responses also bound to the surface of live cells, suggesting that the inclusion of live DMS79 cells in the immunization protocol was of essential value (Figure 8).

[0129] After obtaining data showing a promising anti-Fuc-GM1 IgG response, mouse 2R was sacrificed and its splenocytes were fused with NS0 bone marrow cells. Five days before fusion, 10 μg of Fuc-GM1 and α-GalCer in liposomes were administered iv to boost the mouse immune response. Once established, individual hybridoma colonies were screened for production of IgG antibodies binding to purified Fuc-GM1 by ELISA. Four hybridomas with the highest antibody binding were identified and cloned twice at 0.3 cells / well. After each round of cloning, hybridoma supernatants were rescreened for secretion of IgG antibodies binding to purified Fuc-GM1 by ELISA (results not shown). After cloning, hybridomas FL133.63, FL133.67 and FL134.33 were selected, expanded and mAbs were purified from hybridoma supernatants. Using an isotype testing kit, mAbs FL133.63 and FL133.67 were found to be IgG3, kappa, while mAb FL134.33 was identified as IgG1, kappa.

[0130] After cloning, expansion and purification, it was important to evaluate the binding of these mAbs to the cell surface of Fuc-GM1 positive cancer cell lines. All antibodies were used at the same concentration of 10 μg / ml to allow direct comparison. MAb FL133.63 bound strongly to the cell line DMS79 (GM ≈ 1000) but weakly to the cell line DMS53 (GM ≈ 100). MAb FL133.67 bound strongly to DMS79 (GM ≈ 1000) but did not bind to any of the other Fuc-GM1 positive cell lines. MAb FL134.33 bound strongly to DMS79 (GM ≈ 1000), moderately to H128 (GM ≈ 300) and weakly to DMS53 (GM ≈ 100). The positive control mAb F12 bound to all four cell lines showing very strong staining intensity, with GM≈2000 for DMS79, GM≈700 for DMS53, GM≈500 for H128, and GM≈100 for cell line H69; of all mAbs, only mAb F12 bound to cell line H69. MAb FL134.33 showed unusual behavior in that it bound cell line H128 with greater intensity than it bound to cell line DMS53; however, DMS53 was reported to express greater amounts of Fuc-GM1 than cell line H128, a result confirmed herein by mAb F12 staining (Figure 9).

[0131] All three mAbs were screened for binding to whole blood of healthy human volunteers and showed no binding to any of the nucleated cells present. Isotypes IgG1, IgG3 and F12 were used as negative controls and an anti-MHC class 1 mAb was used as a positive control (Figure 10).

[0132] Example 2 - Definition of the epitope recognized by Fuc-GM1 mAb The specificity of the mAbs, FL133.63, FL133.67 and FL134.33, against Fuc-GM1 was assessed by glycolipid ELISA, testing the mAbs for binding to a series of available purified gangliosides, namely Fuc-GM1, GD3, GM1, GM3, GD1a, GT1b, Gb3 and lactosylceramide. All three mAbs recognized only ganglioside Fuc-GM1 at concentrations between 1 and 0.01 μg / ml. The positive control mAb F12 also recognizes Fuc-GM1 at concentrations between 1 and 0.01 μg / ml. Ganglioside GD3 was detected by the positive control mAb R24 at concentrations between 1 and 0.01 μg / ml. Gangliosides Fuc-GM1, GM1 and GD1a were detected by the positive control cholera toxin B subunit (CTxB) at 1 μg / ml. IgG mAbs specific for GM3, GT1b, Gb3 and LacCer have not yet been generated (FIG. 11).

[0133] To further validate the binding of the mAbs FL133.63, FL133.67 and FL134.33 to Fuc-GM1, they were evaluated in a competition assay between purified Fuc-GM1 and this ganglioside expressed on the cell line DMS79. In this experiment, the mAbs were preincubated with purified Fuc-GM1 and then used as the primary reagent in flow cytometry. Preincubation of mAb FL133.63 with ganglioside Fuc-GM1 reduced its binding to the DMS79 cell line from GM=523 to GM=4, that of mAb FL133.67 from GM=201 to GM=3, that of mAb FL134.33 from GM=434 to GM=29, and that of the positive control mAb F12 from GM=1195 to GM=6. Preincubation of the negative control mAb anti-MHC class 1 with ganglioside Fuc-GM1 did not affect its binding to DMS79 cells (FIG. 12).

[0134] To further characterize the fine specificity of FL133 / 134mAbs, they were screened against >600 natural and synthetic glycans by the Consortium for Functional Glycomics. Binding of FL133.63 to glycan arrays (Figure 13A) showed that, unexpectedly (Table 2), it was unable to bind to FucGM1sp0 (Chart #63), in which FucGM1 (free sugar) was directly attached to the chip, or to Fuc-Gm1sp9 (Chart #64), in which FucGM1 (free sugar) was attached to the chip via a 9-carbon spacer. [Table 2]

[0135] Binding of FL134.33 to the glycan array (Figure S13B) showed that, unexpectedly (Table 3), it was unable to bind to FucGM1sp0 (Chart No. 63), in which FucGM1 (the free sugar) was directly attached to the chip, and it bound very weakly to Fuc-Gm1sp9 (Chart No. 64), in which FucGM1 (the free sugar) was attached to the chip via a nine-carbon spacer. [Table 3]

[0136] Finally, the specificity of these mAbs for Fuc-GM1 was also demonstrated by TLC analysis. Purified ganglioside Fuc-GM1, purified ganglioside GM1 and DMS79 PM TGL were loaded onto a silica plate. All three Fuc-GM1 mAbs recognized purified Fuc-GM1, and the band present in DMS79 PM TGL migrated the same distance as purified Fuc-GM1. None of the antibodies bound to purified ganglioside GM1. The recognition pattern of the Fuc-GM1 mAbs generated here was identical to that of the positive control mAb F12, which recognized purified Fuc-GM1 and DMS79 PM TGL. Surprisingly, mAb F12 also bound weakly to purified ganglioside GM1, a binding that had not been detected in previous assays (Figure 14).

[0137] The affinity of anti-Fuc-GM1 mAbs to their antigens was determined by SPR with Biacore X. The HSA-Fuc-GM1 conjugates originally used for the generation of the three anti-Fuc-GM1 mAbs were used as targets for analysis. The binding of anti-Fuc-GM1 mAbs to the conjugates was confirmed by ELISA (Figure 15).

[0138] To couple the HSA-Fuc-GM1 conjugate to the CM5 biosensor chip, the chip was first washed with HSB-P buffer, after which the crosslinker EDC-NHS was applied. The HSA-Fuc-GM1 conjugate, diluted in acetate 4.5 coupling buffer, was then injected into the flow cell at 20 μg / ml. After a volume of 5 μl was injected, 686 response units of the conjugate were coupled to the chip. Finally, unreacted coupling agent was blocked with ethanolamine. The HSA-Fuc-GM1 coated flow cell was used to determine binding kinetic parameters from several known concentrations of antibody diluted in HBS-P buffer. Curve fitting software (BiaEvaluation) provided with the Biacore instrument was used to generate estimates of the binding and dissociation rates, from which the affinity was calculated using the best-fit bivalent analyte model. This model calculates affinity for bivalent proteins, but is only capable of calculating meaningful data for the binding of the first arm of the antibody. According to this model, the Fuc-GM1 mAb exhibits an average binding rate constant k(3.56x10 for Ab FL133.63) 4 Ms -1 , 9.43x10 for mAb FL133.67 3 Ms -1 and 2.01 × 10 for mAb FL134.33. 3 Ms -1 ), but the dissociation rate constant koff is very fast (0.0637 s for mAb FL133.63). -1 , 0.0783s for mAb FL133.67 -1and 0.117 s-1 for mAb FL134.33), so the functional affinity for these mAbs is overall low (1.8 × 10 for mAb FL133.63). -6 M, 8.3 × 10 for mAb FL133.67 -6 M and 5.8 × 10 for mAb FL134.33 -5 M (Table 4). [Table 4]

[0139] EC of the three Fuc-GM1 antibodies 50 The equilibrium dissociation constants (Kd) were determined by titrating them in purified Fuc-GM1 and DMS79 cells, respectively (Figure 16). EC from Fuc-GM1 ELISA 50 The functional affinity revealed by flow cytometry analysis in DMS79 cells and Fuc-GM1 ELISA was higher than that given by SPR evaluation. MAb FL133.63 was detected at 1 × 10 -9 E-commerce 50 and Kd=7.5×10 in DMS79 cells. -9 This is 1000-fold higher than the affinity established by SPR. MAb FL133.67 had a 2.1 × 10 affinity in the Fuc-GM1 ELISA. -9 E-commerce 50 and Kd=1.2×10 in DMS79 cells. -8 This is 100-1000 times higher than the affinity established by SPR. MAb FL134.33 had a 9.2 × 10 -10 E-commerce 50 and Kd=6.3×10 in DMS79 cells. -9 This is 10,000-fold higher than the affinity established by SPR. These results suggest that the SPR analysis measured monovalent antibody binding. The avidity of the antibody is greatly enhanced when it binds to an antigen on the cell surface.

[0140] Example 3 - Functional Assays It was also important to establish their effector functions, since all three Fuc-GM1 mAbs bound to the DMS79 cell line. Mab FL134.33 (IgG1) demonstrated 50%-80% cytotoxicity (donor dependent) by ADCC, but only 20% by CDC. Both mAbs, FL133.63 (IgG3) and FL133.67 (IgG3), demonstrated 20% cytotoxicity by ADCC, but 60-80% cytotoxicity was mediated by CDC. These results are consistent with previously reported studies that showed that mAbs of IgG1 isotype were more potent in inducing ADCC and mAbs of IgG3 isotype were more potent in inducing CDC (Lopez et al., 1983, Niwa et al., 2005, Bruggemann et al., 1987, Natsume et al., 2008). In contrast, the positive control murine mAb SC101 (IgG1) demonstrated 70% ADCC-mediated and 80% CDC-mediated cytotoxicity of DMS79 cells (FIG. 17).

[0141] Example 4 - Chimeric mAb MAbs FL133.63, FL133.67 and FL134.33 were sequenced and the sequences of the VH and VL chains were compared to those of germline IgG antibodies using the program IMGT / V_QUEST. Overall, all data demonstrated evidence of somatic hypermutation and also suggested the presence of some affinity maturation. The VH chains of all three Fuc-GM1 antibodies were mapped to gene V3, subgroup 1 and allele 02. Compared to the germline sequence, the VH region of mAb FL134.33 contained 14 substitution mutations and 7 amino acid changes, mAb FL133.63 contained 8 substitution mutations and 6 amino acid changes, while mAb FL133.67 contained 6 substitution mutations and 4 amino acid changes (Figure 18 and Table 5).

[0142] In FR2, all three mAbs contained the transition mutation a157>g resulting in the amino acid change M53>V. In CDR2, all three mAbs contained the transversion mutation a157>c resulting in the amino acid change S59>R, and the transition mutation g191>a resulting in the amino acid change S64>N. In FR3, all three mAbs contained the silent transversion mutation t231>a, the transversion mutation c248>a resulting in the amino acid change S83>Y, and finally the silent transition mutation c309>t.

[0143] MAb FL134.33 also contained in FR1 a transversion mutation a2>c resulting in the amino acid change D1>A, a silent transition mutation g9>a, and a silent transition mutation c69>t; in FR3 it contained a silent transition mutation c198>t, a transition mutation a226>g resulting in the amino acid change I76>V, a silent transition mutation g267>a, a silent transition mutation g273>a, and a transversion mutation g280>c resulting in the amino acid change V94>L.

[0144] MAb FL133.63 also contained the transition mutation t259>c, which resulted in the amino acid change F87>L, and the transition mutation a269>g, which resulted in the amino acid change Q90>R. [Table 5]

[0145] The VL chains of all three Fuc-GM1 antibodies were mapped to gene K5, subgroup 39 and allele 01. Compared to the germline sequence, the VL region of mAb FL134.33 contained 10 substitution mutations and 6 amino acid changes, mAb FL133.63 contained 7 substitution mutations and 6 amino acid changes, while mAb FL133.67 contained 7 substitution mutations and 6 amino acid changes (Figure 19 and Table 6). [Table 6]

[0146] In CDR1, all three mAbs contained a transversion mutation t112>g resulting in the amino acid change Y38>D. In CDR2, all three mAbs contained a transition mutation c170>t resulting in the amino acid change A57>V. In FR3, all three mAbs contained a transversion mutation g269>c resulting in the amino acid change S90>T and a silent transition mutation g303>a.

[0147] MAb FL134.33 also contained a transversion mutation c26>a resulting in the amino acid change A9>D in FR1; a silent transition mutation g153>a in FR2; a silent transition mutation c195>t in CDR2; transition mutations g250>a and g252>a resulting in the amino acid change G84>R in FR3, and a transversion mutation g301>c resulting in the amino acid change V101>L.

[0148] MAb FL133.63 also contained a transversion mutation c14>g resulting in the amino acid change T5>S in FR1; a transition mutation a106>g resulting in the amino acid change S36>G in CDR1, and a transversion mutation a256>t resulting in the amino acid change D86>Y in FR3.

[0149] MAb FL133.67 also contained a transversion mutation c14>g resulting in the amino acid change T5>S in FR1; a transversion mutation c200>a resulting in the amino acid change S67>Y, and a transversion mutation c276>g resulting in the amino acid change N92>K.

[0150] Among these three antibodies for which the variable region sequences were available, the antibody selected for chimerization was FL134.33. To allow cloning of both sequences of the antibody, primers had to be designed specifically for them and restriction enzyme sites had to be incorporated into them. The dual expression vector contains restriction sites for the enzymes BamHI and BsiWI for the light chain and HindIII and AfeI for the heavy chain. The primer variable region insert must also have a site that generates an overhang after digestion that is compatible with the overhang of the vector. However, in the case of FL134.33, the restriction site for digestion with BamHI could not be added to the primer, since said site was also present in the internal sequence of the light chain. Therefore, an alternative digestion site that generates an overhang compatible with the overhang of BamHI was required. The restriction enzyme with said characteristics was found to be BglII.

[0151] The primers designed and used to perform the PCR amplification and subsequent cloning procedures were the light (kappa) chain forward primer 5'-ATTAAGATCTAAGATGGTGTCCACTTCTCAGCTC-3' containing a BglII restriction site and the light (kappa) chain reverse primer 5'-AATTCGTACGTTTGATTTCCAGC TTGGTGCCT-3' containing a BsiWI restriction site. Additionally, 5'-TAATAAGCTTAAGATGAGAGTGCTGATTCTTTTG-3' was the heavy chain forward primer containing a HindIII restriction site and 5'-AGAGCAGCGCTGGAGACGGTGACT GAGGT-3' was the heavy chain reverse primer containing an AfeI restriction site. PCR reactions were set up with both the forward and reverse primers and using a proofreading polymerase. Amplification of both the light and heavy chains was confirmed by the presence of products in agarose gel electrophoresis. The heavy and light chains were cloned into TOPO vector and transformed into chemically competent cells supplemented with ampicillin for selective growth. Six colonies carrying the light chain and six colonies carrying the heavy chain were selected to grow in liquid culture and plasmid DNA was prepared by miniprep. By virtue of the internal restriction site of BamHI, enzymatic digestion could prove a confident way to know if the correct sequence was actually cloned into the vector. Enzymatic digestion was then performed using EcoRI (two sites present at both the 5' and 3' ends of the cloning site) and BamHI+BsiWI (internal site in the middle of the sequence and primer integration site, respectively) and evaluated by agarose gel electrophoresis. EcoRI digestion of the vector showed sequences of the expected size (400 bp) in two colonies screened for the light chain and three colonies screened for the heavy chain. BamHI+BsiWI showed sequences of the expected size (200 bp) in two colonies screened for the light chain and three colonies screened for the heavy chain.After confirmation of correct cloning via sequencing, one colony for each heavy and light chain was selected and grown overnight in ampicillin-supplemented media for subsequent plasmid DNA preparation via maxiprep.

[0152] Although direct cloning of PCR products into dual expression vectors has previously been shown to be relatively inefficient, we attempted it nonetheless in the hope that it would work properly. After cloning of the light chain PCR product into the pDCOrig-hIgG1 vector, transformation was performed and cells were grown overnight in Zeocin-supplemented medium. Plasmid DNA was prepared by miniprep, followed by enzymatic digestion of the plasmid with BamHI and BsiWI. The expectation was that if the correct heavy chain was integrated into the vector, a small 200bp band would appear on the gel (from the internal BamHI present in the sequence). However, if the heavy chain was not integrated and the original chain remained in the vector, this would show a 400bp band (the original sequence lacked an internal BamHI site). A few colonies showed the expected band (bad) of the correct insert, and subsequent sequencing confirmed the successful direct cloning of the light chain into the vector. After this, one colony was selected and grown overnight in Zeocin-supplemented medium for subsequent plasmid DNA preparation via maxiprep.

[0153] By the time the light chain was confirmed to be integrated into the pDCOrig-hIgG1 vector, TOPO cloning of the heavy chain was also prepared and the plasmid was prepared. Then, instead of trying to directly clone the PCR product, the heavy chain was obtained through agarose gel purification and extraction, followed by enzymatic digestion of the previously prepared TOPO vector with the enzymes HindIII and AfeI. After cloning the gel purified sequence of the heavy chain into the pDCOrig-hIgG1 vector containing the light chain, transformation was performed and cells were cultured overnight in Zeocin-supplemented medium. Plasmid DNA was prepared by miniprep, followed by enzymatic digestion with HindIII+AfeI and HindIII+BamHI+AfeI. The expectation was that digestion with only two enzymes would show a band of about 400 bp, and with three enzymes a band of about 200 bp (as the heavy chain also contains a BamHI site around its middle). Colonies that showed the small 200bp were sent for sequencing and after confirmation of correct cloning, one of them was selected and grown overnight in Zeocin-supplemented medium for subsequent plasmid DNA preparation via maxiprep. The yield of the plasmid preparation was determined to be 776ng / μl by spectroscopy. After confirming the sequences of both the light and heavy chains of antibody FL134.33, the plasmid was used to transfect CHO cells.

[0154] After confirming that the sequence contained the predicted mouse heavy and light chain variable regions coupled to human constant regions, transient transfection was performed. 15 μg of vector containing the chimeric antibody CH134.33 was incubated with CHO-S cells in the presence of lipofectamine and grown in suspension in 500 ml of medium for 1 week. The supernatant containing the mAb was purified on a protein G column and eluted at pH 8. The final yield of antibody was 0.4 mg.

[0155] Antibody Functional Assays To make a direct comparison between the murine and chimeric forms, as well as between the three murine antibodies, the mAbs tested in the assay were: FL134.33, FL133.63, FL133.67, and Ch134.33. All antibodies bound well to Fuc-GM1 (Figure 20). These antibodies bound to various Fuc-G m1 Binding to four cell lines showing expression levels was also tested: DMS79, DMS53 (ATCC. Middlesex, UK), H128 and H69 (ECACC. Salisbury, UK). The positive control was a commercial anti-Fuc-Gm1 (Fujirebio. Tokyo, Japan) and the negative control was a mouse IgG1 isotype (Dako. Stockport, UK). The highest signal was given by the positive control F12 in all cell lines except DMS79, where FL134.33 was shown to be stronger. The chimeric ch134.33 showed good binding to DMS79 cells (Figure 21).

[0156] References [ka] TIFF0007672395000008.tif228159TIFF0007672395000009.tif231159TIFF0007672395 000010.tif237159TIFF0007672395000011.tif231159TIFF0007672395000012.tif67159

Claims

1. 1. An isolated antibody or antigen-binding fragment thereof capable of specifically binding to Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc-ceramide (Fuc-GM1 glycolipid) but not to Fucα1-2Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβ1-4Glc (free sugar), comprising the following CDRs: (a) GYSITSGYS (CDRH1), IHYRGNT (CDRH2) and AQEPVRNYAMDY (CDRH3) and QSIGDD (CDRL1), YVS (CDRL2) and QNGHGFPPT (CDRL3); (b) GYSITSGYS (CDRH1), IHYRGNT (CDRH2) and AQEPVRNYAMDY (CDRH3) and QSISDD (CDRL1), YVS (CDRL2) and QNGYSFPPT (CDRL3); or (c) GYSITSGYS (CDRH1), IHYRGNT (CDRH2), and AQEPVRNYAMDF (CDRH3) and QSISDD (CDRL1), YVS (CDRL2), and QNGHGFPPT (CDRL3); An antibody or antigen-binding fragment thereof comprising:

2. (a) the amino acid sequence: DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWVGYIHYRGNTNYNPSLKSRISITRDTYKNQLFLRNSVTTEDTATYYCAQEPVRNYAMDYWGQGTSVTVSS and the amino acid sequence: DIVMSQSPATLSVTPGDRVSLSCRASQSIGDDLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSGSYFTLTINSVEPEEDVGVYYCQNGHGFPPTFGGGTK; (b) the amino acid sequence: DVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWVGYIHYRGNTNYNPSLKSRISITRDTYKNQFFLQLNSVTTEDTATYYCAQEPVRNYAMDYWGQGTSVTVSS and the amino acid sequence: DIVMSQSPATLSVTPGDRVSLSCRASQSISDDLHWYQQKSHESPRLLIKYVSQYISGIPSRFSGSGSGSDFTLTIKSVEPE DVGVYYCQNGYSFPPTFGGGTK; or (c) amino acid sequence: AVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWVGYIHYRGNTNYNPSLKSRVSITRDTYKNQFFLQLNSLTTEDTATYYCAQEPVRNYAMDFWGQGTSVTVSS and amino acid sequence: DIVMTQSPDTLSVTPGDRVSLSCRASQSISDDLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSRSDFTLTINSVEPEEDVGLYYCQNGHGFPPTFGGGTKLEIKR; The antibody or antigen-binding fragment thereof of claim 1 .

3. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the CDRs are retained by a human antibody framework.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a human constant region.

5. Heavy chain amino acid sequence: AVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWVGYIHYRGNTNYNPSLKSRVSITRDTYKNQFFLQLNSLTTEDTATYYCAQEPVRNYAMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, and Light chain amino acid sequence: DIVMTQSPDTLSVTPGDRVSLSCRASQSISDDLHWYQQKSHESPRLLIKYVSQSISGIPSRFSGSGSRSDFTLTINSVEPEDVGLYYCQNGHGFPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The antibody or antigen-binding fragment thereof of claim 4 .

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, conjugated or otherwise associated with a chemotherapeutic or cytotoxic agent.

7. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, and a pharma- ceutically acceptable excipient, diluent, carrier, buffer or stabilizer.

8. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 for use in medicine.

9. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 for use in a method for treating or preventing a tumor.

10. A product comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and an active agent as a combined preparation for simultaneous, separate or sequential use in the treatment of tumors.

11. The antibody or antigen-binding fragment thereof of claim 9 or the product of claim 10, wherein the tumor is small cell lung cancer.

12. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

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