Abstract:
The present invention refers to compounds of formula (I) as useful melanoma vaccines&#39; active ingredients. As a matter of fact, compounds of formula (1) are immunogenic artificial antigens, mimetics of GM3 and GM3 lactone, well known tumor associated antigens (TAAs). In addition, the present invention refers to specific antibodies for compounds of formula (I) which are able to bind to the TAAs. The hereinabove reported compounds and antibodies are therefore potentially useful respectively for the active and passive immunization against tumors over-expressing GM3 ganglioside, in a preferred embodiment melanoma and colon cancer, and for their diagnosis and prognosis.

Description:
FIELD OF THE INVENTION 
     The invention relates to the synthetic chemistry domain and in particular the synthesis of immunogenic glycoconjugate derivatives, their synthetic methods and their use for active and passive immunotherapy or for the prognosis of cancers characterized by the overexpression of GM 3  ganglioside and GM 3  lactone. 
     STATE OF THE ART 
     Immunotherapy against tumours and against melanoma in particular has a long history but only recently this therapeutic approach has found a reliable scientific rationale. The main target of this biological therapy consists of teaching the tumour bearing host&#39;s immune system how to recognize antigens express on tumoral cells and destroy them, without damaging healthy cells. The success of this therapy greatly depends on the choice of the antigens responsible for the growth and proliferation of the tumour. GM 3  ganglioside 1 ( FIG. 1 ), a glycosphingolipid found in essentially all types of mammalians&#39; cells and tissues, is the major ganglioside in normal melanocytes. Ganglioside 1 and especially its metabolite, the GM 3  lactone 2 ( FIG. 1 ), are overexpressed in melanoma cells with metastatic potential. These antigens are wrongly recognized as “self” by patient&#39;s immune system and are potential targets for the immunotherapy of this tumours. Although more immunogenic than GM 3  ganglioside 1, GM 3  lactone 2 failed as immunostimulant because unstable under physiological conditions, in unsutable to be used in immunotherapy as melanoma&#39;s associate antigen. Tom et al.  ChemBioChem,  2007, 8, 1646 describes the rational design and the synthesis of the hydrolytically stable mimetic 3 ( FIG. 1 ) which is remarkably simpler than the native 2. The mimetic 3, as reported after its publication, has been demonstrated to be scarcely immunogen. 
     Aim of the present invention is to provide a synthetic derivative of GM 3  lactone which is hydrolytically stable under physiological conditions and good to raise the immune system to elicit antibodies able to recognize antigens express on tumoral cells which present overexpression of GM3 lactone (i.e. melanoma cells or colon tumoral cells). 
     DESCRIPTION AND SHORTENINGS 
     KLH: Keyhole Limpet Hemocyanin 
     CRM: Cross-Reacting Material 197 (non toxic mutation of difteric toxin) 
     OMP: Outer Membrane Protein 
     BSA: Bovine Serum Albumin 
     cBSA: cationized Bovine Serum Albumin 
     HSA: Human Serum Albumin 
     BCG Bacillus Calmette and Guerin 
     MPL: Membrane-Patched Proteoliposomes 
     MDP: Muramyl DiPeptide 
     Pal3CysSer: N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-Cys-Ser-OH “Pal3 Cys: N-α-palmitoly-S-[2,3-bis(palmitolyoxy)-(2RS)-propyl]-L-cysteine” 
     QS-21: Quillaja Saponaria Molina based immunological adjuvant 
     CpG: Cytosine linked to a Guanine by a phosphate bond 
     RAFT: Regioselectively Addressable Functional Template 
     ODN: OligoDeoxyNucleotides 
     SUMMARY OF THE INVENTION 
     The present invention solves the hereinbefore mentioned problems thanks to compounds of formula (I) 
                                
wherein:
 
X is O, NH, S, CH2;
 
L is a linker that is a divalent spacer;
 
A is an adjuvant or a carrier that is an immunogenic molecule or particle;
 
and where bonds between linker and carbohydratic portion and between linker and carrier are covalent.
 
     Surprisingly, compound of formula (I) as above reported, revealed to be immonogen. Therefore, for an aspect, the present invention relates to compounds of formula (I) for their use as drugs, that is as antigens in active immunization. For another aspect, the invention concerns pharmaceutical preparations containing at least one compound of formula (I) as above described and at least another pharmaceutically acceptable ingredient. 
     For a further aspect, the present invention concerns antibodies obtainable by immunization of mice treated with a compound of formula (I) as above described. These antibodies are specific, that is specifically bind cells which overexpress GM 3  ganglioside and GM 3  lactone and therefore for a further aspect the invention relates to antibodies as above described, as drugs, that is as active ingredient in pharmaceutical preparations for the passive immunization and as diagnostic and/or prognostic devices. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       FIG.  1 —Structure of the GM 3  ganglioside 1, GM 3  lactone 2, thioether mimetic 3 and GM 1 . 
       FIG.  2 —Synthesis of a glycoconjugate of formula (I) according to the invention, in particular of compound 5 KLH-conjugate. 
       FIG.  3 —ELISA tests performed for isotype determination. The hybridoma IV1:4C5C4 supernatant results to be composed of two isotypes: IgM and IgG3. 
       FIG.  4 —ELISA test to compare antibody immunoreactivity towards either GM 3 - or GM 1 -ganglioside. 
       FIG.  5 —Melanoma cell lines expressing (A375) and not expressing (WM-266-4) GM 3  antigen. Signals were obtained using hybridoma population IV1:4C5C4 supernatant. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In the scope of the present invention, the linker L is a divalent spacer with a structure suitable for maintaining a correct distance between the carbohydratic portion and the carrier/adjuvant avoiding the undesired masking of their antigenic regions: as first choice the linker presents a chemical structure suitable to space the carbohydratic portion and the carrier by 7-30 atoms or better by 12-16 atoms when the adjuvant/carrier A is a biomacromolecule or by 19-30 atoms when the adjuvant/carrier A is a metallic nanoparticle. 
     In the scope of the present invention, the adjuvant/carrier A is an immunogenic species i.e. able to induce an immune response when administered to an animal subject; the latter carrier/adjuvant A can be selected among a macromolecule, a macrocycle, a dendrimer, a liposome, a nanoparticle, an oligosaccharide featuring from two to five monosaccharidic units or cells like dendritic cells (DC). 
     Macromolecule means a molecule containing at least 100 atoms, among these are included the bio-macromolecules like proteins, lipids and polisaccharides. 
     Macrocycles means compound featuring a seven- (or larger-) membered ring like calixarenes, phthalocyanines, cyclodestrins, porfirines and cyclo-oligopepdides. 
     Dendrimers of G0, G1 and G2 are included. PAMAM (polyethylenamine) is a good example of dendrimer. 
     Nanoparticles are particles whose dimensions range between 1 and 100 nm; they are formed by metals or metal oxides selected among Au, Fe, Ti. 
     Optionally, if A is a carrier with a modest immunogenicity a compound of formula (I) can be associated with another suitable adjuvant able to increase the immunogenicity of the carrier. In this case object of the invention is a composition comprising a compound of formula (I) as above described and another adjuvant B able to enhance the immunogenity of carrier A. 
     Preferably, the carrier/adjuvant A and B is selected in the group consisting of KLH, CRM, OMP, BSA, HSA, BCG, MPL, MDP, Pal3CysSer, Pal3Cys, QS21, trehalose-6,6′-dimycolate, synthetic CpG, CpG-DNA,  Neisseria meningitidis , tetanus toxin, bacterial toxin, liposome, gold nanoparticles, Fe(II) oxide nanoparticles, calixarenes, dendrimers, RAFT, DC, as single species or combined. 
     When the carrier/adjuvant is proteic the linkage between linker and carrier/adjuvant is chosen in the group of amidic, aminic, iminic, ethereal, thioethereal, disulfide, ester, thioester, phosphate, phosphonate, ureidic, thioureidic, carbamate, carbonate, where at least one atom belongs to the protein. 
     When the carrier A is a nanoparticles the linker is bond to the nanoparticles with a pseudo-covalent linkage though a sulfur atom or a disulfide group. 
     When A is a protein the linker presents in preference the structure R1-Z-W1-R2-W2 where R1 and R2 independently from each other are a C2-C8-alkyl, C2-C8-alkenyl, C5-C7-cycloalkyl, 1-4-phenyl; Z is O, S, NH; W1 is missing, C═O, C═NH, S; W2 is missing, C═O, C═NH, S, 1,4-succinamide. The above reported alkyls are in preference linear. 
     Examples of possible linkers are: 
     
       
                 
         
             
             
         
      
     
     More preferred are those compounds wherein: 
     X=O; 
     A is a protein; 
     L is a bivalent spacer of structure (CH2)n-NH—CO—(CH2)m-CO—Y where n and/or m are integer ranging from 2 to 8 and Y is an NH which is part of A. 
     A preferred compound is the compound 5 ( FIG. 2 ) of structure (I) where X is O, linker L is —(CH2)6NHCO—(CH2)4-CO— and carrier A is KLH linked through lysine —NH2 residues to —COOH residues of the linker. 
     Compounds of formula (I) as above described can be obtained starting from compounds of structure 
                                
where:
 
G is F, Br or Cl; SR, SAr or trichloroacetimide, phosphate where R is Et or C1-C4 alkyl; Ar is Ph, substituted Ph, Pyridine, quinoline or other aromatic heterocycles; P independently are suitable hydroxyls protecting groups selected among those protecting groups known to those skilled in the art and such as and preferably acetyl, benzoyl, silyl and benzyl; prepared by known synthetic methods for the insertion of a linker L or L-A.
 
     In a preferred embodiment a compound of formula (II) has groups P all the same. In the event a compound of formula (II) as above described is coupled to the linker only it will follow, after optional suitable deprotection of hydroxyl groups, coupling, through known synthetic strategies, with a carrier/adjuvant A. 
     In a particular, as example embodiment, is herein reported ( FIG. 2 ) the synthetic scheme for the compound 5 obtained from the compound 15, i.e. a compound of formula (II) where G is F. The compound 15 was prepared as already reported in Toma et al.  ChemBioChem,  2007, 8, 1646. The ω-amino glicosyl derivative 4 ( FIG. 2 ) was prepared from the fluoro derivatively by treatment with azido hexanol under Ley&#39;s glycosylation conditions. The pseudo-α azido derivative 17 obtained after removal of acetyl groups, was reduced under Staudinger&#39;s reaction conditions to give 4 which was reacted with the bis-p nitrophenyl adipic ester 18 (Wu, X., Ling, C.-C., Bundle, D.  Org. Lett.  2004, 6, 4407) to form the activated carboxylic derivative 19 (87% over two steps) (Scheme 3). Finally, 19 was conjugated to the proteic carrier KLH. The KLH-thioether conjugate 5 was prepared by mixing 19 with KLH (phosphate buffer, room temperature, 20 h). Saccharide molecule/KLH loading was determined by trinitrobenzensulfonic (TNBS) test (Habeeb, F. S. A.  Anal. Biochem.  1966, 14, 328) to be ˜27%. 
     Compounds of formula (I) as above described are artificial antigens, mimetic of the GM 3  lactone and therefore useful as active ingredient for vaccines against tumoral cells over-expressing GM 3  ganglioside and GM 3  lactone, in particular they are useful in the treatment and/or the prevention and/or the diagnosis of melanoma and cancers of colon. 
     Compounds of formula (I) as above described can also be useful as diagnostic devices for tumors whose cells over-express GM 3  ganglioside and GM 3  lactone. 
     Surprisingly indeed, the compound in the embodiment 5, when is administrated to mice, produced a strong immune response and the antibodies obtained are able to bind to the GM 3  ganglioside overexpressed on melanoma tumoral cells or colon tumoral cell lines. 
     To determine the immunogenicity of the mimetic 5, BALB/c mice were immunized with the KLH conjugate. Following Milstein procedure (Galfrè, G., Milstein, C., “Preparation of monoclonal antibodies: strategies and procedures” in  Methods Enzymol.,  73(Pt B), 3-46, (1981)) for the production of monoclonal antibodies, after sequential immunizations spleen cells from immunized mice were collected and fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma cell line (NS0). The ensuing hybridomas were screened by testing supernatants for binding to GM 3  ganglioside 1 by enzyme-linked immunosorbent assay (ELISA). The supernatant displaying the highest absorbance (IV1:4C5C4) was then chosen for further analyses. An ELISA test aimed at isotype determination was also performed. The IV1:4C5C4 supernatant turned out to be composed by two isotypes: IgM and IgG3, with the IgM quantitatively prevailing on the IgG3 ( FIG. 3 ). 
     The immunoreactivity of the IV1:4C5C4 supernatant towards either GM 3 - or GM 1 -ganglioside was then screened through an ELISA test. Results ( FIG. 4 ) clearly indicated that the supernatant indeed binds GM 3  whereas no appreciable binding to GM 1  was observed. Finally, to evaluate the cross-reactivity of the polyclonal serum for GM 3  expressed on melanoma cells, immunocytochemistry with B16F1 melanoma cells treated with the hybridoma population IV1:4C5C4 supernatant was performed. Reference samples were treated with the commercially available anti-GM 3  monoclonal antibody (M2590) (positive control) and with an hybridoma supernatant resulted negative to the first ELISA test (taken as negative control). Results indicated a clear positivity of the cell line tested evidenced by the brown staining. Note that there is no detectable signal at the nuclear level but only at the cytoplasmatic membrane level. It is evident ( FIG. 5 ) that the IV1:4C5C4 supernatant is able to recognize the GM 3  ganglioside and the GM 3  lactone expressed on melanoma cells better than the antibody anti-GM 3  M2590. 
     All together, data herein reported indicate that the glycoconjugate 5 is a real immunogen “in vivo” since it is capable to induce the formation of specific GM 3  antibodies. These antibodies cross react with the native antigen in ELISA tests as well as with melanoma cell lines. 
     Two main points must be considered: compounds of formula (I) as hereinabove described, and in particular the KLH-conjugate 5, overcomes the problem of the low immunogenicity typical of gangliosides and droops the immunotolerance generally preserved by the GM 3  ganglioside covalently linked to carriers such as the KLH (Bitton, R. J., et al.  Oncology Report  2002, 9, 267; Gabri, M. R.; et al.  Clin. Cancer Res.  2006, 92, 7092). 
     These results prompt to further clonings of the IV1:4C5C4 hybridoma and purification of the IV1:4C5C4 supernatant to obtain the monoclonal antibody anti GM 3  and GM 3  lactone to be used in the future for the passive immunotherapy of tumours whose cells overexpress GM3 ganglioside and GM3 ganglioside lactone, such as melanoma cells and colon tumour cell lines. 
     In addition the invention relates to specific antibodies against the artificial antigen of formula (I). For an aspect the present invention relates to hybridomas obtainable by immunization of mice with compounds of formula (I). 
     For another aspect the invention relates to chimeric or humanized antibodies including the variable or hyper variable sequences of the monoclonal antibodies achievable by immunization of mice with compounds of formula (I). 
     The above said antibodies can be useful active ingredient in pharmaceutical compositions for passive immunization in particular for the immunization against tumours whose cells overexpress GM3 ganglioside and GM3 lactone and in preferred embodiments are useful in the treatment of melanoma and colon cancers. 
     The above said antibodies can also be useful as diagnostic agents and/or prognostic of the above mentioned tumours. 
     The term “tumours whose cells overexpress GM 3  ganglioside and GM 3  lactone” as used herein means those tumours which analyzed by antigen-antibody (commercially available antibody anti-GM 3 ) and by immunocytochemical tests result to be positive to said overexpression. 
     The present invention could be better understood upon reading of the following preferred embodiments. 
     EXPERIMENTAL SECTION 
     Synthesis of Compound 20 
     
       
                 
         
             
             
         
      
     
     To a mixture of 15 (160 mg, 0.25 mmol), bis(cyclopentadienyl)hafnium(IV) dichloride (Cp 2 HfCl 2 ) (170 mg, 0.45 mmol), AgOTf (250 mg, 0.97 mmol) and 6-azido-1-hexanol (70 mg, 0.5 mmol) cooled to −40° C., dry CH 2 Cl 2  (30 mL) was added. After 40 min at −40° C. the solution was neutralized with NEt 3  and filtered through a pad of Celite® and the filtrate was washed with a saturated solution of NaHCO 3  (1×50 mL) then with brine (1×50 mL). The organic phase was dried over Na 2 SO 4  and concentrated to dryness to give 200 mg of crude. The crude was purified by flash column chromatography on silica gel (petroleum ether/EtOAc 1:1) to give 20 (150 mg, 80%), as a glassy solid.  1 H NMR (400 MHz, CDCl 3 ): δ 5.37 (d, 1H, J d-e =2.4 Hz), 5.32 (bs, 1H), 5.26-5.21 (m, 1H), 5.12-5.09 (m, 1H), 4.97 (s, 1H), 4.50-4.46 (m, 1H), 4.25-4.16 (m, 2H), 4.18-4.14 (X part of an AXY system, 1H, J XA =6.4 Hz, J XY =11.6 Hz), 4.11-4.06 (Y part of an AXY system, 1H, J YA =7.6 Hz, J YX =11.6 Hz), 3.96 (ad, 1H, J 5-6 =10.0 Hz), 3.75-3.69 (m, 1H), 3.53-3.47 (m 1H), 3.24 (t, 2H, J=7.2 Hz), 3.02-2.98 (A part of an AB system, 1H, J AB =13.0 Hz), 2.97-2.94 (B part of an AB system, 1H, J BA =13.0 Hz), 2.15 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.99-1.98 (m 2H), 1.95 (s, 3H), 1.62-1.56 (m, 4H), 1.43-1.36 (m, 4H).  13 C NMR (50 MHz, CDCl 3 ): δ 170.8, 170.2, 170.1, 169.6, 169.5 (2C), 137.4, 109.0, 95.7, 93.0, 68.6, 68.0, 67.4, 67.3, 66.4, 64.4 (2C), 62.0, 61.7, 51.3, 33.8, 33.6, 29.3, 28.7, 26.4, 25.7, 20.7 (3C), 20.6 (3C). [α] D   23 +22.31 (c 1.09, CH 2 Cl 2 ). Elem. Anal. for C 32 H 45 N 3 O 16 S: Calc. C, 50.59; H, 5.97; N, 5.53. Found: C, 50.62; H, 6.01; N, 5.42. ESI-MS 782.2 [M+Na] + , 798.2 [M+K] + . 
     Synthesis of Compound 17 
     
       
                 
         
             
             
         
      
     
     To a stirred solution of 20 (120 mg, 0.158 mmol) in CH 3 OH (3.2 mL), MeONa (2 mg, 0.032 mmol) was added. The mixture was stirred at RT for 2.5 h then the pH adjusted to neutrality with HCl (10% in MeOH). Evaporation of the solvent under vacuum gave a crude which was purified by flash chromatography on silica gel (EtOAc/CH 3 OH 5:1) to give 17 (72 mg, 90%) as glassy solid.  1 H NMR (400 MHz, CD 3 OD): δ 4.97 (s, 1H), 4.17-4.12 (m, 1H), 4.09-4.06 (m, 1H), 4.02 (dd, 1H, J 4-3 =3.2 Hz, J 4-5 =1.2 Hz), 3.94 (dd, 1H, J 5-6 =9.6 Hz, J 5-4 =1.2 Hz), 3.84-3.79 (A part of an ABX 2  system, 1H, J A-B =10.0 Hz, J A-X =6.4 Hz), 3.77-3.62 (m, 6H), 3.52-3.47 (B part of an ABX 2  system, 1H, J B-A =10.0 Hz, J B-X =6.4 Hz), 3.31-3.28 (m, 2H), 3.05-3.01 (A part of an AB system, 1H, J AB =13.0 Hz), 2.99-2.96 (B part of an AB system, 1H, J BA =13.0 Hz), 1.94-1.92 (m, 2H), 1.64-1.57 (m, 4H), 1.46-1.41 (m, 4H).  13 C NMR (50 MHz, CD 3 OD): δ 141.6, 106.4, 96.3, 92.3, 72.6, 70.9, 68.5, 67.8, 66.5, 66.3, 64.6, 62.8, 61.1, 51.1, 36.3, 33.4, 29.2, 28.6, 26.3, 25.6. [α] D   23 +51.64 (c 0.25, CH 3 OH). ESI-MS 530.3 [M+Na] + . 
     Synthesis of Compound 19 
     
       
                 
         
             
             
         
      
     
     To a stirred solution of 17 (13.2 mg, 0.026 mmol) in a mixture of dry TI-IF (1 mL) and dry DMF (0.1 mL), Ph 3 P (13.6 mg, 0.052 mmol) and H 2 O (2.3 μL, 0.13 mmol) were added. The reaction mixture was warmed to 40° C. and stirred for 18 h. Evaporation of the solvent under vacuum gave the crude 4 which was used without purification for the following reaction. 
     
       
                 
         
             
             
         
      
     
     To a stirred solution of 4 in dry DMF (1 mL), 18 (50 mg, 0.130 mmol) was added and the reaction mixture was stirred at RT for 6 h. Evaporation of the solvent under vacuum gave a crude which was purified by flash chromatography on silica gel (CH 2 Cl 2 /CH 3 OH 2:1) to give 19 (16.5 mg, 87%) as glassy solid. 
       1 H NMR (400 MHz, CD 3 OD): δ 8.30-8.29 (AA′ part of an AA′MM′ system, 2H, J A-M =4.8 Hz, Ph), 7.38-7.37 (MM′ part of an AA′MM′ system, 2H, J M-A =4.8 Hz, Ph), 4.96 (s, 1H, H a ), 4.15 (adt, 1H, J 3-4 =2.8 Hz, J 3-2 =J 3-2′ =8.8 Hz, H 3 ), 4.09-4.05 (m, 1H, H e ), 4.03-4.02 (dd, 1H, J 4-3 =2.8 Hz, J 4-5 =0.8 Hz, H 4 ), 3.94 (dd, 1H, J 5-6 =9.6 Hz, J 5-4 =0.8 Hz, H 5 ), 3.82-3.77 (A part of an ABX 2  system, 1H, J A-B =10.0 Hz, J A-X =6.4 Hz, H x ), 3.76-3.61 (m, 6H, H d , H 6 , H 7 , H 7′ , H f , H f′ ), 3.51-3.45 (B part of an ABX 2  system, 1H, J B-A =10.0 Hz, J B-X =6.4 Hz, H x′ ) 3.184 (t, 2H, J=7.0 Hz, CH 2y ), 3.03-3.00 (A part of an AB system, 1H, J A-B =13.0 Hz, H 1′a ), 2.98-2.95 (B part of an AB system, 1H, J B-A =13.0 Hz, H 1′b ), 2.67 (t, 2H, J=7.0 Hz, CH 2 CO), 2.25 (t, 2H, J=7.0 Hz, CH 2 CO), 1.93-1.91 (m, 2H, H 2 , H 2′ ), 1.78-1.72 (m, 4H, CH 2 CH 2 ), 1.62-1.49 (m, 4H, CH 2 CH 2 ), 1.44-1.33 (m, 4H, CH 2 CH 2 ).  13 C NMR (50 MHz, CD 3 OD): δ 174.2, 171.1, 155.7, 141.7, 124.7 (2C), 122.5 (2C), 106.4, 96.3, 92.3, 72.5, 70.8, 68.4, 67.8, 66.4, 66.2, 64.5, 62.7, 60.9, 38.9, 36.1, 35.2, 33.2, 33.1, 29.2, 28.9, 26.3, 25.5, 24.8, 23.8. Elem. Anal. for C 32 H 46 N 2 O 15 S: Calc. C, 52.59; H, 6.34; N, 3.83. Found; C, 52.79; H, 6.61; N, 5.51. ESI-MS 753.3 [M+Na] + . 
     Synthesis of Compound 5 
     
       
                 
         
             
             
         
      
     
     To a solution of KLH (20 mg) in 20 mL of buffer (0.1 M sodium phosphate and 0.15 M sodium chloride, pH 7.2) 19 (15 mg, 0.020 mmol) was added and the mixture was stirred overnight at RT. The solution was lyophilized and the solid was dialysed to give 18.5 mg of glycoconjugate 5. 
     Thioether-protein ratios were evaluated by determination of free lysine amino groups before and after conjugation by titration with trinitrobenzensulfonic acid. 
     Immunization and Fusion. 
     Two Female, 8 weeks old, BALB/c mice (Ce.S.A.L., Firenze) were immunized with intraperitoneal (mouse A) and intravenous (mouse B) injections with antigen-KLH conjugate solution four times, twice a week. In the first immunization the antigen was injected as a 1:1 (v/v) emulsion in Freund&#39;s complete adjuvant (mouse A) to a final volume of 0.2 mL and as a 1:1 (v/v) solution in sterile PBS (mouse B) to a total volume of 0.2 mL. The second and third immunizations were performed in the way, except that an incomplete Freund&#39;s adjuvant was used in mouse A. The final immunization was given 3 days prior to cell fusion, through intravenous injection in both mouse A and B. On the day of fusion mice were killed by cervical dislocation, spleens were harvested, and 1×10 8  cells were collected and used for each fusion. 
     Spleen cells were fused with a hypoxanthine-aminopterin-thymidine (HAT) sensitive mouse myeloma cell line, NS0, by the polyethylene glycol (PEG) method (29). NS0 cells were maintained in DMEM with 10% Fetalclone I (HyClone) and split 1:2 the day before fusion. 1×10 8  NS0 cells were mixed in serum-free DMEM, centrifuged and, after removing the supernatant, were placed in a water bath at 37° C. and 1 mL of pre-warmed PEG (Sigma-Aldrich) were added, “drop to drop”. At the end of PEG addition, pre-warmed serum-free medium was added and the tube was centrifuged to remove supernatant. The fusion product was resuspended in DMEM containing 20% Fetalclone 1 and 1×HAT, and the ensuing cell suspension was plated in 24 well plates; cells were incubated at 37° C. in a CO 2  incubator. 
     ELISA Tests 
     Hybridoma supernatants were screened for binding to GM 3  ganglioside by enzyme-linked immunosorbent assay (ELISA). The ELISA 96 well plate (Corning) was first treated with 100 μL of GM 3  (30 μg/mL in 100% ethanol) overnight at room temperature. To minimize nonspecific adsorption 300 μL of PBS containing 0.05% Tween (TPBS) and 3% BSA were added for 1 h, and washed three times with TPBS. Hybridoma culture supernatants (100 μL/well) were added to the coated plate and incubated at room temperature for 2 h. Subsequently, the plate was washed and incubated with secondary antibody (peroxidase-labeled anti-mouse, 1:500 in TPBS) for 1 h. The plate was washed three times with TPBS, then 100 μL tetramethylbenzidine (TMB, Sigma) were added to each well. The plate was further incubated for 5 min, and hence the reaction was stopped with HCl 0.5 M. Absorbance was measured using a microplate reader (ELX800, Bio-Tek Instruments, Inc.) at 450 nm. 
     Immunocitochemistry (ICC) 
     ICC was performed on B16F1 melanoma. Cultured B16 melanoma cells were grown in DMEM supplemented with 10% FCS and 2 mM L-glutamine. Cells were mounted on polylysine-coated slides and fixed with formaline solution. After blocking endogenous peroxidases, cells were treated with proteinase K (Roche; 5 g/ml in PBS) and UltraVBlock solution (LabVision) and then incubated with the primary antibody (hybridomas supernatant) and with the anti-GM3 antibody (M2590) diluted 1:100 in PBS-UltraVBlock (10:1 v/v) overnight at 4° C. immunostaining was carried out using a commercially available kit (PicTure Plus kit; Zymed). For the negative control hybridomas medium replacing the primary antibody was used.