PROCESS FOR PRODUCING GM2 SPECIFIC ANTIBODIES

Abstract: The invention relates to the production of ganglioside-specific antibodies. These antibodies are produced following immunization with a lipopolysaccharide antigen of Campylobacter jejuni. The antibodies bind to GM2.

Description: PROCESS FOR PRODUCING GM2 SPECIFIC ANTIBODIES FIELD OF THE INVENTION This invention relateε to the making of antibodies against glycolipids, and the uses of these antibodieε. More particularly, it relates to processes for making antibodies specific to the monosialogangliosides known as "GM2" and "GM1" , as well as the uses of these antibodies. The uses include diagnostic and screening applications, aε well aε therapeutic modalities. BACKGROUND AND PRIOR ART Gangliosides are a class of moleculeε which are glycolipids. Different gangliosides have been identified as prominent cell surface constituents of various transformed cells, including melanoma, as well as other tumors of neuroectodermal origin. See, e.g., Ritter and Livingston, et al., Sem. Cane. Biol. 2: 401-409 (1991). Oettgen, VCH Verlags Gesellschaft (Weinheim Germany 1989), incorporated by reference in their entirety. Gangliosides are known as mono-, di-, tri or polysialoganglio- sides, depending upon the degree of glycosylation with sialic acid residueε. Abbreviationε employed to identify these molecules include "GM1", "GD3", "GT1" , etc., with the "G" standing for ganglioside, "M" , "D" or "T", etc. referring to the number of sialic acid residues, and the number or number plus letter (e.g., "GTla"), referring to the binding pattern observed for the molecule. See Lehninger, Biochemistry, pg. 294-296 (Worth Publishers, 1981); Wiegandt, in Glycolipids: New comprehensive Biochemistry (Neuberger et al., ed. , Elsevier, 1985), pp. 199-260. The onosialoganglioside GM2 has the structure: GalNAcBl → 4GalBl → 4GlcBl → Ceramide 3 2αNeuAc while GM1 has structure GalBl - 3GalNAcBl - 4GalBl - 4Glcβl→Ceramide 3 2αNeuAc As noted, supra, the gangliosides are prevalent cell surface markers on transformed cells, such as melanoma. This has made them attractive targets for cancer research. Livingston, et al., Proc. Natl. Acad. Sci. USA 84: 2911-2915 (1987) incorporated by reference supra , describe resultε of a vaccine based trial, wherein subjects afflicted with melanoma received, as vaccines, either whole cellε which present high levels of GM2, pure GM2 or pure GM2 plus bacterial adjuvant. Attention is also drawn to Livingston, et al., J. Clin. Oncol. 12(5): 1036-1044 (1994), and Irie, et al. , U.S. Patent No. 4,557,931, both of which are incorporated by reference, and deal with the use of GM2 as a vaccine. There has also been interest in the manufacture and use of antibodies, such as monoclonal antibodies, which bind to ganglioεides. Such antibodies share the common characteristics of all antibodies, including antigenic specificity, and are of interest both diagnostically and therapeutically. See, e.g., Kjeldsen, et al., U.S. Patent No. 5,229,289; Nudelmann, et al., U.S. Patent No. 5,240,833; Hakomori, et al., U.S. Patent No. 5,308,614, and Hakomori, et al., U.S. Patent No. 5,389,530. All of these patents are incorporated by reference, and all deal in large part with the generation of ganglioside specific antibodies. There are difficulties unique to the immunology of gangliosides, which are touched upon briefly here. First, while these molecules are prevalent on transformed cells, they are also common on certain normal cells, such aε neural cellε. There is a risk, in administering gangliosides to a subject, that the resulting antibody response will damage normal cells. Indeed, certain autoimmune pathologies, such as Guillain- Barre' Syndrome, are characterized by autoimmune antibodies reactive with GM1 or GQlb. See, e.g., Yuki, et al., J. Exp. Med. 178: 1771-1775 (1993); Aεpinall, et al., Infect & Immun. 62(5): 2122-2125 (1994). There iε an additional practical problem in that ganglioεideε are extremely difficult to εecure in amountε sufficient for immunization protocols. No practical εynthetic method is presently available. As a result, gangliosides are secured via purification from tissue, such as bovine cranial tissues. Even under optimum conditionε, the yieldε of pure ganglioεideε are vaniεhingly εmall. Further, purification from mammalian tissue carries with it the riεk of tranεmitting contaminantε εuch aε viruεeε, prion particleε, and so forth. Alternate methodologies for securing ganglioside εpecific antibodieε are thuε highly deεirable. Lipopolyεaccharide, or "LPS" moleculeε are found on the surface of Gram negative bacteria. Many of theεe moleculeε are quite toxic, leading to toxic shock syndrome, endotoxemia, and other conditions. There is a great deal of diversity in the LPS molecules of different bacteria. Indeed, even within the confines of a particular type of bacteria, the LPS molecule may differ, between variouε serotypes/serovars. The LPS molecules of Campylobacter jejuni ("C. jejuni") have been studied in some detail. Representative, but by no means exhaustive, of the studies on these LPS moleculeε, are Aspinall, et al., Eur. J. Biochem. 213: 1017-1027 (1993); Aspinall, et al., Biochem. 33: 241-249 (1994); Yuki, et al., Infect. & Immun. 62(5): 2101-2103 (1994); Aspinall, et al., Infect. & Immun. 62(5): 2122-2125 (1994). All of these references are incorporated by reference. The Aspinall 1993 paper is of particular intereεt in that it presents LPS structures for C. jejuni serovars 0:1, 0:4, 0:23, and 0:36. Aspinall et al., state that OS:l, OS:23 and OS:36 are mutally indistinguishable, and have chain termini identical to those of GM2, while the OS:4 chain terminus is identical to GDla. Some of these papers discuss certain similarities between portions of the 0:19 LPS molecule and GM1, an idea also discussed by Wirguin, et al., Ann. Neurol. 35(6): 698-703 (1994), incorporated by reference, as well as by Aspinall, et al., Infect. & Immun. 52(5): 2122-2125 (1994); Yuki, et al. , Infect. & Immun. 62(5): 2102-2103 (1994); Yuki, et al., J. Exp. Med. 178: 1771-1775 (1993). C. jejuni bacteria are themεelveε implicated in gastrointestinal disorderε, and there haε been some interest and activity in developing diagnostic teεtε for determining the preεence of C_j_ jejuni in εamples. See PCT Application WO86/01808 to Wright et al., U.S. Patent No. 5,200,344 to Blaεer, et al., U.S. Patent No. 5,169,757, to Yamazaki, et al., and Japaneεe Patent Application 63-273497 on this point. One notes that in all of these references, the manner in which the antibodies are generated involves the use of whole cells. Blaser, et al., speak of an antigen unique to C. jenuni, but it is a protein, not a glycolipid. There has been no linking of C. jejuni LPS antigens are a source for producing antibodies againεt GM2. Further, there haε been no suggestion that the antibodies produced in this way may be used in diagnostic and therapeutic approaches to pathological conditions, such aε cancer. Theεe are all features of the invention described herein, in detail, in the diεcloεure which followε. BRIEF DESCRIPTION OF THE FIGURES Figure 1 iε a dot blot εhowing reεults obtained, testing various ganglioεide εpecies, with antiserum obtained following immunization of rabbits with LPS prepared from various Campylobacter jejuni serovars. Laneε 1 and 2 uεed antiserum following immunization with 0:1, lanes 3 and 4 used 0:19, lanes 5 and 6 used 0:23, lanes 7 and 8 used 0:36, and lanes 9 and 10 used bovine brain ganglioside GM2. Odd numbered lanes are tests with pre-immune serum, and even numbers are immune seru . Figures 2A and 2B preεent data obtained in immune thin layer chromatography tests. Figure 2A shows results obtained by testing pre-immune serum. The pre-immune serum was from a rabbit prior to immunization, tested with LPS 0:1, diluted 1:100 in PBS. The TLC was developed in two solvents (i.e., CH 3 C1 and acetone, at 1:1 ratio is the first solvent, the second solvent is CHC1 3 , methanol, and 0.2% CaCl 2 , at 55:45:10 ratio) prior to overlaying with serum. Moving acrosε the lanes, lane 1 shows a teεt with mixed bovine brain ganglioεideε (10 ul) lane 2 teεted bovine brain ganglioεide GM2 (1 ug) , lanes 3-5 tested, respectively, human melanoma (5 ul), human neuroblastoma (4 ul), and human renal carcinoma (5 ul) cell lines, while lane 6 tested fresh human melanoma (3 ul). In figure 2B, immune serum from the same subject animal was tested. The serum was taken from the fifth bleed, and diluted 1:500 in PBS, as were the materials tested. Note that the upper bandε, in laneε 5 and 6, were also found in these bands when pre-immune serum waε tested, and thus are not a result of the immunization. In contrast, the lower bands in lanes 5 and 6 are the result of the immunization, while the upper bands are not. Figures 3A and 3B depict the results obtained in antibody dependent cellular cytotoxicity tests. The serum used was obtained following immunization with LPS serotype 0:1. In figure 3A, renal cell carcinoma cell line SK-RC-9, which is known to present GM2 on its surface was tested, while in figure 3B, colon carcinoma cell line SW 1222, which does not present GM2, was tested. In both figures, the open circles present data pointε where effector cellε, target cells, and immune serum were combined. Filled in circles present data points from tests involving effector cells, target cells, and pre-immune serum, while filled in squares represent the combination of target cells and immune serum without effector cells. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1 All bacteria used were obtained from the American Type Culture Collection. Samples of Campylobacter ieπuni ("C. jejuni" hereafter) of serotype 0:1, 0:19, 0:23, and 0:36 were used. For each sample, the lipopolysaccharide antigens ("LPS" antigens hereafter) were extracted from heat inactivated bacteria, using the hot phenol-water methodology of Westphal, et all, Z. Naturforschlag. 7b: 148-155 (1952), incorporated by reference herein with minor modificationε. Specifically, RNA waε removed by digestion with commercially available bovine pancreas RNA, followed by size exclusion chromatography on Sephadex G-25 in accordance with Goldman, Meth. Enzymol. 138: 267-275 (1987). The extracted LPS was lyophilized, and stored at -20°C until ready for use. Example 2 The LPS preparations were then used to prepare immunogenic compositions. In each case, the LPS antigen was mixed with adjuvant. For each composition, two rabbits were used as subject animals (female New Zealand white rabbits, 2- 2.5 kg in weight). On day 0, the animals were injected, intradermally, with 500 ug of LPS in Complete Freunds Adjuvant. Fourteen days later, the animalε received a second injection of 500 ug of LPS, this time in Incomplete Freund's Adjuvant. This waε followed up with injectionε at day 28 and day 42, in each caεe with 250 ug of LPS in Incomplete Freund's adjuvant. Two animals were injected with pure bovine brain GM2, (200 ug, 200 ug, 100 ug, 100 ug, at the same time points in the experiment). All animalε were bled at dayε 0, 14, 28, 42 and 64, to determine antibody titer and to conduct additional analyεeε. Some rabbitε were alεo given booεter injections of 250 ug LPS in IFA, 13 and 17 weekε after the fourth injection, and were bled two weekε after the laεt booεter injection. Example 3 With reference to the immunization protocol deεcribed, supra, following each bleed, sera from the immunized rabbits were analyzed, via a standard ELISA, to determine (i) if antibodies against GM2 were present, and (ii), if so, were these of class IgG, or IgM. This was done using commercially available antirabbit IgM or IgG antibodies. Immunization with C. eiuni serovar 0:1 yielded IgG antibodies, and high titer serum (1:3200, and 60,000 after booster injections), as well as low titer IgM antibodies (1:100), both of which " recognized GM2. The immunization with C. jeiuni 0:36 yielded moderate titer IgM antibodies (1:800), and low titer IgG antibodies (1:100). These also recognized GM2. The immunization with . 0:19 yielded high titer IgG antibodies (peak: 1:12800), and moderate to high titer IgM antibodies (peak: 1:1200); however, ♦5 theεe antibodieε recognized ganglioεide GM1. The teεt with pure, bovine brain GM2 yielded low titer IgM (1:200), and IgG (1:200) . Example 4 The sera obtained as deεcribed, εupra, was analyzed 10 thoroughly for specificity in dot blot assays. The sera were stained for IgM and IgG antibodies to GM2, and also to a panel of other gangliosideε derived from bovine brain material, including GM3, GMl, GD3, GD2, GDla, GDlb, GTlb, and GQlb, immobilized on nitrocellulose paper. The resultε follow, in 15 Table l. The dot blot assay was a standard one, uεing εerum dilutionε of 1:100 and commercially available anti-rabbit IgG and IgM antibodies. In table 2, which follows, a grade of "3+" means that there was strong staining, "2+" a medium- strong εtain, "1+", a weak εtain, and "+/", a trace of 20 εtaining. Figure 1, which follows, presents the dot blots. 8 TABLE 1 IgG reactivity of immune εera with ganglioεides and LPS aε determined by dot blot immune stains Animal Vaccine Serum* Ganglioside GM3 GM2 GMl GD3 GD2 GDla GDlb GTlb 1 LPS 0:1 pre-vacc. - - - - - - - - 1 " 4. vacc. 3+ - - - - - 2 " pre-vacc. - - - - - - - - 2 " 4. vacc. . 2+ - - - - - - 3 LPS0.19 pre-vacc. - - - - - - - - 3 " 4. vacc. - 3+ 1+ 2+ - 4 " pre-vacc. - - - - - - - - 4 " 4. vacc. - - 3+ - - - 3+ - 5 LPS0.23 pre-vacc. - - - - - - - - 5 " 4. vacc. - - - - - - - - 6 " pre-vacc. - - - - - - - - 6 " 4. vacc. - - - - - - - - 7 LPS0:36 pre-vacc. - - - - - - - - 7 " 4. vacc. - - - - - - - - 8 " pre-vacc. - - - - - - - - 8 " 4-vacc. - - - - - - - - 9 GM2 pre-vacc. - - - - - - - - 9 " 4. vacc. - 1+ - - - 10 " pre-vacc. - - - - - - - - 10 " 4. vacc. . 2+ - - - - - - *serum dilution 1:100 As will be εeen in Table 1, there waε εtrong εerum IgG reactivity with GM2 from animalε immunized with C. ieiuni serovar 0:1 LPS, while the other serum sampleε tested negative. The sera from C. ieiuni serovar 0:19 immunized animals showed strong reactivity with GMl, and moderately strong activity with GDlb. Weak activity with GDla was alεo seen. Example 5 In further experiments, the sera were teεted, in immune thin layer chromatography, for reactivity with purified bovine brain GM2, and with GM2 extracted from freεh human melanoma, a human renal cell carcinoma cell line, a human melanoma cell line, and from a human neuroblaεtoma cell line. The immune thin layer chromatography testε were carried out using standard methodologies. The IgG antibodies obtained from sera * of animals immunized with C. jejuni serovar 0:1 reacted strongly with all sourceε of GM2. Figure 2A and 2B εhow theεe .5 results. The IgG antibodies obtained from εera obtained from C. jejuni serovar 0:19 immunized animals reacted strongly with purified bovine brain GMl. Example 6 The ability of the GM2 specific sera to bind to cell 10 surfaces was tested in a standard mixed hemadsorption aεεay. In thiε aεsay, human renal carcinoma cell line SK-RC-9, which expresseε high levels of cell surface GM2 on its surface, was combined with the sera obtained from animals immunized with C. jejuni εerovar 0:1 LPS. the antibodieε in the sera were 15 surface reactive (peak titer 1:600). See following Table 2. TABLE 2 Cell surface reactivity of immune sera with SK-RC-9 as determined by mixed hemasorption assays Animal Vaccine Serum Titer* IgM IgG 1 LPS C. jejuni 0: 1 pre-vacc. - +/- 1 2. wks post 1. vacc. +/- 1:100 1 2. wks post 2. vacc. - 1:300 1 2. wks post 4. vacc. - 1:600 2 pre-vacc. - +/- 2 2. wks post 1. vacc. 1:100 +/- 2 2. wks post 2. vacc. - +/- 2 2. wks post 4. vacc. +/- 1:150 2 2 wks. post 6. vacc. 1:2000 1:1600 9 GM2 gangl ioside pre-vacc. +/- - 9 2. wks post 1. vacc. +/- - 9 2. wks post 2. vacc +/- - 9 2. wks post 4. vacc 1:300 1:400 10 pre-vacc. - +/- 10 2. wks post 1. vacc. +/- +/- 10 2. wks post 2. vacc. 1:100 +/- 10 2. wks post 4. vacc. +/- +/- Starting dilution of serum: 1:100 Example 7 Further experiments were carried out to test for the presence of antibodies which mediate antibody-dependent cellular cytotoxicity ("ADCC"). These experiments involved the well known 51 Cr release assay. In these experimentε, εerum obtained from rabbits which had received six vaccinations with LPS isolated from C. ieiuni serotype 0:1 was combined with various tumor cell lines. The antibodies were cytotoxic to GM2 expressing tumor cell lineε, including renal cell carcinoma cell line SK-RC-9 (up to 60% εpecific killing, at serum dilutions of 1:800), and neuroblastoma cell line 1MR-32 (up to 80% specific killing, at serum dilutions of 1:800); SK-Mel-31 (a melanoma cell line; up to 35% specific lysis), and renal cell carcinoma SK-RC-49 (35% specific lysis, at 1:400 serum dilutions. There was no specific lysiε found for those cell lineε which did not preεent GM2 on their εurfaceε. Normal rabbit εerum did not mediate cell lysiε either. Thiε will be εeen in figure 3. The foregoing exampleε set forth a process for making antibodies specific for a ganglioside, i.e., GM2 or GMl. The methodology iε surprising in that the immunogen is not a ganglioside. As iε seen, supra. immunization with a molecule of εtructure GalNAcBl-4Gal(II 3 NeuAc)-Hex, generated anti-GM2 specific antibodies, while immunization with an immunogen of εtructure GalBl-3GalNAcBl-4Gal(II 3 NeuAc) generated anti-GMl εpecific antibodieε. These molecules are not gangliosides. The immunogen used is preferably a lipopolyεaccharide molecule, most preferably an LPS molecule secured from C. jejuni. As will be seen from the discussion, supra, different εerovars of C. jejuni have different LPS molecules on their surface. It is well within the skill of the artisan to carry out analyses to determine if a molecule, such as an LPS molecule has the desired structure as per Aspinall, et al. , 1993, supra , e.g. Thus, the invention is not limited to the use of the specific serovarε diεcussed herein. It is preferred that the immunogens be administered together with an adjuvant, such as the described Complete and Incomplete Freund's Adjuvant. Other adjuvants, including, e.g., Bortadella pertussis. aluminum hydroxide, QS-21, BCG, Salmonella innesota R595, adjuvant, MPL and other adjuvantε well known to the art may be used. Similarly, the immunogen may be used "as is", such as described herein, or it may be 12 coupled to materials which enhance itε immunogenicity, εuch aε Keyhole limpet hemocyanin, bovine serum albumin, etc. The subject animal may be any of the standard mammalian hostε uεed in the art for generating antibodieε, including mice, ratε, and other rodentε; εheep, goatε, and other ruminantε, and εo forth. The antibodieε described herein are polyclonal in nature. It is aεsumed, however, that one of ordinary skill is fully able to work with materialε which generate the diεclosed polyclonal antibodies so as to develop hybridomas and monoclonal antibodies. Such methodologies, including the well known Kδhler-Milstein method and other wayε to "immortalize" antibody producing B cells, need not be set forth here. While one does not use humans as test animalε, the ability to generate antibodieε againεt these LPS structureε doeε provide the artisan with an appropriate way to vaccinate individuals to achieve active, rather than passive immunization. The art is fully familiar with methodologies which eliminate the toxic effect of LPS molecules without εignificantly modifying their immunogenicity. It haε been εhown, that mammalian immune εystems can, and do react to these LPS molecules to produce antibodieε againεt GM2. It haε alεo been εhown that variouε types of cancer, such aε renal carcinoma, neuroblastoma carcinoma, sarcoma, glioma, seminoma and melanoma, are characterized in part by expression of GM2 molecules on their surface. Vaccination with non-toxic forms of LPS, including those used in the examples described herein, should lead to immunological clearance of GM2 presenting cancer cells. Hence one aspect of the invention relates to a method for treating a subject with a pathological condition, such as a cancer, where abnormal cellε present GM2 on their surfaces, by administering an effective amount of a non-toxic LPS molecule to the subject, so as to provoke production of anti-GM2 antibodies in said subject. As iε shown in the examples, supra, antibodies produced in the manner described are useful diagnostically, as in the determination of whether or not cells are preεent in a sample 13 which present ganglioεideε GMl or GM2 on their εurface. Thuε, one is able to screen a sample for irregularitieε, εuch as the onset of tumorigenicity, where tumor cells present GMl or GM2 on their surface, while non-tranεformed cells do not. Melanoma and renal carcinoma are exampleε of εuch transformed cells. Similarly, samples can be analyzed generally, using the antibodies deεcribed herein. For example, a bovine brain fraction can be aεεayed to determine whether or not deεired ganglioεideε GMl and/or GM2 are preεent, and if εo, whether or not they are preεent in an amount to warrant further analyεis and purification. The work described supra as it relates to vaccines alεo provides support for another aspect of the invention, which is a method for treating a pathological condition, such aε melanoma, neuroblastoma, sarcoma, glioma, seminoma, and other types of cancer wherein the pathology is characterized by appearance of GM2 or GMl on abnormal cells. One may adminiεter a therapeutically uεeful dose of the antibodies of the invention sufficient to provide a therapeutically positive effect to the subject εo treated. Other aspects of the invention will be clear to the skilled artisan and need not be repeated here. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expresεionε of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modificationε are possible within the scope of the invention.

Claims: Claims : 1. Process for making an antibody which binds to mono¬ sialoganglioside GM2, comprising: (i) immunizing a εubject animal with an antigen compriεing the εtructure: GalNAcβl→4Gal(II 3 NeuAc)Hex in an amount sufficient to provoke production of antibody by said animal with the proviso that the antigen is not GM2, and (ii) isolating the antibodies produced by said animal. 2. The method of claim 1, wherein said antigen is a lipo-polysaccharide. 3. The proceεε of claim 2, wherein εaid lipopolysaccharide is purified lipopolysaccharide from a gram negative bacteria. 4. The method of claim 3, wherein said gram negative bacteria are Campylobacter jejuni. 5. The process of claim 4, wherein said Campylobacter jejuni are of serotype 0:1. 6. The process of claim 4, wherein said Campylobacter iejuni are of serotype 0:36. 7. The process of claim 2, wherein said lipopolysaccharide is combined with an adjuvant. 8. The process of claim 7, wherein said adjuvant is Complete Freund's Adjuvant or Incomplete Freund's Adjuvant. 9. Method for determining presence of monosialoganglioside GM2 in a sample, comprising contacting said sample with the antibody produced in accordance with claim 1, and determining binding of said antibody as a determination of GM2 in said sample. 10. The method of claim 9, wherein said sample contains transformed cells. 11. The method of claim 10, wherein said transformed cellε are melanoma cellε, neuroblaεtoma cells, sarcoma cells, glioma cells, or seminoma cells. 12. Method for treating a subject with a pathological condition characterized by abnormal expresεion of GM2 molecules on cellε, comprising administering to said subject a therapeutically effective amount of the antibody produced in accordance with claim 1.

Inventors: OLD LLOYD J, RITTER GERD, OLD, LLOYD, J., RITTER, GERD
Applicants: LUDWIG INST CANCER RES, LUDWIG INSTITUTE FOR CANCER RESEARCH
Application year: 1996
Main classification: G01N33
IPC codes: A61K39, A61P31, A61P35, C07K16, C12P21, C12R1, G01N33