Title: Molecular mimetics of unique Neisseria meningitidis serogroup B epitopes
United States Patent: 6,642,354
Issued: November 4, 2003
Inventors: Granoff; Dan M. (Berkeley, CA); Moe; Gregory R. (Alameda, CA)
Assignee: Chiron Corporation (Emeryville, CA); Children's Hospital Medical Center of Northern California (Oakland, CA)
Appl. No.: 910552
Filed: July 23, 2001
Novel bactericidal antibodies against Neisseria meningitidis serogroup B ("MenB") are disclosed. The antibodies either do not cross-react or minimally cross-react with host tissue polysialic acid and hence pose minimal risk of autoimmune activity. The antibodies are used to identify molecular mimetics of unique epitopes found on MenB or E. coli K1. Examples of such peptide mimetics are described that elicit serum antibody capable of activating complement-mediated bacteriolysis of MenB. Vaccine compositions containing such mimetics can be used to prevent MenB or E. coli K1 disease without the risk of evoking autoantibody.
SUMMARY OF THE INVENTION
The present invention is based on the discovery of functionally active antibodies directed against MenB PS derivatives, wherein the antibodies do not cross-react, or are minimally cross-reactive, with host tissues as determined using the assays described herein. These antibodies therefore pose minimal risk of evoking autoimmune disease and are termed "non-autoreactive" herein. Assays used herein to determine autoreactivity include binding assays against a neuroblastoma cell line expressing long chain polysialic acid residues on the cell surface. Specifically, antibodies that are negative in these assays are considered to lack autoreactivity. The non-autoreactive antibodies are particularly useful for identifying molecular mimetics of unique MenB PS epitopes that can be used in vaccine compositions. Furthermore, the antibodies, humanized versions of the antibodies, fragments and functional equivalents thereof, will also find use in passive immunization against, and/or as an adjunct to therapy for, MenB and E. coli K1 disease. Since such molecules do not bind to polysialic acid in host tissue as determined by the autoreactivity assays described herein, they provide a safe and efficacious method for the treatment and/or prevention of MenB and E. coli K1 disease.
Accordingly, in one embodiment, the subject invention relates to antibodies directed against MenB PS derivatives, wherein the antibodies are not autoreactive with host tissue. Such antibodies may further be characterized as being capable of eliciting functional activity against MenB bacteria. One particular group of such antibodies is also characterized as non cross-reactive with Neisseria meningitidis serogroup B capsular polysaccharide (NAc-MenB PS) in an ELISA. However, these antibodies are anti-capsular in that they can bind to the cell surface of a Group B encapsulated bacteria, but not to capsular-deficient mutants.
Another embodiment of the invention relates to monoclonal antibodies directed against MenB PS derivatives, and hybridomas producing those monoclonal antibodies.
Other embodiments of the invention relate to unique Neisseria meningitidis serogroup B epitopes that are capable of being bound by the antibody molecules of the present invention.
Still further embodiments of the subject invention are related to methods for isolating molecular mimetics of unique epitopes of MenB PS and molecular mimetics identified using the methods. The methods comprise:
(a) providing a population of molecules including a putative molecular mimetic of a unique epitope of MenB PS;
(b) contacting the population of molecules with the antibodies described above under conditions that allow immunological binding between the antibody and the molecular mimetic, if present, to provide a complex; and
(c) separating the complexes from non-bound molecules.
In another embodiment, the subject invention is directed to a vaccine composition comprising a unique epitope of MenB in combination with a pharmaceutically acceptable excipient.
In yet another embodiment, the invention is directed to a vaccine composition comprising a molecular mimetic of a unique epitope of MenB in combination with a pharmaceutically acceptable excipient.
In still a further embodiment, the invention is directed to a vaccine composition comprising an anti-idiotypic antibody molecular mimetic of a unique epitope of MenB in combination with a pharmaceutically acceptable excipient.
In yet further embodiments, the invention relates to pharmaceutical compositions comprising the antibodies described above.
In another embodiment, the subject invention is directed to a method for treating or preventing MenB and/or E. coli K1 disease in a mammalian subject comprising administering an effective amount of the above pharmaceutical compositions to the subject.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); and Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., 1986, Blackwell Scientific Publications).
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
Modes of Carrying Out the Invention
The present invention is based on the discovery of novel functional antibodies directed against MenB. The antibodies do not cross-react, or are minimally cross-reactive with polysialic acid in host tissue as determined using the assays described herein, and hence the antibodies have a lower risk of evoking autoimmune activity than antibodies that are highly cross-reactive with host tissue. The antibodies can be used to identify molecular mimetics of unique epitopes found on the surface of MenB. The antibodies and/or mimetics can be used in vaccine compositions to treat and/or prevent MenB and E. coli K1 disease, as well as in diagnostic compositions for the identification of MenB and E. coli K1 bacteria.
As explained above, the native capsular polysaccharide of MenB, termed "MenB PS" herein, is poorly immunogenic in humans and other mammalian subjects. Furthermore, native MenB PS can elicit the production of autoantibodies and, hence, may be inappropriate for use in vaccine compositions. Thus, the present invention uses antibodies prepared against MenB PS derivatives. These antibodies are selected based on their ability to exhibit functional activity against MenB bacteria, wherein the functional activity is important in conferring protection against MenB disease. The antibodies are also selected on the basis of showing minimal or undetectable autoimmune activity.
More particularly, MenB PS derivatives were prepared for use in obtaining the antibody molecules of the present invention. The derivatives generally comprise C3 -C8 acyl substitutions of sialic acid residue N-acetyl groups of the native molecule. Particularly preferred MenB PS derivatives comprise the substitution of N-propionyl groups for N-acetyl groups of native MenB PS and are termed "NPr-MenB PS" herein. Such derivatives and methods for synthesizing the same are described in e.g., U.S. Pat. No. 4,727,136 and EP Publication No. 504,202 B, both to Jennings et al.
The C3 -C8 acyl derivatives can be made by first treating native MenB (obtained from e.g., N. meningitidis cultures) in the presence of a strong base to quantitatively remove the N-acetyl groups and to provide a reactive amine group in the sialic acid residue parts of the molecule. The deacylated MenB PS fragments are then N-acylated. For example, in the case of NPr-MenB PS, the deacylated molecule is N-propionylated using a source of propionyl groups such as propionic anhydride or propionyl chloride, as described in U.S. Pat. No. 4,727,136 to Jennings et al. The extent of N-acylation can be determined using, for example, NMR spectroscopy. In general, reaction conditions are selected such that the extent of N-acylation is at least about 80%.
In order to increase the immunogenicity of the MenB PS derivatives, the derivatives can be conjugated to a suitable carrier molecule to provide glycoconjugates. Particularly, N-acylated MenB PS glycoconjugate preparations having well defined and controlled structural configurations can be formed from intermediate sized N-acylated MenB oligosaccharides as described below.
Thus, a group of N-acylated MenB PS glycoconjugates, an example of which is termed "CONJ-2" herein, can be prepared as follows. An N-acylated MenB PS preparation, having substantially 100% N-acylated sialic acid residues, as determined by, e.g., NMR analysis, can be fragmented under mild acidic conditions to provide a population of oligosaccharide molecules of varying sizes. The fragmented products are size fractionated, using for example, standard ion exchange chromatographic techniques combined with e.g., stepwise salt gradients, to provide fractions of N-acylated MenB molecules of homogenous sizes. Fractions containing intermediate sized oligosaccharides e.g., with an average Dp of about 5 to about 22, preferably 10 to about 20, and more particularly about 12 to about 18, are chemically end-activated at the non-reducing termini and conjugated to protein carriers by a reductive amination technique to provide the CONJ-2 glycoconjugates. Successful conjugation can be determined by, e.g., gel filtration, and the final saccharide to protein ratio (w/w) assessed by colorimetric assay.
Glycoconjugates formed from MenB PS derivatives, such as the CONJ-2, are then used herein to elicit the formation of anti-saccharide antibodies in an immunized host. A subset of such antibodies should bind to MenB bacteria, should not cross-react, or be minimally cross-reactive with host tissue sialic acid residues as determined using the binding assays described herein. The antibodies can be fully characterized with respect to isotype, fine antigenic specificity, functional activity and cross-reactivity with host tissue.
For example, mammalian subjects, conveniently, standard laboratory animals such as rodents and rabbits, can be immunized with compositions containing the glycoconjugates along with a suitable adjuvant to elicit the production of polyclonal sera. Groups of animals are generally immunized and boosted several times with the compositions. Antisera from immunized animals can be obtained, and polyclonal sera that does not cross-react with host tissue can be obtained using in-situ absorption or conventional affinity chromatography techniques. Successful glycoconjugate antigens can be identified by their ability to elicit a substantial IgG anti-MenB PS derivative antibody response, characteristic of a T-cell dependent antigen. Conjugates that are found to be highly immunogenic and produce predominantly IgG antibodies are particularly preferred for use in the methods of the present invention.
MenB PS derivatives that are capable of eliciting the formation of bactericidal antisera are suitable for use in the production of monoclonal antibodies. More particularly, the process used to provide the various MenB PS derivative conjugates is designed to produce superior immunogens presenting unique saccharide-associated epitopes that mimic those found on the surface of MenB organisms and are expressed minimally in the host. The MenB PS derivatives described herein are thus capable of eliciting the production of MenB-specific antibodies which can be used directly in protective or therapeutic pharmaceutical preparations or, preferably, used to search for mimetics of MenB polysaccharide antigens that will provide unique epitopes for anti-MenB vaccines.
Thus, in one embodiment of the invention, selected MenB derivatives are used to provide monoclonal antibodies and functional equivalents thereof. The term "functional equivalent" with respect to a particular monoclonal antibody, as used herein, means a molecule that: (a) cross-blocks an exemplified monoclonal antibody; (b) binds selectively to the MenB PS derivative or glycoconjugate in question; (c) does not cross-react, or minimally cross-reacts, with host PSA as determined using the binding assays described herein; and, optionally, activity (e.g., complement-mediated bactericidal and/or opsonic activity) against MenB bacterial cells as determined by standard assays described below. Further, as used herein with regard to a particular monoclonal antibody producing hybridoma of the invention, the term "progeny" is intended to include all derivatives, issue, and offspring of the parent hybridoma that produce the monoclonal antibody produced by the parent, regardless of generation or karyotypic identity.
Monoclonal antibodies are prepared using standard techniques, well known in the art, such as by the method of Kohler and Milstein, Nature (1975) 256:495, or a modification thereof, such as described by Buck et al. (1982) In Vitro 18:377. Typically, a mouse or rat is immunized with the MenB PS derivative conjugated to a protein carrier, boosted and the spleen (and optionally several large lymph nodes) removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and will not be rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas. Representative murine myeloma lines for use in the hybridizations include those available from the American Type Culture Collection (ATCC).
More particularly, somatic cell hybrids can be prepared by the method of Buck et al., (supra), using the azaguanine resistant, non-secreting murine myeloma cell line P3X63-Ag8.653 (obtainable from the ATCC). The hybridoma cell lines are generally cloned by limiting dilution, and assayed for the production of antibodies which bind specifically to the immunizing antigen and which do not bind to unrelated antigens. The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).
Hybridoma supernatant can be assayed for anti-MenB PS derivative reactive antibody using, for example, either solid phase ELISA or an indirect immunofluorescence assay with the immunizing MenB PS derivative or with native MenB PS (NAc-MenB PS). The selectivity of monoclonal antibodies secreted by the hybridomas can be assessed using competitive specific binding assays, such as inhibition ELISA, or the like. For example, antibody molecules, either diluted in buffer, or buffer containing soluble MenB PS derivatives or NAc-MenB PS, are reacted in an ELISA vessel in the presence of bound MenB PS derivatives. After washing, bound antibody is detected by labeled anti-Ig (anti-IgM, IgG and IgA) as the secondary antibody. Antibodies that are inhibited by the soluble MenB PS derivatives can be considered specific and, thus are selected for further study including, isotyping and additional screening for cross-reactivity, functional activity, and autoreactivity.
Specifically, partially purified monoclonal antibody molecules can be individually evaluated for their ability to bind to host cells which express polysialic acid residues on their cell surfaces. Such cells represent surrogate targets for the detection of antibodies that exhibit autoimmune activity. One target comprises the human neuroblastoma cell line, CHP-134, which expresses long chain .alpha.2-8 polysialic acid (NCAM) on its cell surface, as described by Livingston et al. (1988) J. Biol. Chem. 263:9443. Other suitable targets include, but are not limited to, newborn brain cells, tissues derived from e.g., kidney, heart and the olfactory nerve, cultured saphenous vein endothelial cells, cytotoxic T lymphocytes and natural killer (NK) cells. See, e.g., Brandon et al. (1993) Intl. J. Immunopathology and Pharmacology 6:77. Monoclonal antibody molecules obtained from the hybridomas can be added to suitable test cell populations in culture, and the potential binding of the monoclonals to the cellular targets detected and quantified directly using labeled monoclonals, or indirectly using an appropriately labeled secondary reagent that reacts specifically with each monoclonal antibody (e.g., Staphylococcal Protein A and G and anti-murine antibody molecules). Antibodies that do not cross-react with test host tissue PSA or that display minimal reactivity are not considered autoreactive for purposes of the present invention. Thus, these antibodies are appropriate for further use. In addition, some antibodies that show binding with test tissue, which binding is not affected by pre-treatment of the test cells with neuraminidase, may also be appropriate for further use. Autoreactivity of such antibodies is termed "indeterminate" herein.
Functional activity can be determined by assessing complement-mediated bactericidal activity and/or opsonic activity. In particular, complement-mediated bactericidal activity of the antibodies can be evaluated using standard assays such as those described by Gold et al. (1970) Infect. Immun. 1:479, Westerink et al. (1988) Infect. Immun. 56:1120, Mandrell et al. (1995) J. Infect. Dis. 172:1279, and Granoff et al. (1995) Clin. Diagn. Laboratory Immunol. 2:574. In these assays, N. meningitidis is reacted with a complement source as well as with the antibody to be tested. Bacterial counts are done at various sampling times. Those antibodies that demonstrate complement-mediated bactericidal activity, asdemonstrated by a minimum of a 50% reduction in viable bacterial cell counts determined after sixty minutes incubation with antibody and complement, as compared to colony counts at time zero, are considered to exhibit bactericidal activity for purposes of the present invention and are suitable for further use.
Complement-mediated bacteriolysis is thought to be the major mechanism responsible for host protection against invasive Meningococcal disease. However., evidence also supports an important protective role for opsonization (see, e.g., Bjerknes et al. (1995) Infect. Immun. 63:160). Accordingly, the opsonic activity of the antibodies produced herein can be evaluated as a second measure, or as an alternative measure, to assess functional activity. Results from opsonic assays can be used to supplement bactericidal data, and to help in the selection of antibodies capable of conferring protection. Evaluation of opsonic activity is also particularly useful herein for the evaluation of the murine monoclonal antibodies of the invention which have an IgG1 isotype. Murine IgG1 (in contrast to human IgG1) is ineffective in activation of complement. Thus, murine IgG1 antibodies do not activate complement-mediated bacteriolysis of MenB in the above-described assays. However, functional activity of IgG1 anti-NPr-MenB PS monoclonal antibodies can be assessed by opsonization in the absence of complement.
A variety of opsonic assay methods are known in the art, and can be used to evaluate functional activity of the monoclonal antibodies of the present invention. Such standard assays include those described by Sjursen et al. (1987) Acta Path. Microbiol. Immunol. Scand., Sec. C 95:283, Halstensen et al. (1989) Scand. J. Infect. Dis. 21:267, Lehmann et al. (1991) APMIS 99:769, Halstensen et al. (1991) NIPH Annals 14:157, Fredlund et al. (1992) APMIS 100:449, Guttormsen et al. (1992) Infect. Immun. 60:2777, Guttormsen et al. (1993) J. Infec. Dis. 167:1314, Bjerknes et al. (1995) Infect. Immun. 63:160, Hayrinen et al. (1995) J. Infect. Dis. 171:1481, de Velasco et al. (1995) J. Infect. Dis. 172:262, and Verheul, A. F. M. (1991) "Meningococcal LPS Derived Oligosaccharide-Protein Conjugate Vaccines, Immunochemical and Immunological Aspects," Thesis, Utrecht University, The Netherlands, pp. 112-135.
Selected monoclonal antibodies of interest can be expanded in vitro., using routine tissue culture methods, or in vivo, using mammalian subjects. For example, pristane-primed mice can be inoculated with log phase hybridoma cells in PBS for ascites production. Ascites fluid can be stored at -70oC. prior to further purification.
It may be desirable to provide chimeric antibodies, especially if the antibodies are to be used in preventive or therapeutic pharmaceutical preparations, such as for providing passive protection against MenB., as well as in MenB diagnostic preparations. Chimeric antibodies composed of human and non-human amino acid sequences may be formed from the mouse monoclonal antibody molecules to reduce their immunogenicity in humans (Winter et al. (1991) Nature 349:293; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA 86:4220; Shaw et al. (1987) J Immunol. 138:4534; and Brown et al. (1987) Cancer Res. 47:3577; Riechmann et al. (1988) Nature 332:323; Verhoeyen et al. (1988) Science 239:1534; and Jones et al. (1986) Nature 321:522; EP Publication No. 519,596, published Dec. 23, 1992; and U.K. Patent Publication No. GB 2,276,169, published Sep. 21, 1994).
Antibody molecule fragments, e.g., F(ab')2, FV, and sFv molecules, that are capable of exhibiting immunological binding properties of the parent monoclonal antibody molecule can be produced using known techniques. Inbar et al. (1972) Proc. Nat. Acad. Sci. USA 69:2659; Hochman et al. (1976) Biochem 15:2706; Ehrlich et al. (1980) Biochem 19:4091; Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879; and U.S. Pat. Nos. 5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778, to Ladner et al.
In the alternative, a phage-display system can be used to expand the monoclonal antibody molecule populations in vitro. Saiki, et al. (1986) Nature 324:163; Scharf et al. (1986) Science 233:1076; U.S. Pat. Nos. 4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392; Barbas, III et al. (1995) Methods: Comp. Meth Enzymol 8:94; Barbas, III et al. (1991) Proc Natl Acad Sci USA 88:7978.
Once generated, the phage display library can be used to improve the immunological binding affinity of the Fab molecules using known techniques. See, e.g., Figini et al. (1994) J. Mol. Biol. 239:68.
The coding sequences for the heavy and light chain portions of the Fab molecules selected from the phage display library can be isolated or synthesized, and cloned into any suitable vector or replicon for expression. Any suitable expression system can be used, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Expression systems in bacteria include those described in Chang et al. (1978) Nature 275:615, Goeddel et al. (1979) Nature 281:544, Goeddel et al. (1980) Nucleic Acids Res. 8:4057, European Application No. EP 36,776, U.S. Pat. No. 4,551,433, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25, and Siebenlist et al. (1980) Cell 20:269.
Expression systems in yeast include those described in Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929, Ito et al. (1983) J. Bacteriol. 153:163, Kurtz et al. (1986) Mol. Cell. Biol. 6:142, Kunze et al. (1985) J. Basic Microbiol. 25:141, Gleeson et al. (1986) J. Gen. Microbiol. 132:3459, Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302, Das et al. (1984) J. Bacteriol. 158:1165, De Louvencourt et al. (1983) J. Bacteriol. 154:737, Van den Berg et al. (1990) Bio/Technology 8:135, Kunze et al. (1985) J. Basic Microbiol. 25:141, Cregg et al. (1985) Mol. Cell. Biol. 5:3376, U.S. Pat. Nos. 4,837,148 and 4,929,555, Beach et al. (1981) Nature 300:706, Davidow et al. (1985) Curr. Genet. 10:380, Gaillardin et al. (1985) Curr. Genet. 10:49, Ballance et al. (1983) Biochem. Biophys. Res. Commun. 112:284-289, Tilburn et al. (1983) Gene 26:205-221, Yelton et al. (1984) Proc. Natl. Acad. Sci. USA 81:1470-1474, Kelly et al. (1985) EMBO J. 4:475479; European Application No. EP 244,234, and International Publication No. WO 91/00357.
Expression of heterologous genes in insects can be accomplished as described in U.S. Pat. No. 4,745,051, European Application Nos. EP 127,839 and EP 155,476, Vlak et al. (1988) J. Gen. Virol. 69:765-776, Miller et al. (1988) Ann. Rev. Microbiol. 42:177, Carbonell et al. (1988) Gene 73:409, Maeda et al. (1985) Nature 315:592-594, Lebacq-Verheyden et al. (1988) Mol. Cell. Biol. 8:3129, Smith et al. (1985) Proc. Natl. Acad. Sci. USA 82:8404, Miyajima et al. (1987) Gene 58:273, and Martin et al. (1988) DNA 7:99. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts are described in Luckow et al. (1988) Bio/Technology 6:47-55, Miller et al. (1986) GENERIC ENGINEERING, Setlow, J. K. et al. eds., Vol. 8, Plenum Publishing, pp. 277-279, and Maeda et al. (1985) Nature 315:592-594.
Mammalian expression can be accomplished as described in Dijkema et al. (1985) EMBO J. 4:761, Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79:6777, Boshart et al. (1985) Cell 41:521, and U.S. Pat. No. 4,399,216. Other features of mammalian expression can be facilitated as described in Ham et al. (1979) Meth. Enz. 58:44, Barnes et al. (1980) Anal. Biochem. 102:255, U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655 and Reissued U.S. Pat. No. RE 30,985, and in International Publication Nos. WO 90/103430, WO 87/00195.
Any of the above-described antibody molecules can be used herein to provide anti-MenB therapeutic or preventive pharmaceutical agents. Additionally, "humanized" antibody molecules, comprising antigen-binding sites derived from the instant murine monoclonal antibodies, can be produced using the techniques described above.
The anti-MenB antibodies of the present invention, described above, are conveniently used as receptors to screen diverse molecular libraries in order to identify molecular mimetics of unique epitopes from MenB. Methods for identifying mimetics in molecular libraries generally involve the use of one or more of the following procedures: (1) affinity purification with an immobilized target receptor; (2) binding of a soluble receptor to tethered ligands; and (3) testing soluble compounds directly in antigen competition assays or for biological activity. Molecules screened for molecular mimics include but are not limited to small organic compounds, combinatorial libraries of organic compounds, nucleic acids, nucleic acid derivatives, saccharides or oligosaccharides, peptoids, soluble peptides, peptides tethered on a solid phase, peptides displayed on bacterial phage surface proteins, bacterial surface proteins or antibodies, and/or peptides containing non-peptide organic moieties.
For example, libraries of diverse molecular species can be made using combinatorial organic synthesis. See, e.g., Gordon et al. (1994) J. Med. Chem. 37:1335. Examples include but are not limited to oligocarbamates (Cho et al. (1993) Science 261:1303); peptoids such as N-substituted glycine polymers (Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367); and vinylogous polypeptides (Hagihara et al. (1992) J. Am. Chem. Soc. 114:6568).
A variety of approaches, known in the art, can be used to track the building blocks as they are added during synthesis so that the history of individual library members can be determined. These approaches include addressable location on a photolithographic chip (oligocarbamates), a deconvolution strategy in which "hits" are identified through recursive additions of monomers to partially synthesized libraries (peptoids, peptides), and coding combinatorial libraries by the separate synthesis of nucleotides (Nielsen et al. (1993) J. Am. Chem. Soc. 115: 9812) or other organic moieties (Ohlmeyer et al. (1993) Proc. Natl. Acad. Sci. USA 90:10922) ("tags"). The coded tags associated with each library member can then be decoded after a mimetic has been selected. For example, nucleic acid tags can be decoded by DNA sequencing.
Peptoid combinatorial libraries are particularly useful for identifying molecular mimetics of unique MenB epitopes. Peptoids are oligomers of N-substituted glycine (Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367) and can be used to generate chemically diverse libraries of novel molecules. The monomers may incorporate t-butyl-based side-chain and 9-fluorenylmethoxy-carbonyl .alpha.-amine protection. The assembly of monomers into peptoid oligomers can be performed, for example, on a solid phase using the "submonomer method" of Zuckermann et al. (1991) J. Am. Chem. Soc. 114:10646. In this method, syntheses are conducted with Rink amide polystyrene resin (Rink et al. (1987) Tetrahedron Lett. 28:3787). Resin-bound amines are bromoacetylated by in situ activation of bromoacetic acid with diisopropylcarbodiimide. Subsequently, the resin-bound bromoacetamides are displaced by addition of an amine. The amines may incorporate t-butyl-based protection of additional reactive groups. This two-step cycle is repeated until the desired number of monomers is added. The oligopeptide is then released from the resin by treatment with 95% trifluroacetic acid/5% water. The syntheses are performed, preferably, using a robotic synthesizer. See, e.g., Zuckermann et al. (1992) Pept. Protein Res. 40:498. In the alternative, oligomerization of the peptoid monomers may be performed by in situ activation by either benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorphosphate or bromotris(pyrrolidino)phosphonium hexafluorophosphate. In this alternative method, the other steps are identical to conventional peptide synthesis using .alpha.-(9-fluorenylmethoxycarbonyl) amino acids (see, e.g., Simon et al. (1992), supra).
Once the peptoid libraries are generated, they can be screened by, e.g., adding the monoclonal antibodies of the present invention, along with various pools of the combinatorial peptoids, to wells of microtiter plates coated with MenB PS derivatives or MenB bacteria, either alone or as glycoconjugates. After a period of incubation and a wash to remove unbound antibody, the presence of bound antibody is determined by standard ELISA assays. See, e.g., Harlow & Lane, Antibodies: A Laboratory Manual (1988), Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 553. Wells that do not contain bound antibody indicate the presence of peptoid mimetics that bind to the antibody. The particular identities of the peptoid mimetics in the pools are determined by recursively adding back monomer units to partially synthesized members of the libraries. Zuckermann et al. (1994) J. Med. Chem. 37:2678.
Peptide libraries can also be used to screen for molecular mimetics of unique epitopes of MenB using the anti-MenB antibodies of the present invention. Such libraries are based on peptides such as, but not limited to, synthetic peptides that are soluble (Houghten (1985) Proc. Natl. Acad. Sci. USA 82:5131) or tethered to a solid support (Geysen et al. (1987) Immunol. Methods 102:259; U.S. Pat. No. 4,708,871) and peptides expressed biologically as fusion proteins (Scott et al. (1990) Science 249:386). For a review of peptide combinatorial libraries, see, e.g., Gallop et al. (1994) J. Med. Chem. 37:1233.
For example, random soluble peptides, having known sequences, can be synthesized on solid supports and members of the library separated from each other during the repetitive coupling/deprotection cycles in individual labeled polypropylene bags (Houghten (1985) Proc. Natl. Acad. Sci. USA 82:5131). Following synthesis, the peptides are cleaved from the solid support and identified by the label on the polypropylene bag. The synthetic peptide library generated using this method can be screened for binding to an antibody having the desired properties by adsorbing individual peptides to microtiter plate wells and determining antibody binding using standard ELISA assays.
Large libraries of potential peptide mimetics can also be constructed by concurrent synthesis of overlapping peptides as described in U.S. Pat. No. 4,708,871, to Geysen. The synthetic peptides can be tested for interaction with the antibodies by ELISA while still attached to the support used for synthesis. The solid support is generally a polyethylene or polypropylene rod onto which is graft polymerized a vinyl monomer containing at least one functional group to produce polymeric chains on the carrier. The functional groups are reacted to provide primary or secondary amine groups which are sequentially reacted with amino acid residues in the appropriate order to build the desired synthetic peptide using conventional methods of solid phase peptide chemistry. For example, peptide sequences can be made by parallel synthesis on polyacrylic acid-grafted polyethylene pins arrayed in microtiter plates, as described in Geysen et al. (1987) J. Immunol. Methods 102:259. Such libraries can be screened by, e.g., adding antibody to wells containing the peptide-pins. After washing unbound antibody from the wells, the presence of bound antibody can be detected using an ELISA assay.
Peptide mimetics that interact with the antibodies of the present invention can also be identified using biological expression systems. See, e.g., Christian et al. (1992) J. Mol. Biol. 227:711; Devlin et al. (1990) Science 249:404; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378; Gallop et al. (1994) J. Med. Chem. 37:1233. Using such systems, large libraries of random peptide sequences can be screened for molecules that bind the antibodies of the present invention. This approach also allows for simple molecular characterization of identified mimetics since DNA encoding the peptides can be readily sequenced. Additionally, rare mimetics can be amplified through several rounds of selection/amplification.
For example, phage-display libraries can be produced by inserting synthetic DNA pieces, encoding random peptide sequences, near the 5'-end of the gene encoding the pIII or pVIII protein of the filamentous bacterial phage m13, fd, or f1 (Parmley et al. (1988) Gene 73:305; Smith et al. (1993) Meth. Enzymol. 217:228). The phage, phagemid, or plasmid DNA containing the gene and randomized extension is then used to transform a suitable host such as E. coli or E. coli coinfected with a helper phage. The phage isolated from the culture carry pIII (1-5 copies) or pVIII (.about.4000 copies) surface proteins having the randomized peptide sequences extending from the amino terminus. Phage can be purified by, e.g., affinity purification by biotinylating the receptor antibodies of the present invention, incubating the phage with the biotinylated receptor and reacting the phage on streptavidin-coated plates. Bound phage are eluted and amplified by infecting a suitable host on agar medium and subjected to further rounds of affinity purification. Phage from later rounds of affinity purification can be cloned and propagated, their DNAs sequenced to determine the amino acid sequences of their expressed peptide and their binding to MenB antibodies assessed by ELISA or by a variety of other screening procedures, well known in the art.
Combinatorial libraries of human Fab antibodies can also be displayed on phage surface proteins to select useful molecular mimetics for use herein. Preparation of such libraries has been described hereinabove. See, e.g., Burton et al. (1994) Adv. Immunol. 57:191 for a review of such techniques.
Molecular mimetics of MenB unique epitopes can also be identified using the anti-MenB antibodies of the present invention in those methods described by Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865. The Cull technique utilizes the DNA binding protein, LacI, to form a link between peptide and its encoding DNA sequence. In this method, DNA encoding randomized peptides is appended to the 3'-end of the LacI gene present on a plasmid. The plasmid also contains the DNA binding site for LacI, lacO. When LacI is expressed from the plasmid in a suitable host (e.g. E. coli), it binds tightly to lacO. Thus, when the cells are lysed, each copy of LacI that displays a randomized peptide at its carboxyl terminus is associated with the DNA encoding it. Methods for screening, amplifying, and sequencing these "peptides-on-plasmids" libraries are the same as those used in phage display, as described above.
Molecular mimetics can also be identified using the anti-MenB antibodies in in vitro, cell-free systems such as the system described by Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022. In this approach, nascent peptides are displayed in polysome complexes and construction of libraries, expression of the peptides, and screening is carried out in a cell-free system. Peptides displayed on polysomes can be screened using, for example, an affinity purification/amplification screening procedure where the MenB-specific antibody/receptor is immobilized, e.g., on a plastic plate.
Molecules used in the libraries above can be manipulated in order to form more stable conformations and thus enhance identification of useful molecular mimetics. For example, cysteine residues can be incorporated in the randomized sequences to form disulfide loops (O'Neal et al. (1992) Proteins 14:509) and protein scaffolds can be used to display randomized peptides in internal loop segments (Freimuth et al. (1990) J. Biol. Chem. 265:896; Sollazzo et al. (1990) Prot. Engin. 4:215).
Anti-idiotypic antibodies can also be produced using the anti-MenB antibodies of the present invention for use as molecular mimetics of unique epitopes of MenB. For a review of anti-idiotype antibodies, see, e.g., Kieber-Emmons et al. (1986) Int. Rev. Immunol. 1:1. In this regard, the pocket or cleft formed by the heavy and light chains of an antibody is often intimately involved in antigen binding. This region, called the paratope, is an "internal image" of the antigen surface bound by the antibody. An antibody directed against the paratope is one of several potential anti-idiotypic antibodies and can be a mimetic of the antigen. Randomized peptide loops of the heavy and light chains occur naturally as part of the generation of antibody diversity.
Anti-MenB monoclonal antibodies of the present invention can be used to elicit anti-idiotype antibody production and to select anti-idiotypes bearing the "image" of the antigen, using the techniques described in e.g., Westerink et al. (1988) Infect. Immun. 56:1120.
In one embodiment, a combinatorial library of phage-display antibodies, as described above, are screened using the anti-MenB monoclonal antibodies of the present invention to identify mimetic antibodies, i.e. phage-display Fab anti-idiotypic antibodies.
Anti-idiotype antibodies produced can be easily tested for their ability to elicit anti-MenB antibody production in standard laboratory animal models. The variable genes of the anti-idiotype antibodies can be sequenced to identify peptide vaccine candidates.
Additionally, combinatorial libraries of oligonucleotides (DNA, RNA, and modified nucleotides) can be screened to find molecular mimetics that bind to the non-autoreactive, anti-MenB antibodies of the present invention. Techniques for the production and use of such libraries are reviewed in e.g., Gold et al. (1995) Annu. Rev. Biochem. 64:763. A system, known as SELEX for Systematic Evolution of Ligands by Exponential enrichment, can be used for rapidly screening vast numbers of oligonucleotides for specific sequences that have desired binding affinities and specificities toward the anti-MenB antibodies. (Tuerk et al. (1990) Science 249:505). For example, immobilized non-autoreactive MenB monoclonal antibodies can be used to affinity purify specific binding oligonucleotides from a combinatorial library. The bound oligonucleotides are released from the immobilized antibodies by adding a competitive ligand or lowering the pH. The released oligonucleotides are either amplified directly using the polymerase chain reaction or converted to double stranded DNA using reverse transcriptase (Tuerk et al., 1990, supra). This is followed by additional rounds of selection and amplification until the desired mimetic is obtained. The sequences of the oligonucleotide mimetics are determined by DNA sequencing.
Once putative molecular mimetics are identified, they are tested for their ability to elicit functionally active (e.g., bactericidal and/or opsonic) antibodies which lack autoreactivity or have minimal autoreactivity, as described above. Molecular mimetics that have these properties are appropriate for further use, for example, in vaccine compositions.
The anti-MenB monoclonal antibodies can also be used to investigate the bactericidal and/or opsonic function of antibodies of different specificities, as well as to identify the molecular nature of the unique epitopes on the MenB bacterial surface that are not cross-reactive with host PSA. Furthermore, the anti-MenB antibodies can be used to isolate fractions of MenB bacteria or MenB PS derivatives. Once isolated, the critical epitopes reactive with the anti-MenB antibodies can be characterized and employed directly in oligosaccharide protein conjugate vaccines or to model synthetic saccharides or mimetics for use in vaccines.
Molecular mimetics identified using the functionally active anti-MenB antibodies of the invention can be used to generate antibody reagents for use in diagnostic assays. For example, antibodies reactive with the molecular mimetics can be used to detect bacterial antigen in biological samples using immunodiagnostic techniques such as competition, direct reaction, or sandwich type assays. Such assays include Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, and the like.
In addition, molecular mimetics, unique (e.g., non-autoimmune) Men B epitopes identified using the molecular mimetics and anti-id monoclonal antibodies can be used herein in vaccine compositions for the prevention of MenB disease in vaccinated subjects.
The vaccine compositions can comprise one or more of the anti-id monoclonal antibodies, molecular mimetics or non-autoimmune epitopes of MenB. The vaccines may also be administered in conjunction with other antigens and immunoregulatory agents, for example, immunoglobulins, cytokines, lymphokines, and chemokines, including but not limited to IL-2, modified IL-2 (cys125.fwdarw.ser125), GM-CSF, IL-12, .gamma.-interferon, IP-10, MIP1.beta. and RANTES.
The vaccines will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Adjuvants may also be used to enhance the effectiveness of the vaccines. Adjuvants can be added directly to the vaccine compositions or can be administered separately, either concurrently with or shortly after, vaccine administration. Such adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% TWEEN 80 (polyoxyethylenesorbitan monooleate), and 0.5% SPAN 85 (sorbitan trioleate) (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% TWEEN 80 (polyoxyethylenesorbitan monooleate), 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN 80 (polyoxyethylenesorbitan monooleate), and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester, Mass.) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FICA); (5) cytokines, such as interleukins (IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn -glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
In order to enhance the effectiveness of vaccine compositions formed from a molecular mimetic, it may be necessary to conjugate the mimetic to a carrier molecule. Such carrier molecules will not themselves induce the production of harmful antibodies. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), inactive virus particles, CRM197 (a nontoxic mutant diphtheria toxin), and the like. Such carriers are well known to those of ordinary skill in the art. The mimetic conjugates are selected for their ability to express epitopes that closely resemble those found on the surface of MenB bacterial cells. Suitable conjugates thus elicit the formation of antibodies that have functional activity against bacteria, and do not cross-react, or are minimally cross-reactive with polysialic acid in host tissue as determined using the binding assays described herein.
Typically, the vaccine compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes, or adsorbed to particles for enhanced adjuvant effect, as discussed above.
The vaccines will comprise an effective amount of the anti-id monoclonal antibody; molecular mimetic, peptide molecular mimetic or complexes of proteins; or nucleotide sequences encoding the same, and any other of the above-mentioned components, as needed. By "an effective amount" is meant an amount of a molecule which will induce an immunological response in the individual to which it is administered and poses a minimal risk of stimulating an autoimmune response in the individual. Such a response will generally result in the development in the subject of a secretory, cellular and/or antibody-mediated immune response to the vaccine. Usually, such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell and/or .gamma..delta. T cell populations.
Once formulated, the vaccines are conventionally administered parenterally, e.g., by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Polynucleotides encoding DNA or RNA mimetics of the MenB PS can also be used in vaccines for nucleic acid immunization. In the alternative, polynucleotides encoding peptide mimetics can be used in nucleic acid immunization. Such methods generally comprise the introduction of a polynucleotide encoding one or more of the desired molecules into a host cell, for the in vivo expression of the nucleic acid molecules or proteins. The polynucleotide can be introduced directly into the recipient subject, such as by injection, inhalation or the like, or can be introduced ex vivo, into cells which have been removed from the host. In the latter case, the transformed cells are reintroduced into the subject where an immune response can be mounted against the molecule encoded by the polynucleotide. Methods of nucleic acid immunization are known in the art and disclosed in e.g., International Publication No. WO 93/14778 (published Aug. 5, 1993); International Publication No. WO 90/11092 (published Oct. 4, 1990); Wang et al. Proc. Natl. Acad. Sci. USA (1993) 90:4156; Tang et al. Nature (1992) 356:152; and Ulmer et al. Science (1993) 259:1745. Generally, the polynucleotide is administered as a vector which has been encapsulated in a liposome and formulated into a vaccine composition as described above.
The anti-MenB monoclonal antibodies of the present invention, and functional equivalents thereof, can be used in pharmaceutical compositions to treat and/or prevent MenB and E. coli K1 disease in mammals. Such disease includes bacterial meningitis and sepsis, in infants, children and adults. In this regard, the administration of a highly-active, anti-MenB monoclonal antibody preparation to an individual who is at risk of infection, or who has been recently exposed to the agent will provide immediate passive immunity to the individual. Such passive immunizations would be expected to be successful in both normal and immunocompromised subjects. Further, administration of such monoclonal antibody compositions can be used to provide antibody titer to MenB in a mammalian subject, either alone, or in combination with known anti-MenB therapeutics.
The pharmaceutical compositions of the present invention generally comprise mixtures of one or more of the above-described anti-MenB monoclonal antibodies, including Fab molecules, Fv fragments, sFv molecules and combinations thereof. The compositions can be used to prevent MenB disease or to treat individuals following MenB infection.
Therapeutic uses of the pharmaceutical compositions involve both reduction and/or elimination of the MenB infectious agent from infected individuals, as well as the reduction and/or elimination of the circulating MenB agent and the possible spread of the disease.
As described above in regard to the vaccine compositions of the present invention, the pharmaceutical compositions can be administered in conjunction with ancillary immunoregulatory agents such as IL-2, modified IL-2 (cys125.fwdarw.ser125), GM-CSF, IL-12, .gamma.-interferon, IP-10, MIP1.beta. and RANTES.
The preparation of pharmaceutical compositions containing one or more antibodies, antibody fragments, sFv molecules or combinations thereof, as the active ingredient is generally known to those of skill in the art. Once formulated, the compositions are conventionally administered parenterally, e.g., by injection (either subcutaneously, intravenously or intramuscularly). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications.
The pharmaceutical compositions are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective. The amount of the composition to be delivered, generally in the range of from about 50 to about 10,000 micrograms of active agent per dose, depends on the subject to be treated, the capacity of the subject's immune system to mount its own immune-responses, and the degree of protection desired. The exact amount necessary will vary depending on the age and general condition of the individual to be treated, the severity of the condition being treated and the mode of administration, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, "an effective amount" of the pharmaceutical composition will be sufficient to bring about treatment or prevention of MenB disease symptoms, and will fall in a relatively broad range that can be determined through routine trials.
In addition, the pharmaceutical compositions can be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of administration may be with 1-10 separate doses, followed by other doses given at subsequent time intervals needed to maintain or reinforce the action of the compositions. Thus, the dosage regimen will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgement of the reasonably skilled practitioner.
Claim 1 of 7 Claims
1. An isolated molecular mimetic of a unique epitope of Neisseria meningitidis serogroup B (MenB), wherein said mimetic comprises a peptide having an amino acid sequence as depicted in SEQ ID NO:8.