Title: Methods for isolating
molecular mimetics of unique Neisseria meningitidis serogroup B epitopes
United States Patent: 7,063,949
Issued: June 20, 2006
Inventors: Granoff; Dan M.
(Berkeley, CA); Moe; Gregory R. (Alameda, CA)
Corporation (Emeryville, CA)
Children's Hospital Medical Center of Northern California (Oakland, CA)
Appl. No.: 643465
Filed: August 19, 2003
George Washington University's Healthcare MBA
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.
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).
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
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 C.sub.3 C.sub.8 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
The C.sub.3 C.sub.8 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
Thus, a group of N-acylated MenB PS glycoconjugates, an example of which
is termed "COMJ-2" herein can be prepared as follows. An N-acylated MenB
PS preparation, having substantially 100% N-acylated MenB 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 or 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
glyoconjugation. Successful conjugation can be determined by, e.g., gel
filtration, and the final saccharide to protein ratio (w/w) assessed by
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 the binding assays described herein.
The antibodies can be fully characterized with respect to isotype the
antigenic specificity, functional activity and cross reactivity with host
For example, mammalian subjects, conveniently standard laboratory animals
such as rodents and rabbits, can be summarized 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 and thus capable of eliciting the
production of MenB-specific antibodies which can be use directly in the
selective or therapeutic pharmaceutical preparation or, preferably, used
to search for mimetics of MenB polysaccharide antigens that will provide
unique epitopes for anti-MenB vaccines.
Thus is 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 that: (a)
cross-blocks an exemplified monoclonal antibodies kinds 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
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
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, 2:57. 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, as demonstrated 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 optimal activity is also particularly
useful herein for the evaluation of the murine monoclonal antibodies of
the invention which have an IgGl isotype. Murine IgGl (in contrast to
human IgGl) is ineffective in activation of complement. Thus, murine IgGl
antibodies do not activate complement-mediated bacteriolysis of MenB in
the above described assays. However, functional activity of IgGl anti-NPr-MenB
PS monoclonal antibodies can be accessed by opsonization in the absence of
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
-70.degree. C. 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 23 Dec. 1992, and U.K.
Patent Publication No. GB 2,276,169, published 23 Sep. 1994).
Antibody molecule fragments, e.g., F(ab').sub.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 at al. (1973) Proc. Nat. Acad. Sci. USA 69:2659; Hochman
et al. (1975) Biochem 15:2706; Ehrlich et al. (1980) Biochem 19:4091;
Huston et al. (1986) 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. Salkl, et al. (1986)
Nature 324:163; Scharf et al. (1985) Science 133:1076; U.S. Pat. Nos.
4,683,195 and 4,683,202; Yang et al. (1995) J Mol Biol 254:392; Barbus,
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., Fagina et al. (1994) J. Mol. Biol. 239:68.
The ongoing sequences for the heavy and light chain portions of the Fab
molecules selected from the image 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.
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.
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 Geyser. 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 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 Geyser 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 cells, 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. Acad. Sci. USA 87:6378;
Gallop et al. (1994) J. Med. Chem 37:1233. Using such systems, large
libraries of 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 Lacd 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
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
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 of buffering substances, and the like, may be present
is any 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 11Y 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-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, CRM.sub.197 (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
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
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 5 Aug. 1993); International Publication No. WO 90/11092
(published 4 Oct. 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 infection 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 (cyc125.fwdarw.ser125), GM-CSF, IL-12, .gamma.-interferon, IP-10,
MIP1.beta. and RANTES.
The preparation of pharmaceutical composition containing 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
The pharmaceutical compositions are administered to the subject on 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 4 Claims
1. A method for identifying a
molecular mimetic of a unique epitope of Neisseria meningitidis serogroup B
(MenB), said method comprising: (a) providing a library of molecules
comprising a putative molecular mimetic of a unique epitope of MenB, wherein
said library of molecules is selected from the group consisting of a peptoid
library, a peptide library and a phage-display library; (b) contacting said
library of molecules with an isolated antibody immunologically reactive with
an N-acyl-substituted Neisseria meningitidis serogroup B capsular
polysaccharide, wherein said antibody is not autoreactive with Neisseria
meningitidis serogroup B capsular polysaccharide as determined by measuring
the ability of said antibody to react with human neuroblastoma cell line
CHP-134, under conditions that allow immunological binding between said
antibody and said molecular mimetic, if present, to provide a complex; and
(c) separating the complex from non-bound molecules; and (d) identifying the
molecular mimetic present in the complex.
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