Note: Descriptions are shown in the official language in which they were submitted.
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RICIN VACCINE AND METHODS OF MAKING AND USING THEREOF
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[Ol] This invention was made by employees of the United States Army. The
government has rights in the invention.
BACKGROUND OF THE INVENTION
1. FIELD OF THE INVENTION.
[02] The present invention generally relates to ricin toxin. In particular,
the present
invention relates to ricin vaccines as well as methods of making and using
thereof.
2. DESCRIPTION OF THE RELATED ART.
[03] Ricin is a very toxic protein obtained from the castor bean, Ricinus
cofnnaunis,
Euphorbiaceae. Ricin is a heterodimer comprising an A chain and a B chain
joined by a
disulfide bond. Ricin A chain (RTA) is an N-glycosidase enzyme that
irreversibly
damages a specific adenine base from 28S rRNA. Once the rRNA has been damaged,
the
cell cannot make protein and will inevitably die (cytotoxicity). As RTA
exhibits this type
of destructive catalytic activity, RTA is cormnonly referred to as a type II
ribosome
inactivating protein (RIP). See Lord, et al. (1991) Semin. Cell Biol. 2(1):15-
22. RTA has
been coupled with a targeting moiety to selectively destroy target cells such
as tumor
cells. See U.S. Pat. Nos. 4,80,457; 4,962,188; and 4,689,401; see also Vitetta
et al.
(1993) Trends Pharmacol. Sci. 14:148-154 and Ghetie ~z Vitetta (1994) Cancer
Drug
Delivery 2:191-198.
[04] The toxic consequences of ricin are due to the biological activity of
RTA. Ricin B
chain (RTB) binds the toxin to cell surface receptors and then RTA is
transferred inside
the cell where inhibition of ribosome activity occurs. The human lethal dose
of ricin
toxin is about 1 ~,g/kg. As highly purified ricin is commercially available,
the use of ricin
toxin in biological warfare and terrorism is highly possible and probable.
Unfortunately,
there is no effective antidote for toxic exposure to ricin. Thus, attempts
have been made
to provide vaccines against ricin intoxication.
[OS] Ricin vaccines have been prepared by isolating the natural toxin from
castor
beans, and treating the toxin with harsh chemicals, such as typically
formaldehyde, to
reduce the toxic activity. See Hewetson, et al. (1993) Vaccine 11(7):743-746;
Griffiths,
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et al. (1995) Hum. Exp.,Toxicol. 14(2):155-164; Griffiths, et al. (1999)
Vaccine 17(20-
21):2562-2568; and Yan, et al. (1996) Vaccine 14(11):1031-1038. These first
generation
vaccines are called "toxoid" vaccines as they are made directly from natural
toxin itself.
The current toxoid vaccine suffers from several important limitations that
include: (1) the
presence of both RTA and RTB; (2) the presence of trace amounts of active RTA
and
RTB; (3) the possibility that denatured toxoid could refold and, thereby,
revert to the
active form; (4) side-effects that arise from the presence of the harsh
chemicals used in
the vaccine formulation; (5) heterogeneity in the final vaccine product
arising from the
process of formaldehyde treatment; and (6) heterogeneity in the final vaccine
resulting
from the natural structural variations of ricin protein found in castor beans.
See
Despeyroux, et al. (2000) Anal. Biochem. 279(1):23-36.
[06] The second generation ricin vaccines comprise wild-type (wt) RTA, but not
RTB.
See U.S. Patent No. 5,453,271. Early efforts centered on isolating the whole
toxin from
castor beans, and then purifying RTA from RTB. Unfortunately, there are major
problems associated with the use of natural or wt RTA as a vaccine such as:
(1)
heterogeneity at the level of protein composition (Despeyroux, et al. (2000)
supra); (2)
heterogeneity at the level of sugar composition (N-linked and/or o-linlced
carbohydrates
covalently attached to the polypeptide backbone) (Despeyroux, et al. (2000)
sups°a); (3)
retention of naturally toxic N-glycosidase-rRNA enzymatic activity; (4) the
poor
solubility of isolated RTA in the absence of RTB, as evidenced by protein
aggregation
under physiological conditions; (5) rapid clearance circulation by the liver,
thereby
reducing the effectiveness of the vaccine (Wawrzynczak, et al. (1991) Int. J.
Cancer
47:130-135); and (6) causing lesions of the liver and spleen.
[07] Heterogeneity (variability) in a vaccine is undesirable as standardized
and
reproducible vaccine lots are necessary for regulatory compliance and
approval. To
reduce the heterogeneity of RTA vaccines, deglycosylated RTA (dgRTA) vaccines
were
produced. See International patent publication WO 00/53215. Unfortunately,
dgRTA is
still poorly soluble. Additionally, both dgRTA and wt recombinant RTA (rRTA)
retain
toxic N-glycosidase-rRNA enzymatic activity, which poses a safety risk. See
Blalcey, et
al. (1987) Cancer~Res. 47(4):947-952; Foxwell, et al. (1987) Biochim. Biophys.
Acta.
923(1):59-65; Soler-Rodriguez, et al. (1992) Int. J. Immunopharmacol.
14(2):281-291;
and Schindler, et al. (2001) Clin. Cancer Res. 7(2):255-258. Attempts at
eliminating the
toxic enzymatic activity of wt RTA gave rise to the third generation of ricin
vaccines.
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[08] The third generation ricin vaccines are based on the active site of RTA
that
comprises the amino acid residues that interact directly with, or are within
about five
angstroms from, the bound ribosomal RNA substrate. Specifically, these mutant
substitution RTA vaccines are based on recombinant DNA technology and
substitute
amino acids of wt RTA in order to reduce N-glycosidase-rRNA activity. See
Ready, et
al. (1991) Proteins 10(3):270-278; Kim, et al. (1992) Biochem. 31:3294-3296;
Roberts, et
al. (1992) Targeted Diag. Ther. 7:81-97; Frankel, et al. (1989) Mol. Cell.
Biol. 9(2):415-
420; and Gould, et al. (1991) Mol. Gen. Genet. 230(1-2):81-90.
[09] Unfortunately, these mutant substitution RTA vaccines are problematic
because
unwanted changes often occur in the protein structure and render the protein
unstable.
Self organization of the native RTA tertiary fold is optimized by the
electrostatic charge
balance of the active site cavity. See Olson (2001) Biophys. Chem. 91(3) 219-
229. Thus,
amino acid substitutions that alter the charge balance lead to structural
reorganization
coupled with a reduction in protein-fold stability. For example, disrupting
the ion-pair
between amino acid residues, Glu-177 and Arg-180, at the active site cavity by
replacing
the arginine with a histidine affects the global stability of the protein if
the imidazole ring
is deprotonated. See Day, et al. (1996) Biochem. 35(34):11098-11103. The more
stable
form of mutant substitution RTA R180H, reduces the overall enzymatic activity
about
500-fold, yet remains cytotoxic.
[10] Another example of a failed substitution was mutant substitution RTA
E177A.
The x-ray crystallographic structure of RTA E177A demonstrates a remarl~able
rescue of
electrostatic balance in the active site, achieved by the rotation of a
proximal glutamic
acid into the vacated space. See Kim, et al. (1992) supra. Despite the non-
conservative
substitution, the free energy of denaturation for RTA E177A was anticipated
from
modeling studies to be two-fold more favorable than the conservative
replacement of
mutant substitution RTA E177Q See Olson (2001) supra. In terms of expression
levels,
RTA E177Q is far less well behaved than wt RTA. See Ready, et al. (1991)
supra.
Additionally, both RTA E177A and RTA E177Q remain active enzymes, thereby
indicating plasticity in obtaining the catalytic transition-state. See
Schlossman, et al.
(1989) Mol. Cell. Biol. 9(11):5012-5021; and Ready, et al. (1991) supra..
[ll] Thus, a need still exists for a vaccine that is stable, safe and
effective against ricin
intoxication.
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SUMMARY OF THE INVENTION
[12] The present invention generally relates to polypeptides related to
ricin.toxin A
chain.
[13] In some embodiments, the present invention relates to an isolated
polypeptide or
variant thereof comprising a polypeptide sequence having substantial identity
to a wild
type ricin A chain first globular domain sequence and which laclcs detectable
N-
glycosidase-rRNA activity or exhibits reduced N-glycosidase-rRNA activity as
compared
to a control. The polypeptide may retain the functional integrity of the
neutralizing
immunological epitope of wild type ricin A chain. In some preferred
embodiments, the
polypeptide has a solubility that is greater than the solubility of wild type
ricin A chain in
aqueous solutions of physiological pH and ionic strength.
[14] The wild type ricin A chain first globular domain sequence may be SEQ ID
N0:2
or a variant thereof and the polypeptide of the present invention may comprise
SEQ ID
NO:3, SEQ ID N0:4, or variants thereof. The polypeptide of the present
invention may
be substantially identical to SEQ ID N0:3 or SEQ m NO:4.
[15] In some embodiments, the polypeptide of the present invention lacks a
hydrophobic loop that corresponds to the hydrophobic loop of wild type ricin A
chain.
The polypeptide of the present invention may comprise at least one amino acid
mutation,
substitution, deletion, or a combination thereof.
[16] The polypeptides of the present invention may be made by recombinant DNA
techniques or by proteolytically cleaving the first globular domain and the
second
globular domain of ricin A chain and then purifying the first globular domain.
[17] In other embodiments, the present invention relates to isolated
polynucleotides
that encode the polypeptides or variants of the present invention.
[18] In some embodiments, the present invention relates to antibodies raised
against
the polypeptides or variants of the present invention. In some embodiments,
the antibody
is a neutralizing antibody that is capable of neutralizing ricin, ricin A
chain, or both.
[19] In some embodiments, the present invention relates to a pharmaceutical
composition comprising at least one polypeptide or variant of the present
invention, or at
least one polynucleotide of the present invention or at least one antibody of
the present
invention a pharmaceutically acceptable vehicle. The pharmaceutical
composition may
further comprise an adjuvant. The pharmaceutical composition may be capable of
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eliciting an immune response when administered to a subject. The immune
response may
be a protective irmnune response against ricin intoxication.
[20] The present invention also relates to vaccines comprising an immunogenic
amount
of at least one polypeptide or variant of the present invention.
[21] In some embodiments, the present invention relates to a method of
inducing an
immune response in a subject which comprises administering to the subject at
least one
immunogenic amount of the polypeptide or variant of the present invention.
Preferably,
the subj ect is mammalian, more preferably, the subj ect is human. The method
may
further comprise administering to the subject at least one booster dose.
[22] In some embodiments, the present invention relates to a method of
providing
passive immunity against ricin intoxication in a subject comprising
administering to the
subject a therapeutically effective amount of at least one antibody raised
against the
polypeptides or variants of the present invention.
[23] In other embodiments, the present invention relates to a method of
treating or
preventing ricin intoxication in a subject comprising administering to the
subject an
immunogenic amount of the polypeptide or variant of the present invention, or
administering to the subject a therapeutically effective amount of an antibody
raised
against the polypeptide or variant of the present invention.
[24] In some embodiments the present invention relates to kits comprising the
polypeptides or variants of the present invention, or antibodies raised
against the
polypeptides or variants of the present invention, or polynucleotides of the
present
invention packaged together with instructions for use. The kits may further
comprise
diagnostic reagents such as labeling compounds for detecting the presence of
ricin toxin
or for diagnosing exposure of a subject to ricin. The kits may comprise drug
delivery
devices for administering the compositions of the present invention to a
subject.
[25] It is to be understood that both the foregoing general description and
the following
detailed description are exemplary and explanatory only and are intended to
provide
further explanation of the invention as claimed. The accompanying drawings are
included to provide a further understanding of the invention and are
incorporated in and
constitute part of this specification, illustrate several embodiments of the
invention and
together with the description serve to explain the principles of the
invention.
DESCRIPTION OF THE DRAWINGS
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[26] This invention is further understood by reference to the drawings
wherein:
[27] Figure 1 is a ribbon diagram of wt RTA. .
[28] Figure 2 is an SDS-PAGE that compares the heterogeneity of dgRTA, wt RTA,
and RTA198 and RTA 1-33/44-198.
[29] Figure 3 is an SDS-PAGE that exhibits the consistency and reproducibility
of
making the polypeptides of the present invention as exemplified with RTA1-
33/44-198.
DETAILED DESCRIPTION OF THE INVENTION
[30] The present invention provides polypeptides derived from native or wt RTA
amino acid sequences, such as the amino acid sequence set forth in SEQ ID NO:1
(SwissProt accession no. P02879), for use in compositions and methods for
treating or
preventing ricin intoxication in a subj ect.
[31] The structure of RTA comprises two globular domains according to a widely
accepted method of evaluating protein secondary structure elements and
paclcing called
PDBSum, a web-based database of summaries and analyses of all PDB structures.
See
Laskowski R A, et al. (1997) Trends Biochem. Sci. 22:488-490. The first
globular
domain of RTA comprises amino acid residues from about 1 to about 179
(counting from
the amino terminus) of native RTA which is set forth in SEQ ID N0:2. The
second
globular domain of RTA comprises amino acid residues from about position 180
to about
267 of native RTA. The second globular domain of RTA is the part that mainly
interfaces with RTB.
[32] Detailed computer-aided structural models of RTA were constructed using
InsightlI Biopolymer, Discover, and Delphi programs which are available from
Accelrys
Inc. (San Diego, CA), and the program GRASP developed by B. Honig of Columbia
University (New York, NY). Starting structures for these models included: (A)
the three-
dimensional crystal structures of ricin toxin, as well as natural RTA and RTB,
and wt
rRTA (Villafranca, et al. (1981) J. Biol. Chem. 256(2):554-556; Montfort, et
al. (1987) J.
Biol. Chem. 262(11):5398-5403; Katzin, et al. (1991) Proteins 10(3):251-259;
Rutenber,
et al. (1991) Proteins 10(3):240-250; and Weston, et al. (1994) J. Mol. Biol.
244(4):410-
422, which are herein incorporated by reference); (B) the three-dimensional
crystal
structures of several ribosome inactivating proteins (RIPS) related to RTA,
including
pokeweed antiviral protein (PAP) (Monzingo, et al. (1992) J. Mol. Biol.
277(4):1136-
1145, which is herein incorporated by reference); and (C) published computer-
aided
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theoretical models of RTA in solution (Olson (1997) Proteins 27(1):80-95;
Lebeda (1997)
Int. J. Biol. Macromol. 24(1):19-26; and Olson (1999) COMPUTATIONAL CHEMISTRY:
REVIEWS of CURRENT TRENDS. J. Leszczynski. Singapore, World Scientific
Publishing C.
Pte. Ltd. 4:153-190, which are herein incorporated by reference). The computer-
aided
models were used to design and develop the polypeptides of the present
invention for use
as safe and efficacious ricin vaccines.
[33] Analysis of computer-aided models suggested that native RTA could be
converted
into a much smaller, stably folded vaccine scaffold by careful placement of a
break in the
polypeptide backbone within a specific coiled region near the start of the
second globular
domain of RTA. This specific coiled region is referred to as the "linker
region" of RTA
and comprises amino acid residues from about position 195 to about 202 of
native RTA.
A ribbon diagram depicting the three-dimensional topology of RTA derived from
the
solved crystal structure is shown in Figure 1. See Weston, et al. (1994)
supra, which is
herein incorporated by reference. The first globular domain comprises sections
1, 2, and
3 and the second globular domain comprises sections 4 and 5. The neutralizing
immunogenic epitope, section 2, is believed to be the amino acid residues from
about
position 95 to about 110 of native RTA. See Aboud-Pirak, et al. (1993) Med.
Def. Biosci.
Rev., Baltimore, MD, U.S. Army Medical Research and Materiel Command, which is
herein incorporated by reference. Based on structural analysis and protein
chemistry, the
first globular domain was determined to relate to imrnunogenicity and the
second globular
domain was determined to include the hydrophobic interfacial region that
interacts
directly with RTB.
[34] The aqueous solubility of native RTA was determined to be limited by the
absence of RTB. Moreover, major structural changes were identified that could
be made
in native RTA to overcome limited solubility by optimizing solute-solvent
interactions.
The significant changes described herein were surprisingly successful as
significant
changes to a polypeptide sequence usually results in an unfolded protein and,
therefore,
loss of utility as a biological therapeutic. Specifically, as disclosed
herein, the engineered
polypeptides do not retain any natural catalytic activity of RTA.
Additionally, the
engineered polypeptides retained the favorable thermodynamic equilibrium
between
folded and unfolded RTA. Most surprisingly, the functional integrity of the
neutralizing
immunological epitope of RTA was retained in the engineered polypeptides.
Therefore,
the present invention provides polypeptides that: (1) do not retain any
natural catalytic
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activity of RTA; (2) retain the favorable thermodynamic equilibrium between
folded aald
unfolded RTA; (3) retain the functional integrity of the neutralizing
immunological
epitope of RTA; or (4) a combination thereof.
[35] As shown in the Examples below, significant changes in the linker region
of wt
RTA provided polypeptides having reduced surface hydrophobicity (increased
solubility
in aqueous solutions of physiological pH and ionic strength) or a reduced
ability to form
aggregates as well as reduced N-glycosidase-rRNA activity, if any, as compared
to wt
RTA. In particular, the engineered polypeptides that lack most of the second
globular
domain of RTA. Specifically, the second globular domain was deleted at
position 198 of
RTA since the second residue following the cleavage site is a proline (residue
200), and
this residue was thought to be restrictive in the torsional backbone of the
deleted domain.
[36] Thus, the polypeptides of the present invention comprise at least one
amino acid
mutation, substitution, deletion, or a combination thereof, in the linker
region of RTA.
The polypeptides of the present invention comprise an amino acid sequence that
has
substantial identity to amino acid residues from about position 1 to about
210, preferably
about 1 to about 202 of wt RTA, more preferably about 1 to about 198. In
preferred
embodiments, the polypeptides of the present invention comprise an amino acid
sequence
that has a substantial identity to amino acid residues from position 1 to 198
of wt RTA.
[37] Further, as disclosed herein, amino acid residues in the first globular
domain that
do not change its overall stability and do not change the neutralizing
immunogenic
epitope of wt RTA may be removed. Specifically, deletion of amino acid
residues
between positions 33 and 44 of wt RTA still provided a stable polypeptide that
retained
immunogenicity.
[38] Thus, the polypeptides of the present invention comprise at least one
amino acid
mutation, substitution, deletion, or a combination thereof, in the linker
region and in the
first globular domain of wt RTA. The polypeptides of the present invention
comprise an
amino acid sequence that has substantial identity to amino acid residues from
position 1
to about 210, preferably about 1 to about 200, more preferably about 1 to
about 198 of wt
RTA and include at least one amino acid mutation, substitution, deletion, or a
combination thereof, in the first globular domain, preferably in the loop
region, more
preferably from about position 33 to about 44 of wt RTA. In preferred
embodiments, the
amino acid mutation, substitution, deletion, or a combination thereof, is a
deletion of
amino acid residues from about position 34 to about 43 of wt RTA.
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[39] The polypeptides of the present invention are stable, non-toxic, and
capable of
eliciting an immune response in a subject. Thus, the polypeptides of the
present invention
may be used to prevent or treat systemic side effects of locally administered
ricin toxin.
In preferred embodiments, the polypeptides of the present invention are
capable providing
a protective immune response in a subject. Preferably, the subject is
mammalian, more
preferably, the subject is human. As used herein, an "immune response" refers
to a
humoral or cellular response caused by exposure to an antigenic substance.
Thus, an
immune response against ricin or "ricin immune response" refers to a humoral
or cellular
response in a subject that is caused by exposing the subject to an antigenic
substance such
as polypeptides of the present invention. A "protective immune response"
against ricin
refers to humoral immune responses, cellular immune responses, or both, that
are
sufficient to inhibit or prevent ricin intoxication in a subj ect.
[40] In some embodiments, the polypeptides of the present invention comprise
at least
one amino acid mutation, substitution, deletion, or a combination thereof, in
the linker
region of the second globular domain of LTA such as the following:
A. RTA198 (designated from the protein amino terminus to the protein carboxyl
terminus):
IFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRF
ILVELSNHAELSVTLALDVTNAYWGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNR
YTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIIC
IQMISEAARFQYIEGEMRTRIRYNRRS (SEQ ID N0:3); and
B. RTA1-33/44-198 (designated from the protein amino terminus to the protein
carboxyl-terminus) The bold type face of SEQ ID N0:4 indicates the position
where the
hydrophobic loop would be when compared with wt RTA.:
IFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTVLPNRVGLPINQRFILVELSNHAE
LSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYD
RLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARF
QYIEGEMRTRIRYNRRS (SEQ ID N0:4)
[41] The polypeptides of the present invention need not be identical to those
exemplified herein so long as the subject polypeptides .are able to induce an
immune
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response against RTA, ricin, or both. Thus, as used herein "variants" of the
polypeptides
of the present invention refer to polypeptides having insignificant changes
such as a
methionine as the first amino acid residue at the amino terminus, conservative
amino acid
substitutions, deletion of or insertion of up to about 10 amino acid residues
in the linker
or loop region of wt RTA, and co-translational or post-translational surface
modifications
such as the addition of covalently attached sugars or lipids. Insignificant
changes refer to
modifications in the amino acid sequence of a given polypeptide that do not
change the
solubility, N-glycosidase-rRNA activity, or immunogenicity of the polypeptide.
[42] Examples of such variants include:
a. MIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR
FILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQN
RYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFII
CIQMISEAARFQYIEGEMRTRIRYNRRS (SEQ ID NO:S)
b. MIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTVLPNRVGLPINQRFILVELSNHA
ELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNY
DRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAAR
FQYIEGEMRTRIRYNRRS (SEQ ID N0:6)
C. MVPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRF
ILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNR
YTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIIC
IQMISEAARFQYIEGEMRTRIRYNRRS (SEQ m N0:7)
d. MVPKQYPIINFTTAGATVQSYTNFIRAVRGRLTVLPNRVGLPINQRFILVELSNHAE
LSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYD
RLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARF
QYIEGEMRTRIRYNRRS (SEQ ID NO:~)
e. IFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTNRVGLPINQRFILVELSNHAELSV
TLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLE
QLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYI
EGEMRTRIRYNRRS (SEQ ID N0:9)
f. MIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQR
FILVELSNHAELSVTLALDVTNAYWGYRAGNSAYFFHPDNQEDAEAITHLFTDVQN
RYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFII
CIQMISEAARFQYIEGEMRTRIRYNRRSA (SEQ ID NO:10)
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g. MIFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTVLPNRVGLPINQRFILVELSNHA
ELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNY
DRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAAR
FQYIEGEMRTRIRYNRRSA (SEQ ll~ NO:11)
[43] The polypeptides of the present invention may also be modified to provide
a
variety of desired attributes, e.g., improved pharmacological characteristics,
while
increasing or at least retaining substantially all of the immunological
activity of the RTA.
By using conventional methods in the art, one of ordinary skill will be
readily able to
make a variety of polypeptides having mutated linker regions and then screen
the
polypeptides for stability, toxicity, and immunogenicity according to the
present
invention.
[44] Additionally, single amino acid substitutions, deletions, or insertions
can be used
to determine which residues are relatively insensitive to modification. Amino
acid
substitutions are preferably made between relatively neutral moieties, such as
alanine,
glycine, proline, and the like. Substitutions with different amino acids, of
either D or L
isomeric forms, or amino acid mimetics can be made. The number and types of
substitutions, deletions, and insertions depend on the functional attributes
that are sought
such as hydrophobicity, immunogenicity, three-dimensional structure, and the
like.
[45] An "amino acid mimetic" as used herein refers to a moiety other than a
naturally
occurnng amino acid residue that conformationally and functionally serves as a
suitable
substitute for an amino acid residue in a polypeptide of the present
invention. A moiety is
a suitable substitute for an amino acid residue if it does not interfere with
the ability of the
peptide to elicit an immune response against ricin. Examples of amino acid
mimetics
include cyclohexylalanine, 3-cyclohexylpropionic acid, L-adamantyl alanine,
adamantylacetic acid, and the like. See e.g. Morgan and Gainor, (1989) Ann.
Repts. Med.
Chem. 24:243-252.
[46] Individual amino acid residues may be incorporated in the polypeptides of
the
present invention with peptide bonds or peptide bond mimetics. A peptide bond
mimetic
include peptide backbone modifications of the amide utrogen, the a-carbon,
amide
carbonyl, complete replacement of the amide bond, extensions, deletions or
backbone
crosslinks. See e.g. Spatola (1983) CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS,
PEPTIDES AND PROTEINS, Vol. VII, Weinstein ed. The polypeptides of the present
invention may include an additional methionine as the first amino acid residue
on the
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protein amino terminus. The polypeptides may be truncated by up to about ten
(10)
amino acid residues from the carboxyl terminus of RTA198, RTAl-33/44-198, or
the
like. Similarly, up to about ten (10) amino acid residues from wt RTA
bordering the
hydrophobic loop, amino acid residues at about position 34 to about 43 may be
deleted.
Additionally, co-translational or post-translational surface modifications,
such as the
addition of covalently attached sugars or lipids, may be made to the
polypeptides of the
present invention.
[47] In preferred embodiments, the polypeptides of the present invention have
a
substantial sequence identity to the amino acid sequence set forth in SEQ ID
NOs:2, 3,
and 4. As used herein "sequence identity" means that two sequences are
identical over a
window of comparison. The percentage of sequence identity is calculated by
comparing
two optimally aligned sequences over the window of comparison, determining the
number of positions at which the identical residues occurs in both sequences
to yield the
number of matched positions, dividing the number of matched positions by the
total
number of positions in the window of comparison (i.e., the window size), and
multiplying
the result by 100 to yield the percentage of sequence identity.
[48] The structural models and conclusions deduced for RTA may be applied to
the
solved three-dimensional structure of related proteins, such as other RIPS.
This could be
done by a structure-structure alignment that minimizes the root-mean-square-
deviation of
atomic coordinates among the aligned structure (for example, optimal
superposition of the
position of all atoms, or the protein backbone atoms or the alpha-carbon
atoms). Such
methods are now largely automated, see for example, the homology module of
Insight II
(Accelrys, Inc., San Diego, CA); the Combinatorial Extension (CE) of the
optimal path
method (Shindyalov and Bourne (1998) Protein Engineering 11:739-747
http://cl.sdsc.edu/cd.html); and the Vector Alignment Search Tool (VAST)
(Gibrat,
Madej and Bryant (1996) Current Opiuon in Structural Biology 6:377-385), which
are
incorporated herein by reference. Preferably, the amino acid residues in
corresponding
positions among aligned structures are identical or differ only by
"conservative amino
acid substitutions".
[49] A "conservative amino acid substitution" is one in which the amino acid
residue is
replaced with an amino acid residue having a similar side chain. Families of
amino acid
residues having similar side chains have been defined in the art. These
families include
amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic
side chains
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(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-
branched side chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g.,
tyrosine, phenylalanine, tryptophan).
[50] Additionally, the structural information and conclusions deduced for RTA
could
be applied to other protein sequences, such as other RIPS, by an aligmnent of
known
structures (e.g., RTA and PAP), and then a subsequent alignment of the
unknowxn
structure with the structurally aligned sequences. Such methods are now
largely
automated. See e.g., SAS program suite of Milburn, D., et al. (1998) Protein
Engineering
11:855-859, which is herein incorporated by reference; see also Homologous
Structure
Alignment Database (HOMSTRAD). Mizuguchi, Deane, Blundell, and Overignton
(1998) Protein Science 7:2469-2471 (http://www-
cryst.bioc.cam.ac.uk/~homstrad/),
which is herein incorporated by reference.
[51] The polypeptides of the present invention may be made by conventional
methods
known in the art. The polypeptides of the present invention may be manually or
synthetically synthesized using conventional methods and devices known in the
art. See
e.g., Stewart and Young (1984) SOLID PHASE PEPTmE SYNTHESIS, 2 ed. Pierce,
Rockford,
IL, which is herein incorporated by reference. Ricin or RTA may be obtained by
conventional methods. See LT.S. Patent No. 5,547,867, which is herein
incorporated by
reference. The ricin or RTA may then be exposed to proteolytic enzymes that
cleave the
amino acid residues to obtain polypeptides of the present invention. Ricin and
RTA may
be purified from natural sources using conventional protein purification
techniques such
as reverse phase high-performance liquid chromatography (HPLC), ion-exchange
or
immunoaffinity chromatography, filtration or size exclusion, or
electrophoresis. See
Olsnes, S. and A. Pihl (1973) Biochem. 12(16):3121-3126; and see e.g., Scopes
(1982)
PROTEIN PURIFICATION, Springer-Verlag, NY, which are herein incorporated by
reference.
[52] Prior art methods for purifying RTA rely upon the separation of RTA and
RTB by
disulfide reduction and subsequence lectin binding, or affinity chromatography
with
specialized affinity resins. See Fulton et al. (1986) J. Biol. Chem. 261:5314-
5319 and
Emmanuel et al. (1988) Anal. Biochem. 173:134-141, which are herein
incorporated by
reference. Since the biophysical properties of the polypeptides, i. e.
isoelectric points, of
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the present invention are different from wt RTA, the polypeptides of the
present invention
may be purified without the use of sulfhydryl reduction or costly, specialized
affinity
resins. Specifically, conventional ion-exchange chromatography based upon the
isoelectric points of the polypeptides may be used to purify the polypeptides
of the
present invention.
[53] In preferred embodiments, the polypeptides of the present invention are
substantially purified. As used herein, a "substantially purified" compound
refers to a
compound that is removed from its natural environment and is at least about
60% free,
preferably about 75% free, and most preferably about 90% free from other
macromolecular components with which the compound is naturally associated.
[54] Alternatively, the polypeptides of the present invention may be made by
conventional recombinant DNA techniques such as those disclosed in the
Examples
below. Thus, the present invention provides polynucleotides that encode the
polypeptides
of the present invention. In preferred embodiments, the polynucleotides are
isolated. As
used herein "isolated polynucleotides" refers to polynucleotides that are in
an
environment different from that in which the polynucleotide naturally occuxs.
[55] A polynucleotide that encodes a polypeptide having substantial identity
to either
SEQ m N0:2 or SEQ m N0:3 can be made by introducing one or more nucleotide
substitutions, insertions, or deletions into the nucleotide sequence that
encodes SEQ ID
NO:1, SEQ m N0:2, or SEQ m N0:3 such that one or more amino acid
substitutions,
insertions, or deletions are introduced into the encoded polypeptide.
Mutations ca~i be
introduced by standard techniques, such as site-directed mutagenesis and PCR-
mediated
mutagenesis.
[56] A polynucleotide encoding a polypeptide of the present invention is then
inserted
in to a vector such as a cloning vector or an expression vector. An expression
vector
allows the polypeptide to be expressed when present in a host. Either the
expression
vector or the host may comprise the regulatory sequences necessary for
expression of the
polypeptide. Where the regulatory sequences are within the expression vector,
the
regulatory sequences are operatively linked to the sequence encoding the
polypeptide. As
used herein, "operably linked" means that the nucleotide sequence of interest
is linked to
at least one regulatory sequence in a manner that allows the polypeptide to be
expressed
in an in vitro transcription/translation system or in a host cell. Regulatory
sequences
include promoters, enhancers and other expression control elements (e.g.,
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polyadenylation signals). See e.g., Goeddel (1990) GENE EXPRESSION TECHNOLOGY:
METHODS IN ENZYMOLOGY, Academic Press, San Diego, CA, which is herein
incorporated by reference.
[57] It will be appreciated by those skilled in the art that the design of the
expression
vector can depend on such factors as the choice of the host cell to be
transformed, the
desired expression levels of the polypeptide, the compatibility of the host
cell and the
expressed polypeptide, and the like.
[58] The vectors can be designed for expressing the polypeptides of the
present
invention of in prokaryotic or eukaryotic host cells such as bacterial cells,
insect cells,
plant cells, yeast cells, or mammalian cells. In preferred embodiments, the
host cells are
bacterial cells. Suitable host cells are discussed further in Goeddel supra;
Baldari, et al.
(1987) EMBO J. 6:229-234; Kurjan and Herskowitz (1982) Cell 30:933-943;
Schultz, et
al. (1987) Gene 54:113-123; Smith, et al. (1983) Mol. Cell Biol. 3:2156-2165;
Lucklow
and Summers (1989) Virology 170:31-39; Seed (1987) Nature 329:840; Kaufman, et
al.
(1987) EMBO J. 6:187-6195; Sambrook, et al. (2000) MOLECULAR CLONING: A
LABORATORY MANUAL. Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory
Press, Cold Spring Harbor, NY; and those available from Invitrogen
Corporation, San
Diego, CA, such as pYES2 and picZ, all of which are herein incorporated by
reference.
[59] Thus, the present invention also provides host cells comprising
polynucleotides
that encode the polypeptides of the present invention. Host cells include the
progeny or
potential progeny of the primary cell in which the polynucleotide was
introduced.
Because certain modifications may occur in succeeding generations due to
either
mutation or environmental influences, such progeny may not, in fact, be
identical to the
parent cell, but are still included within the scope and meaning of host cell.
[60] A polypeptide of the present invention may be used to prepare antibodies
against
ricin by immunizing a suitable subject, e.g., rabbit, goat, mouse or other
marmnal with the
polypeptide by conventional methods knov~m in the art. Large quantities of
neutralizing
antibodies could be generated and then used as an antidote for ricin
intoxication. See
Lemley, et al. (1994) Hybridoma 13(5):417-427 and U.S. Patent No. 5,626,844,
which
are herein incorporated by reference. The antibodies raised against the
polypeptides of
the present invention may be used to prevent or treat systemic side effects of
locally
administered ricin toxin. Thus, the present invention also provides antibodies
that are
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raised against or derived from the polypeptides of the present invention, and
methods of
using thereof.
[61] Antibodies of the present invention may be produced by conventional
methods
known in the art. See e.g., Coligan (1991) CURRENT PROTOCOLS IN IMMUNOLOGY.
Wiley/Greene, NY; and Harlow and Lane (1989) ANTIBODIES: A LABORATORY MANUAL,
Cold Spring Harbor Press, NY; Stites, et al. (1986) BASIC AND CL1IVICAL
IMMUNOLOGY.
4th ed. Lange Medical Publications, Los Altos, CA; Goding (1986) MONOCLONAL
ANTIBODIES: PRINCIPLES AND PRACTICE. 2d ed. Academic Press, New Yorlc, NY; and
Kohler and Milstein (1975) Nature 256:495-497, which are herein incorporated
by
reference. Therapeutic antibodies may be produced specifically for clinical
use in
humans byconventional methods known in the art. See Chadd, H.E. and S.M.
Chamow
(2001) Curr. Opin. Biotechnol. 12:188-194 and references therein, all of which
are herein
incorporated by reference. The present invention has the advantage of allowing
safe
exposure of subjects, such as humans, to the RTA neutralizing epitope. Thus,
the present
invention allows for the safe in vivo production of RTA antibodies directly
subjects.
[62] As used herein, "antibody" refers to immunoglobulin molecules and
immunologically active portions that comprise an antigen binding site which
specifically
binds an antigen, such as ricin. Examples of immunologically active portions
of
immunoglobulin molecules include Flab) and F(ab')2 fragments which may be
generated
by treating the antibody with an enzyme such as pepsin. Polyclonal and
monoclonal
antibodies against the polypeptides of the present invention may be made by
conventional
methods known in the art.
[63] The polypeptides, polynucleotides, or antibodies of the present invention
may be
administered, preferably in the form of pharmaceutical compositions, to a
subject.
Preferably the subject is mammalian, more preferably, the subject is human.
Preferred
pharmaceutical compositions are those comprising at least one immunogenic
composition
against ricin, RTA, or both, in an immunogenic amount or a therapeutically
effective
amount, and a pharmaceutically acceptable vehicle. The immunogenic composition
may
be an active immunizing agent, such as a polypeptide of the present invention,
or a
passive immunizing agent, such as an antibody raised against the polypeptide
of the
present invention. The immunogenic composition may elicit an immune response
that
need not be protective or the immunogenic composition may provide passive
immunity.
A vaccine elicits a local or systemic immune response that is protective
against
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subsequent challenge by the immunizing agent such as the polypeptides of the
present
invention, or an immunologically cross-reactive agent, such as ricin.
Conventional
methods in the art may be used to determine the feasibility of using the
polypeptides of
the present invention as vaccines, against ricin intoxication. Accordingly, as
used herein,
an "irmnunogenic composition" can refer to vaccines as well as antibodies. A
protective
immune response may be complete or partial, i. e. a reduction in symptoms as
compared
with an unvaccinated mammal.
[64] Thus, the present invention provides immunogenic compositions comprising
the
polypeptides or the antibodies of the present invention that may be used to
immunize a
subject against ricin by methods known in the art. See U.S. Patent No.
5,453,271, which
is herein incorporated by reference. As used herein, an "immunogenic amount"
is an
amount that is sufficient to elicit an immune response in a subject and
depends on a
variety of factors such as the immunogenicity of the polypeptide, the manner
of
administration, the general state of health of the subject, and the like. The
typical
immunogenic amounts for initial and boosting immunization for therapeutic or
prophylactic administration ranges from about 0.01 mg to about 0.1 mg per
about 65-70
kg body weight of a subject. For example, the typical immunogenic amount for
initial
and boosting immunization for therapeutic or prophylactic administration for a
human
subject ranges from about 0.01 mg to about 0.1 mg. Examples of suitable
immunization
protocols include initial immunization injections at time 0 and 4 or initial
immunization
injections at 0, 4, and 8 weeks, which initial immunization injections may be
followed by
further booster injections at 1 or 2 years.
[65] As used herein, a "therapeutically effective amount" refers to an amount
of a
polypeptide, a polynucleotide, or an antibody that may be used to treat,
prevent, or inhibit
ricin intoxication in a subject as compared to a control. Again, the skilled
artisan will
appreciate that certain factors may influence the dosage required to
effectively treat a
subject, including the severity of ricin exposure, previous treatments, the
general health
and age of the subject, and the like. A therapeutically effective amount may
be readily
determined by conventional methods known in the art. It should be noted that
treatment
of a subject with a therapeutically effective amount of a polypeptide, a
polynucleotide, or
an antibody of the present invention can include a single treatment or,
preferably, can
include a series of treatments.
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[66] The pharmaceutical compositions may include an adjuvant. As used herein,
an
"adjuvant" refers to any substance which, when administered with or before the
polypeptide, polynucleotide, or antibody of the present invention, aids the
polypeptide,
polynucleotide, or antibody in its mechanism of action. Thus, an adjuvant in a
vaccine is
a substance that aids the immunogenic composition in eliciting an immune
response.
Suitable adjuvants include incomplete Freund's adjuvant, alum, aluminum
phosphate,
aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-
acetyl-nor-muraxnyl-L-alanyl-D-isoglutamine (CGP 11637, nor-MDP), N-
acetylmuramyl-
Lalanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dips-lmitoyl-sn-glycero-3-
hydroxyphosphoryloxy)-ethylamine (CGP 19835A, MTP-PE), and RIBI, which
comprise
three components extracted from bacteria, monophosphoryl lipid A, trehalose
dimycolate
and cell wall slceleton (NPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The
effectiveness of an adjuvant may be determined by conventional methods in the
art.
[67] The compositions of the present invention may be administered to a
subject by
any suitable route including oral, transdermal, intranasal, inhalation,
intramuscular, and
intravascular administration. It will be appreciated that the preferred route
of
administration and pharmaceutical formulation will vary with the condition and
age of the
subject, the nature of the condition to be treated, the therapeutic effect
desired, and the
particular polypeptide, polynucleotide, or antibody used.
[68] As used herein, a "pharmaceutically acceptable vehicle" or
"pharmaceutically
acceptable carrier" refers to and includes any and all solvents, dispersion
media, coatings,
antibacterial and antifungal agents, isotonic and absorption delaying agents,
and the like,
that are compatible with pharmaceutical administration. Pharmaceutically
acceptable
vehicles include those known in the art. See e.g. IZEMINGTON: THE SCIENCE AND
PRACTICE OF PHARMACY. 20th ed. (2000) Lippincott Williams & Wilkins.
Baltimore, MD,
which is herein incorporated by reference.
[69] The pharmaceutical compositions of the present invention may be provided
in
dosage unit forms. "Dosage unit form" as used herein refers to physically
discrete units
suited as unitary dosages for the subject to be treated; each unit containing
a
predetermined quantity of active compotuld calculated to produce the desired
therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the
dosage unit forms of the invention are dictated by and directly dependent on
the unique
characteristics of the active compound and the particular therapeutic effect
to be
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achieved, and the limitations inherent in the art of compounding such an
active compound
for the treatment of individuals.
[70] Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals.
For
example, one may determine the lethal dose of toxin, LCtso (the dose expressed
as
concentration of toxin x exposure time that is lethal to 50% of the
population) or the LDSn
(the dose lethal to 50% of the population), and the EDSO (the dose
therapeutically
effective in 50% of the population) by conventional methods in the art. The
dose ratio
between toxic and therapeutic effects is the therapeutic index and it can be
expressed as
the ratio LDSO/EDSO. Compounds which exhibit large therapeutic indices are
preferred.
While compounds that exhibit toxic side effects may be used, care should be
taken to
design a delivery system that targets such compounds to the site of affected
tissue in order
to minimize potential damage to uninfected cells and, thereby, reduce side
effects.
[71] The data obtained from the cell culture assays and animal studies can be
used in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the EDSO
with little or
no toxicity. The dosage may vary within this range depending upon the dosage
form
employed and the route of administration utilized. For any compound used in
the method
of the invention, the therapeutically effective dose can be estimated
initially from cell
culture assays. A dose may be formulated in animal models to achieve a
circulating
plasma concentration range that includes the ICSO (i.e., the concentration of
the test
compound which achieves a half maximal inhibition of symptoms) as determined
in cell
culture. Such information can be used to more accurately determine useful
doses in
humans. Levels in plasma may be measured, for example, by high performance
liquid
chromatography.
[72] The present invention also provides polypeptides, polynucleotides,
antibodies, or
compositions of the present invention may be provided in kits along with
instructions for
use. A kit comprising a pharmaceutical composition may include the
pharmaceutical
composition as a single dose or multiple doses. The kit may include a device
for
delivering the pharmaceutical composition. The device may be a multi-chambered
syringe for intramuscular delivery, a microneedle or set of microneedle arrays
for
transdermal delivery, a small balloon for intranasal delivery, or a small
aerosol generating
device for delivery by inhalation.
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[73] Ricin exposure is presently detected by medical history and symptoms, and
is
confirmed by antibody- or activity-based measurements of ricin in bodily
fluids. Ricin
detection or medical diagnosis of ricin exposure, therefore, may based upon
immunoassays utilizing the antibodies or polypeptides of the present invention
or
combinations thereof. Since a subject may be safely exposed to the
polypeptides of the
present invention, exposure to ricin may be determined by detecting RTA
antibodies in a
subject iya vivo. Additionally, as the polypeptides of the present invention
are relatively
non-toxic and safe, immunoassays utilizing the polypeptides of the present
invention for
detecting antibodies against ricin would be also be safe. Thus, the present
invention
provides diagnostic assays for detecting ricin toxin, exposure to ricin, or
both. The
diagnostic assays may comprise the polypeptides of the present invention,
antibodies of
the present invention, or a combination thereof. The diagnostic assays may be
provided
in the form of kits that may be used outside of a laboratory setting, such as
in the field.
[74] As disclosed herein, the polypeptides of the present invention are
fundamentally
superior as immunogenic compositions such as ricin vaccines as compared ricin
toxoid,
wt RTAs, dgRTAs, and mutant substitution RTAs as the polypeptides do not
include
RTB, and lack detectable N-glycosidase-rRNA activity or exhibit reduced N-
glycosidase-
rRNA activity as compared to controls. As used herein, "detectable" N-
glycosidase-
rRNA activity refers to N-glycosidase-rRNA activity that may be detected by
assays
conventional in the art. See Hale (2001) Pharmacol. & Toxicol. 88:255-260, and
Langer,
M.M. et al. (1996) Anal. Biochem. 243:150-153, both of which are herein
incorporated
by reference. These conventional assays routinely detect the ribosome
inactivating
activity of native RTA at toxin concentrations of 0.1 to 0.5 nM. Thus, the
polypeptides of
the present invention that lack detectable N-glycosidase-rRNA activity refers
to
polypeptides that do not exhibit ribosome inactivating activity at
concentrations of less
than about 0.5 nM, preferably, less than about 0.1 nM.
[75] The polypeptides of the present invention have an aqueous solubility that
is
greater than the aqueous solubility of wt RTA as evidenced by high expression
yields in
the soluble fraction and by the absence of protein aggregation or
precipitation upon
storage in physiological saline solutions. A direct comparison of dgRTA and
polypeptides of the present invention was made using standard methods of SDS-
PAGE
and isoelectric focusing. Each method showed evidence of higher molecular
weight
species, indicative of aggregation, in the dgRTA samples that were absent from
samples
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of the polypeptides of this invention. Additionally, the polypeptides of the
present
invention are more homogenous than dgRTA as the polypeptides may be
consistently
expressed and substantially purified as explained in Examples 1 and 2 and
evidenced by
the SDS-PAGES in Figures 2 and 3.
[76] The following Examples are intended to illustrate, but not to limit the
present
invention.
Example 1
Construction and Analysis of RTA198
A. DNA Cloning, Sequencing, and Expression of RTA198
[77] DNA cloning and sequencing was conducted using conventional methods in
the
art. See Sambrook, et al. (2000) sups°a, which is herein incorporated
by reference.
Cloning was based upon the published polypeptide sequence of RTA (SEQ ID NO:1,
SwissProt accession number P07829). See Lamb, et al. (1985) Eur. J. Biochem.
148(2):265-270; and Roberts, et al. (1992) supra, which are herein
incorporated by
reference.
[78] To make the polynucleotide that encodes RTA198, a "stop codon" was
incorporated after the codon for serine 198 in wt RTA. Mutagenesis to
introduce the stop
codon was done using a QuickChange kit commercially available from Stratagene
(La
Jolla, CA) based upon the polymerase chain reaction method. Briefly, a BamH 1
endonuclease cleavage site was replaced with a site for Nde I at the 5' end of
the RTA
gene using the following primer: 5' GAA TTC CAT ATG ATC TTC CCA AAG C 3'
(SEQ ID N0:12). Additionally, a stop codon and a Sal I restriction
endonuclease
cleavage site were introduced by use of the following primer: 5' GTC GAC CTA
GGA
TCT ACG GTT GTA TCT AAT TC 3' (SEQ ID N0:13). The reaction mixture,
comprising RTA DNA template, PCR primer pairs, pfu DNA polymerase and dNTP,
was
subjected to PCR as outlined in the commercial kit (Stratagene, La Jolla, CA).
Template
DNA is then eliminated by incubating at 37 °C for 1 hour with Dpn I
restriction
endonuclease. A 1 ~,1 sample of the PCR mixture containing the mutated
polynucleotide
was used to transform E. coli XLl-Blue competent E. coli cells (Stratagene, La
Jolla,
CA). Mutagenesis was conducted under contract by Clinical Research Management
(Frederick, MD). The resultant mutated polynucleotide was purified using a
commercial
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Miniprep kit (Qiagen, Valencia, CA) and sequenced using conventional methods
to
confirm that only the desired changes were made.
[79] The DNA products were purified, ligated to a commercial pET-24 expression
vector based upon the T7 promoter system of Studier, et al. (1990) Methods
Enzyrnol.
185:60-89, which is herein incorporated by reference, and then used to
transform
competent E. coli BL21 DE3 cells (Invitrogen, CA). Plasmid DNA was purified by
conventional methods in the art.
B. Expression of RTA198
[80] The transformed E. coli BL 21 cells were cultured in Ternfic Broth (TB; a
standard bacterial cell culture medium comprising 12 g/L tryptone, 24 g/L
yeast extract,
0.4% (v/v) glycerol, 2.31 g/L I~HH2P04, and 12.54 g/L I~ZHP04) containing 50
p,g/ml of
kanamycin until a cell density of 0.4 to 0.6 OD~oo was reached. Expression of
the
RTA198 polynucleotide was induced by adding 1 mM isopropyl-(3-D-
thiogalactopyranoside (IPTG) at 25°C, for about 18 to about 20 hours.
The cells were
centrifuged in a Sorvall GS3 rotor at 6,000 rpm for 15 minutes at 4 °C.
The cell media
was decanted and discarded. The pellet containing E. coli cells were bathed in
TB
medium with kanamycin and IPTG to create a cell paste that was frozen at -20
°C until
further use. Generally, the frozen cell paste was stored for at least about 24
hours prior to
protein purification.
[81] As an "induction check" to ensure that RTA198 was being expressed, an
aliquot
of E. coli cells was analyzed using polyclonal antiserum specific for RTA and
standard
methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and
Western blot analysis. The antiserum was affinity purified previously using
dgRTA by
conventional methods in the art. Briefly, wet E. coli cells were solubilized
in 50 mM
sodium phosphate buffer, pH 7.3, and centrifuged to give a soluble fraction
(supernatant)
and a pellet. The pellet was then solubilized by boiling in 6M urea, pH 7.
RTA198 was
detected by Western blot analysis in both the phosphate buffer soluble
fraction, and in the
phosphate buffer insoluble fraction.
C. Puf-ification of RTA198
[82] One (1) gram of cell paste was dissolved in 15 ml of an ice cold buffer
solution
comprising 50 mM sodium phosphate buffer and 2 mM ethylene diamine tetra-
acetic acid
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(EDTA), pH 7.3. The cells were then sonicated and the homogenized cells were
centrifuged using a Sorvall SS-34 rotor for 15,000 rpm for 15 minutes at
4°C. The
supernatant was collected and syringe filtered (0.2 ~,m pore size).
[83] A Pharmacia Mono-QOO 10/10 anion exchange column (Amersham-Pharmacia
Biotech, Piscataway, NJ) was pre-equilibrated with 50 mM sodium phosphate, pH
7.3,
and 2 mM EDTA. The supernatant comprising RTA198 was applied and a step
elution
was run to 1M NaCI (0-1.0 min). RTAl98 was found predominantly in the column
flow-
through.
[84] The Mono-Q~ 10/10 flow through was titrated to 0.6 ammonium sulfate
(final
concentration) using a stock solution of 3M ammonium sulfate, pH 7Ø This
material
was filtered (0.2 p,m pore size), and subsequently applied to an equilibrated
TosoHaas~
phenyl hydrophobic interaction chromatography (HIC) column (Tosoh Biosep,
Montgomeryville, PA) pre-equilibrated with 0.6 M ammonium sulfate, 50 mM
sodium
phosphate buffer, and 2 mM EDTA, pH 7. A gradient elution was run from 0.6 M
to 0 M
ammonium sulfate, 50 mM sodium phosphate buffer, 2 mM EDTA, pH 7 (0-30 min).
All
fractions showing protein by ODZBO absorbance were analyzed by reducing SDS-
PAGE.
Multiple fractions (4 x 4 ml) from the major HIC column peak comprised RTA198
which
were then pooled and dialyzed against 50 mM Tris base / 2 mM EDTA, pH 9.2.
[85] The pooled HIC fractions were applied to a Pharmacia Mono-QQ 10/10
(Amersham-Pharmacia Biotech, Piscataway, NJ) anion exchange column pre-
equilibrated
in 50 mM Tris base / 2 mM EDTA, pH 9.2, and a step eluted to 0.4 M NaCI. The
purified
RTA198 was found almost exclusively in the flow-through fraction.
D. Cha~~acterizatioh. of Purified RTA19~
[86] The flow-through fraction from the Mono-Q~ 10/10 anion exchange column at
pH 9.2 comprised greater than about 90% pure RTAl98 as determined by SDS-PAGE
stained with Coomassie blue and Western blots. The buffer was changed to 120
mM
NaCI, 2.7 mM KCI, 10 mM NaP04, pH 7.4, by extensive dialysis (2L x 3 changes
of
greater than about 3 hours at room temperature). Purified RTA198 was filtered
(0.2 qm
pore size) and stored sterile at 4 °C prior to animal studies. Total
protein concentrations
were determined using the Pierce micro-BCA assay (Pierce Chemical, Rockford,
IL)
(Smith, P.K., et al. (1985) Anal. Biochem. 150:76-85, which is herein
incorporated by
reference) relative to a bovine serum albumin standard curve. The purity of
RTA198
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obtained is shown in Figure 2. Specifically, Figure 2 is an SDS-PAGE result
wherein
lane 1 shows the molecular weight standards. To each lane, about 2 to about 4
~.g of
protein (200 kDa standard ladder, dgRTA, wt RTA, RTA198) as determined by a
bicinchoninic acid assay (BCA) standard in the art. The gel is 10% (w/v) total
acrylamide
stained with Coomassie blue. Lane 2 is dgRTA, lane 3 is purified recombinant
wt RTA,
and lane 4 is RTA198. As shown, the RTA198 produced a clear dark band thereby
indicating a high degree of homogeneity.
[87] The purified RTA198 did not possess detectable levels of N-glycosidase
activity
as evidenced by the inability to disrupt protein synthesis in a cell-free,
translation assay
under conditions where either natural ricin (positive control) or isolated wt
RTA (positive
control) did show ribosome inactivation. See Langer, et al. (1996) Anal.
Biochem.
243(1):150-153; and Hale (2001) Pharmacol. Toxicol. 88(5):255-260, which are
herein
incorporated by reference.
[88] The RTA activity assay was conducted as described by Hale (2001)
Pharmacol.
Toxicol. 88(5):255-260. This assay takes advantage of the fact that the N-
glycosidase
activity of RTA can be monitored with an in vitro translation assay using the
rabbit
reticulocyte lysate system. See Barbieri et al. (1989) Biochem. J. 257:801-
807, which is
herein incorporated by reference. The assay measures the in vitro translation
of the
enzyme luciferase, as determined by the fluorescence produced when luciferase
reacts
with the substrate luciferin. Briefly, a translation lysate was prepared at 4
°C that
contained nuclease-treated rabbit reticulocyte lysate, amino acid mixture,
Rnasin
ribonuclease inhibitor, nuclease-treated deionized water, and luciferase mRNA
in a ratio
of (v/v): 35:1:1:35:1, respectively (all available from Promega, Madison, W)].
[89] Aliquots of RTA198 at several dilutions were added to the translation
lysate in
wells of a microtiter plate, and subsequently incubated at 30 °C in a
moist chamber for 90
minutes. As negative controls to observe maximal fluorescence, some wells
received
phosphate buffered saline. As additional negative controls, some wells
received several
dilutions of RTB (ranging in final concentration from 0.06 to 17 nM RTB). As
positive
controls to observe decreased fluorescence, some wells contained the
translation lysate as
described without the luciferase mRNA. As additional positive controls to
observe
graded decrease in fluorescence, some wells received dilutions of RTA (ranging
in final
concentrations from 0.06 to 17 nM RTA). At the end of the incubation period,
the
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luciferin substrate in commercial reaction buffer (Promega, Madison, Wn was
added to
all wells, and the relative fluorescence was recorded using a microtiter plate
fluorimeter.
E. haccine Studies with RTA19~
[90] When RTA198 was administered to mice, purified RTA198 effectively
protected
animals from a challenge with about 5 to about 10 LDSO of ricin by either
intraperitoneal
injection, or by whole-body aerosol delivery of ricin toxin.
[91] Three groups of 20 female BALB/c mice were treated with i.m. injections
of
RTA198, dgRTA (positive control), or phosphate buffered saline (control
vehicle). At 0,
4, and 8 weeks, the mice in each group were injected i.m. with 0.1-ml at the
following
concentration of test/control articles:
a. Group l: 20 mice injected with 10 ~,g of RTA198 protein.
b. Group 2: 10 mice injected with 10 ~g of RTA198 protein + 0.2% alhydrogel.
c. Group 3: 20 mice injected with 10 ~,g of dgRTA protein.
d. Group 4: 20 mice injected with phosphate buffered saline.
[92] At 2 weeks after the third dosing, 20 mice in treatment groups 1, 3, & 4
and 10
mice in treatment group 2 were anesthetized and 0.2 to about 0.3 ml of blood
was
collected by the periorbital sinus method and recorded. The blood was later
used to
measure the specific antibody concentrations and ricin neutralizing antibody
titers.
[93] One week after blood collection, the same mice were weighed and ten mice
from
treatment groups 1, 3, & 4 were injected intraperitoneally on body weight
bases with 0.1
ml of a solution that contained 10 mouse LDso of ricin toxin D. The remaining
10 mice
from each treatment group were exposed over ten minutes in a dynamic system to
a liquid
aerosol that supplied 5 to 10 mouse LDso of ricin toxin D. After exposure to
ricin, daily
cage side observations were made for survival rates.
[94] The results are summarized in the following Table 1:
Table 1
-, RTA198 Protects
Mice from
Lethal Exposure
to Ricin Toxin
Anti en Dosen
Survival AIive/Total
Mean Time
to Death
Intraperitoneal
Injection
of 10 LDsos
of Ricin Toxin
RTA198 10 9/10 ~ 1.67 Da s
d RTA 10 10/10 > 14 Da s
phosphate buffered0.1 ml 0/10 0.83 0.02$ Da s
saline
Aerosol VI/hole
Body Ex osure
to Between
and 10 LDsos
of Ricin Toxin
RTA198 10 10/10 . > 14 Da s
RTA198 10 ~ 10/10 . > 14 Da s
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+ 0.2% Alh
dro el
d RTA 10 10/10 > 14 Da s
phosphate buffered0.1 ml 0/10 3.77 Days
saline
~[ - Three
intramusclar
injection
at 0, 4, &
8 weeks
$ - Average
standard
error
- Significantly
different
(p < 0.01)
from phosphate
buffered saline
controls by
Fisher's Exact
Test
t - Not si
nificantl
different
p > 0.05 from
d RTA b Fisher's
Exact Test
[95] Administration of the purified RTA198 without an adjuvant had no obvious,
untoward effects on the mice as noted by survival from three immunizations,
weight gain,
physical appearance, food consumption, and normal activity in comparison to
phosphate
buffered saline controls. Vaccinated animals survived without co-
administration of an
adjuvant (in these experiments, the adjuvant was Alhydrogel~ 2% AlzOz
available from
Siperfos Biosector). Thus, RTA198 may be used to elicit an immune response in
a
subject and may be used as a safe and effective ricin vaccine.
[96] Serum anti-ricin IgG antibody concentrations were measured by a direct
method
ELISA for the detection of ricin-specific IgG immunoglobulin in mouse sera.
Specifically, ricin stock solution (5 mg/ml) (Vector Laboratories, Inc.,
Burlington, CA)
was diluted 1:1000 in ELISA coating buffer and 100 lul was added to each well
of a plate.
The plate was stored at about 12 to about 48 hours at about 4 °C. Seven
concentrations of
mouse anti-ricin serum (Perirmnune Inc., Rockville, MD) were prepared and
standardized
to provide a suitable standard curve for the ELISA. Positive and negative
mouse anti-
ricin serum controls were prepared and used. Fresh dilutions of unknown sera
were
prepared by adding 12.5 pl of serum to 987.5 pl of MASP buffer. Optimal sera
dilutions
may be determined by conventional methods in the art. The plate was washed 3
times
with 0.2 ml/well of ELISA wash buffer. To each well, 200 p,l of 5% skim milk
buffer
was added. The plate was covered and incubated in a moist chamber at about 37
°C for 1
hour. The plate was then washed 3 times with 0.2 ml/well ELISA wash buffer.
Then
serial dilutions of the unknowns and controls and standards were added to the
wells. The
plate was incubated at about 37 °C for about 1 hour. The plate was then
washed 3 times
with 0.2 ml/well ELISA wash buffer. To each well, 100 ~,1 of goat anti-mouse
IgG (H+L)
conjugate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and
then
incubated at room temperature for about 1 hour. The plate was then washed 3
times with
0.2 ml/well ELISA wash buffer. To each well, 100 p,l of ABTS peroxidase
substrate
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added and then allowed
to
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stand at room temperature for about 40 minutes. Then 100 ~1 of ABTS peroxidase
stop
solution was added to each well. The plate was read using a microplate reader
at 405 nm.
[97] The results are summarized in the following Table 2:
Table 2
RTA198 Protects
Mice from
Lethal Exposure
to Ricin Toxin
Anti en Dosed
Survival AIive/Total
Anti-Ricin
I G m /ml
Intra eritoneal
In'ection
of 10 LDsos
of Ricin Toxin
RTA198 10 9110 . 0.32 0.06
d RTA 10 N 1 OI10 3.04 0.36
phosphate buffered0.1 ml OI10 <0.001
saline
Aerosol Whole
Bod Ex osure
to Between
and 10 LDsos
of Ricin Toxin
RTA198 10 ~ 10110 ~ 0,18 0.04*
RTA198 10 ~g 10/10 t 0.51 0.14+*
+ p.2~/a Alh
dro el
d RTA 10 N 10110 4.64 0.50
phosphate buffered0.1 ml 0110 <0.01
saline
~[ - Three
intramusclar
injection
at 0, 4, &
8 weeks
$ - Average
standard
error
- Significantly
different
(p < 0.01
) from phosphate
buffered saline
controls by
Fisher's Exact
Test
t - Not significantly
different
(p > 0.05)
from dgRTA
by Fisher's
Exact Test
+ - Significantly
different
(p < 0.01)
from chemically
dgRTA controls
by Unpaired
t Test
* - Significantly
different
(p < 0.01)
from non-adjuvant
1-198 & 33-44
deletion wild-type
rRTA by
Unpaired t
Test
Example 2
Construction and Analysis of RTA1-33/44-198
[98] Calculations of solvation free energies indicated that the protein RTA198
is lilcely
to be more stable in solvent water than the native RTA fold. It was noticed,
however, that
water was likely to fill several protein cavities not initially exposed to
solvent in the
native fold. Thus, a structural element of concern was the loop region
comprising the
amino acid residues from about position 33 to about 44. The loop region
comprises a (3-
hairpin and several charged residues and the overall structure has a
hydrophobic
character. Thus, it was hypothesized that this loop region may be deleted to
give a RTA
comprising amino acid residues 1-33 and 44-198 (RTAl-33/44-198) that will
retain the
compactness and activity of RTA198.
A. DNA Cloning, Sequencing, and Expression of RTAI -33/44-198
[99] DNA cloning, sequencing, and expression similar to that disclosed in
Example 1
were used. Briefly, mutagenesis was done using the QuickChange lcii
commercially
available from Stratagene (La Jolla, CA) based upon the polymerase chain
reaction (FCR)
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method. Starting with the RTA198 construct described in Example 1, a pair of
PCR
oligonucleotide primers selected to delete 10 amino acids at position 34-43
were used.
The sense PCR oligonucleotide used was: 5' CTG TCA GAG GTA GAT TGA CTG
TCT TGC CTA ACA GAG TTG G 3' (SEQ m N0:14). The antisense PCR
oligonucleotide used was: 5' CCA ACT CTG TTA GGC AAG ACA GTC AAT CTA
CCT CTG ACA G 3' (SEQ m NO:15). Mutagenesis was conducted under contract by
Clinical Research Management (Frederick, MD).
[100] After eliminating template DNA, 1 pl sample of the PCR mixture
containing the
mutated polynucleotide is used to transform E. coli XL1-Blue competent E. coli
cells
(Stratagene). The resultant mutated polynucleotide was purified using a
commercial
Miniprep kit (Qiagen, Valencia, CA) and sequenced using conventional methods
to
confirm that only the desired changes were made. The polynucleotide products
were
purified, ligated to a commercial expression vector based upon the T7 promoter
system
(Studier, et al. (1990) supra), and then used to transform competent E. coli
BL21 DE3
strain (Invitrogen, CA).
B. Expressiof~ of RTAI -33/44-198
[101] The transformed E. coli BL 21 cells were cultured in TB media comprising
50
pg/ml of kanamycin until a cell density of 0.4 to 0.6 OD6oo was reached.
Expression of
the RTA1-33/44-198 polynucleotide was induced using 1 mM IPTG at 25 °C,
for about
18 to about 20 hours. The cells were centrifuged in a Sorvall GS3 rotor at
6,000 rpm for
15 minutes at 4°C. The cell media was decanted and discarded. The
pellet containing E.
coli cells were bathed in TB medium with kanamycin and IPTG to create a cell
paste that
was frozen at -20°C until further use. Generally, the frozen cell paste
was stored for at
least about 24 hours prior to protein purification.
[102] As an "induction check" to ensure that RTAl-33/44-198 was being
expressed was
performed according to Example 1B above. Itnmunoreactive protein corresponding
to
RTA1-33/44-198 was found in both the phosphate buffer soluble and the
insoluble
fractions.
C. PuYification of RTAI -33/44-198
[103] One (1) gram (wet weight) of cell paste was dissolved in 15 ml of an ice
cold
buffer solution comprising 50 mM sodium phosphate buffer and 2 mM EDTA, pH
7.3.
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The cells were then sonicated and the homogenized cells were centrifuged using
a Sorvall
SS-34 rotor for 15,000 rpm for 15 minutes at 4°C. The supernatant was
collected and
syringe filtered (0.2 ~,m pore size).
[104] A Pharmacia Mono-Q~ 10/10 anion exchange column (Amersham-Pharmacia
Biotech, Piscataway, NJ) was pre-equilibrated with 50 mM sodium phosphate, pH
7.3,
and 2 mM EDTA. The supernatant comprising RTA1-33/44-198 was applied and a
step
elution was run to 1M NaCI (0-1.0 min). RTA1-33/44-198 was found predominantly
in
the column flow-through as determined by SDS-PAGE and Western blots.
[105] The Mono-Q~ 10/10 flow-through was pooled and the buffer was changed by
dialysis to 50 mM MES, 2 mM EDTA, pH 6.4. The material was syringe filtered
(0.2 ~,m
pore size), and loaded onto an equilibrated Pharmacia Mono-S~ 5/5 cation
exchange
column (50 mM MES, 2 mM EDTA, pH 6.4). Protein was eluted from the column
using
a NaCI gradient (0-500 mM NaCI in 30 minutes). Purified RTA1-33/44-198 began
to
elute by 20 mM and was completely eluted by about 150 mM NaCI. All fractions
showing protein by OD28o absorbance were analyzed by reducing SDS-PAGE.
D. ChaYacterizatiofz ofPurified RTAl-33/44-198
[106] Fractions from the Mono-S~ column that comprised greater than about 90%
pure
RTAl-33/44-198, as judged by Coomassie blue stained gels, were pooled. The
buffer
was changed to a buffer solution comprising 120 mM NaCI, 2.7 mM KCI, 10 mM
NaP04,
pH 7.4, by extensive dialysis (2L x 3 changes of greater than about 3 hours at
room
temperature). Purified RTA1-33/44-198 was filtered (0.2 ~.m pore size) and
stored sterile
at 4°C prior to animal studies. Total protein concentrations were
determined using the
Pierce micro-BCA assay, relative to a bovine serum albumin standard curve.
Purity and
consistency of lots of RTA1-33/44-198 obtained by this method is demonstrated
in Figure
3. Specifically, Figure 3 is an SDS-PAGE result wherein lane 1 shows the
molecular
weight standards. Lanes 2-5 show about 2 ~g of RTAl-33/44-198 from four
different
lots that were expressed and purified independent of each other. The gel is
10% (w/v)
total acrylamide stained with Coomassie blue. As shown, the each lane produced
a clear
dark band, which bands were consistently similar to each other. Therefore, the
production and purification of the polypeptides of the present invention are
reproducible
and suitable for vaccine production.
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[107] To determine whether RTAl-33/44-198 exhibited N-glycosidase activity,
the
assay according to Example 1D above was conducted. Purified RTAl-33/44-198 did
not
exhibit any detectable N-glycosidase activity as evidenced by inability to
disrupt protein
synthesis in a cell-free, translation assay under conditions where either
natural ricin
(positive control) or isolated RTA (positive control) did show ribosome
inactivation. See
Langer, et al. (1996) supra; and Hale (2001) supra.
E. ' haccine Studies with RTAI-33/44-198
[108] When RTA1-33/44-198 was administered, purified RTA1-33/44-198
effectively
protected animals from a challenge with about 5 to about 10 LDSO of ricin by
either
intraperitoneal injection, or by whole-body aerosol delivery of ricin toxin.
[109] Three groups of 20 female BALB/c mice were treated with i.m. injections
of
RTAI-33/44-198, dgRTA (positive control), or phosphate buffered saline
(control
vehicle). At 0, 4, and 8 weeks, the mice in each group were injected i.m. with
0.1-ml at
the following concentration of test/control articles:
a. Group 1: 20 mice injected with 10 ~g of RTA1-33/44-198 protein.
b. Group 2: 20 mice injected with 10 ~g of RTA1-33/44-198 protein + 0.2%
alhydrogel.
c. Group 3: 20 mice injected with 10 ~g of dgRTA protein.
d. Group 4: 20 mice injected with phosphate buffered saline.
[110] At 2 weeks after the third dosing, 20 mice were anesthetized and 0.2 to
about 0.3
ml of blood was collected by the periorbital sinus method and recorded. The
blood was
later used to measure the specific antibody concentrations and ricin
neutralizing antibody
titers.
[111] One week after blood collection, same mice were weighed and ten mice
from each
treatment group were injected intraperitoneally on body weight bases with 0.1
ml of a
solution that contained 10 mouse LDSO of ricin toxin D. The remaining 10 mice
from
each treatment group were exposed over ten minutes in a dynamic system to a
liquid
aerosol that supplied 5 to 10 mouse LDSO of ricin toxin D. After exposure to
ricin, daily
cage side observations were made for survival rates.
[112] The results are provided in the following Table 3:
Table 3
RTA1-33144-198 Protects Mice from Lethal Exposure to Ricin Toxin
Antigen Doses Survival (AlivelTotal) Mean Time to Death
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Intraperitoneal
Injection of 10
LDSOS of Ricin
Toxin
RTA1-33/44-198 10 10/10 ,t > 14 Da s
RTA1-33/44-198 10 Ng 10110 ,t > 14 Days
+ 0.2% Alh dro el
d RTA 10 10110 > 14 Da s
phos hate buffered 0.1 ml 0110 0.74 0.01$
saline Da s
Aerosol Whole Bod
Ex osure to Between
and 10 LDsos
of Ricin Toxin
RTA1-33/44-198 10 10110 ,t > 14 Da s
RTA1-33144-198 10 ~g 10110 ,t > 14 Days
+ 0.2% Alh dro el
d RTA 10 10110 > 14 Da s
hos hate buffered 0.1 ml 0110 3.84 0.07 Da
saline s
~[ - Three intramusclar
injection at 0,
4, & 8 weeks
$ - Average standard
error
- Significantly
different (p <
0.01 ) from phosphate
buffered saline
controls by Fisher's
Exact Test
t - Not si nificantl
different > 0.05
from d RTA b Fisher's
Exact Test
[113] Administration of the purified RTA1-33/44-198 without an adjuvant had no
obvious, untoward effects on the mice as noted by survival from three
immunizations,
weight gain, physical appearance, food consumption, and normal activity in
comparison
to phosphate buffered saline controls. Vaccinated animals survived without co-
administration of an adjuvant (in these experiments, the adjuvant was
Alhydrogel~ 2%
(A1z02) available from Siperfos Biosector). Thus, RTA1-33/44-198 may be used
to elicit
an immune response in a subject and may be used as a safe and effective ricin
vaccine.
[114] Serum anti-ricin IgG antibody concentrations were measured by the direct
method
ELISA for the detection of ricin-specific IgG immunoglobulin in mouse sera
described
above. The results are provided in the following Table 4:
Table 4
RTA1-33144-198 Protects
Mice from Lethal
Exposure to Ricin
Toxin
Antigen Doses Survival
AlivelTotal) Anti-Ricin
I G mglml)
Intra eritoneal
Injection of 10
LDSOS of Ricin
Toxin
RTA1-33144-198 10 ~ 10110 ,t 0.28 0.08*
RTA1-33144-198 10 ~g 10/10 ,t 0.56 0.10*
+ 0.2% Alh dro el
d RTA 10 10/10 1.12 0.11
hos hate buffered 0.1 ml 0110 <0.01
saline
Aerosol Whole Bod
Ex osure to Between
5 and 10 LDsos
of Ricin Toxin
RTA1-33/44-198 10 ~ 10/10 ,t 0.15 0.04*
RTA1-33144-198 10 ~g 10/10 ,t 0.81 0.12**
+ 0.2% Alh dro el
d RTA 10 10110 0.95 0.15
hos hate buffered 0.1 ml 0/10 <0.01
saline
1J - Three intramusclar
injection at 0,
4, & 8 weeks
$ - Average standard
error
- Significantly
different (p <
0.01 ) from phosphate
buffered saline
controls by Fisher's
Exact Test
t - Not significantly
different (p >
0.05) from dgRTA
by Fisher's Exact
Test
~- - Significantly
different (p <
0.01 ) from chemically
dgRTA controls
by Unpaired t Test
* - Significantly
different (p <
0.01) from non-adjuvant
1-198 & 33-44 deletion
wild-type rRTA
by Unpaired t Test
. - Not significantly
different (p >
0.05) from chemically
dgRTA by Unpaired
t Test
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[115] Additionally, since RTAl-33/44-98 maintains a similar conformation and
immunogenic activity as RTAl98, polypeptides of the present invention such as
RTA1-
33/44-98 may be used as stable structural scaffolds to present antigenic
determinants of
ricin for use in immunogenic compositions such as ricin vaccines.
Example 3
Protein Ag rg-a ag tLion
[116] To compare the relative amounts of protein aggregation in solutions of
the novel
RTA polypeptides versus dgRTA or recombinant RTA the following experiment may
be
conducted. The amounts and types of aggregates formed by the vaccine
candidates may
be quantified as a function of protein concentration and time under various
conditions of
fixed buffer composition, ionic strength, and pH. A combination of analytical
size-
exclusion chromatography (SEC) may be used with on-line multi-angle light
scattering
(MALS) detection. The SEC-MALS provides a measure of the molar mass of
proteins in
solution because the light scattering response is directly proportional to the
weight-
averaged molar mass (Mw) of the protein sample multiplied by the sample
concentration.
[117] Briefly, solutions of protein samples at a concentration of about 0.4 to
about 1.5
mg/ml in 0.067 M Na/K phosphate, pH 7.5 are separated using standard high
performance
liquid chromatographic methodology with a flow rate of about 0.5 to about 1.0
ml/min.
Protein species are detected with an in-line standard UV/VIS HPLC detector,
and the
relative refractive indices of the sample components are determined from an in-
line
interferometric refractometer. Light scattering data are collected at many
wavelengths,
averaged, and evaluated using an in-line MALS instrument. Aggregates are
quantified by
molar mass for each protein sample.
Example 4
Protein Folding Stability
[118] To compare the relative protein folding stability of the novel RTA
polypeptides
versus dgRTA or recombinant RTA the following may be conducted. Relative
protein
folding stability is measured indirectly by comparing the amount of energy
(proportional
to temperature) required to unfold each polypeptide under various conditions
of ionic
strength or pH. The extent of protein unfolding is assessed indirectly by
circular
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dichroism (CD) spectroscopy. Briefly, CD scans of protein samples are
performed in a
spectropolarimeter, fitted with pettier thermal control unit, using 0.2 mm and
1 cm path
length quartz cuvettes, respectively, for near and far UV measurements.
Solutions of
purified polypeptide are used at a concentration of about 0.5 mg/ml in 0.067 M
Na/K
phosphate, pH 7.5. The initial scans provide baseline spectra and corroborate
that the
protein samples are folded. Subsequently, protein samples are intentionally
and slowly
unfolded by increasing the cuvet temperature by means of the thermal control
unit.
Temperature-induced changes in protein secondary structure are assessed
indirectly by
monitoring the change in mean residue ellipticity at 222 nm. From these data,
one may
calculate and compare the temperature (Tin) at which 50% of the protein is
unfolded. If
the novel RTA polypeptides are found to fold and unfold reversibly, then one
may also
calculate and compare the thermodynamic and enthalpic constants for protein
folding.
Example 5
Hydrophobic Surface Exposure
[119] To compare the relative exposure of hydrophobic surfaces among the novel
RTA
polypeptides of the present invention versus dgRTA, or recombinant RTA the
following
(indirect) assay may be conducted by measuring the amount of 1-anilino-S-
naphthalenesulfonic acid (ANS) bound to each polypeptide under defined
conditions of
buffer composition, pH, ionic strength and temperature. Increased binding of
ANS, is
indicative of increased availability of hydrophobic surfaces, and bound dye
can be
differentiated from unbound dye on the basis of fluorescence. Briefly, 2 pl of
0.1 mM
ANS in acetonitrile is added to 200 ~1 of 0.5 ~M RTA polypeptide in 0.067 M
NaII~
phosphate, pH 7.5. Fluorescence is measured using a spectrofluorimeter at room
temperature; the excitation wavelength is 390 nm, and the emission spectrum is
evaluated
between 400 and 600 nm.
Example 6
Effects of Storage Time on Biophysical Properties
[120] To evaluate how the biophysical properties of the novel RTA polypeptides
of the
present invention may vary as a function of storage time (such as over the
course of 24
months) and storage temperature (such as at about 2 to about ~, 25 and
40°C) in different
storage formulations the following assay may be conducted. The number of
possible
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storage formulations is very large; therefore, only a subset of formulations
using a sparse
matrix that isolates selected variables are tested. These variables might
include the buffer
composition, the ionic strength, the pH, or the presence or absence of
preferred adjuvants.
Biophysical properties of the novel RTA polypeptides to be observed under
different
storage conditions may include, relative molecular mass of polypeptides,
apparent
isoelectric point of polypeptides (net charge), extent of self aggregation,
protein folding
stability, and exposure of hydrophobic surfaces. These can be evaluated as
described
using SDS-PAGE, native PAGE, isoelectric focusing, light scattering, ANS dye
binding,
and circular dichroism spectroscopy.
Example 7
Elucidation of Three-Dimensional Structure
[121] To determine the precise three-dimensional structures of the novel RTA
polypeptides of the present invention near atomic resolution single crystal,
macromolecular X-ray crystallographic methods may be used. Protein crystals of
novel
RTA polypeptides are obtained using the hanging-drop vapour-diffusion method,
and a
sparse matrix of crystallization conditions. Protein crystals are flash-frozen
in liquid
nitrogen prior to data collection at 100 °K. Data may be collected at
the National
Synchrotron Light Source at Brookhaven National Laboratory. Structure solution
will be
attempted using molecular replacement and the coordinates of RTA (Protein
Databank,
PDB) s a starting model. The structures may be refined by a combination of
simulated
annealing and molecular dynamics with a maximum likelihood target function,
using the
CNS program suite. See Adams, P. D., et al. (1997) PNAS USA 94:5018-23; and
Brunger, A.T., et al. (1998) Acta Cryst. D54, 905-921.
[122] To the extent necessary to understand or complete the disclosure of the
present
invention, all publications, patents, and patent applications mentioned herein
are
expressly incorporated by reference therein to the same extent as though each
were
individually so incorporated.
[123] Having thus described exemplary embodiments of the present invention, it
should
be noted by those skilled in the art that the within disclosures are exemplary
only and that
various other alternatives, adaptations, and modifications may be made within
the scope
of the present invention. Accordingly, the present invention is not limited to
the specific
embodiments as illustrated herein, but is only limited by the following
claims.
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SEQUENCE LISTING
<110> Olson, Mark A
Millard, Charles B
Byrne, Michael P
Wannemacher, Robert W
LeClaire, Ross D
<120> Ricin Vaccine and Methods of Making and Using Thereof
<130> P67452US0 (RIID 01-58)
<140>
<141>
<160> 15
<170> PatentIn Ver. 2.1
<210> 1
<211> 576
<212> PRT
<213> Ricinus communis
<400> 1
Met Lys Pro Gly Gly Asn Thr Ile Val Ile Trp Met Tyr Ala Val Ala
1 5 10 15
Thr Trp Leu Cys Phe Gly Ser Thr Ser Gly Trp Ser Phe Thr Leu Glu
20 25 30
Asp Asn Asn Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr
35 40 45
Ala Gly Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg
50 55 60
Gly Arg Leu Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu
65 70 75 80
Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu
85 90 95
Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr
100 105 110
Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe
115 120 125
His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr
130 135 140
Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg
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Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn
165 170 175
Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly
180 185 190
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Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln
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Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg
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Thr Arg Ile Arg Tyr Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile
225 230 .235 240
Thr Leu Glu Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser
245 250 255
Asn Gln Gly Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg Asn Gly
260 265 270
Ser Lys Phe 5er Val Tyr Asp Val Ser Ile Leu Ile Pro Ile Ile Ala
275 280 285
Leu Met Val Tyr Arg Cys Ala Pro Pro Pro Ser 5er Gln Phe Ser Leu
290 295 300
Leu Ile Arg Pro Val Val Pro Asn Phe Asn Ala Asp Val Cys Met Asp
305 310 315 320
Pro Glu Pro Ile Val Arg Ile Val Gly Arg Asn GTy Leu Cys Val Asp
325 330 335
Val Arg Asp Gly Arg Phe His Asn Gly Asn Ala Ile Gln Leu Trp Pro
340 345 350
Cys Lys Ser Asn Thr Asp Ala Asn Gln Leu Trp Thr Leu Lys Arg Asp
355 360 365
Asn Thr Ile Arg Ser Asn Gly Lys Cys Leu Thr Thr Tyr Gly Tyr Ser
370 375 380
Pro Gly Val Tyr Val Met Ile Tyr Asp Cys Asn Thr Ala Ala Thr Asp
385 390 395 400
Ala Thr Arg Trp Gln Ile Trp Asp Asn Gly Thr Ile Ile Asn Pro Arg
405 410 415
Ser Ser Leu Val Leu Ala Ala Thr Ser Gly Asn Ser Gly Thr Thr Leu
420 425 430
Thr Val Gln Thr Asn Ile Tyr Ala Val Ser Gln Gly Trp Leu Pro Thr
435 440 445
Asn Asn Thr Gln Pro Phe Val Thr Thr Ile Val Gly Leu Tyr Gly Leu
450 455 460
Cys Leu Gln Ala Asn Ser Gly Gln Val Trp Ile Glu Asp Cys Ser Ser
465 470 475 480
Glu Lys Ala Glu Gln Gln Trp Ala Leu Tyr Ala Asp Gly Ser Ile Arg
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Pro Gln Gln Asn Arg Asp Asn Cys Leu Thr Ser Asp Ser Asn Ile Arg
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Glu Thr Val Val Lys Ile Leu Ser Cys Gly Pro Ala Ser Ser Gly Gln
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515 520 525
Arg Trp Met Phe Lys Asn Asp Gly Thr Ile Leu Asn Leu Tyr Ser Gly
530 535 540
Leu Val Leu Asp Val Arg Ala Ser Asp Pro Ser Leu Lys Gln Ile Ile
545 550 555 560
Leu Tyr Pro Leu His Gly Asp Pro Asn Gln Ile Trp Leu Pro Leu Phe
565 570 575
<210> 2
<211> 179
<212> PRT
<213> Ricinus communis
<400> 2
Met Lys Pro Gly Gly Asn Thr Ile Val Ile Trp Met Tyr Ala Val Ala
1 5 10 15
Thr Trp Leu Cys Phe Gly Ser Thr Ser Gly Trp Ser Phe Thr Leu Glu
20 25 30
Asp Asn Asn Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr
35 40 45
Ala Gly Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg
50 55 60
Gly Arg Leu Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu
65 70 75 80
Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu
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Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr
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Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe
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His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr
130 135 140
Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg
145 150 155 160
Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn
165 170 175
Gly Pro Leu
<210> 3
<211> 198
<212> PRT
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<213> Ricinus communis
<400> 3
Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn Arg
35 40 45
Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn
50 55 60
His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala Tyr
65 70 75 80
Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro Asp
85 90 95
Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val Gln
100 105 110
Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu Gln
115 120 125
Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro Leu
130 135 140
Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr Gln
145 150 155 160
Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile Ser
165 170 175
Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile
180 185 190
Arg Tyr Asn Arg Arg Ser
195
<210> 4
<211> 188
<212> PRT
<213> Ricinus communis
<400> 4
Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Val Leu Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile
35 40 45
Leu Val Glu Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu
50 55 60
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Asp Val Thr Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala
65 70 75 80
Tyr Phe Phe His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His
85 90 95
Leu Phe Thr Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn
100 105 110
Tyr Asp Arg Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu
115 120 125
Leu Gly Asn Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr
130 135 140
Ser Thr Gly Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile
145 150 155 160
Cys Ile Gln Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly
165 1'70 175
Glu Met Arg Thr Arg Ile Arg Tyr Asn Arg Arg Ser
180 185
<210> 5
<211> 199
<212> PRT
<213> Ricinus communis
<400> 5
Met Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly
1 5 10 15
Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg
20 25 30
Leu Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn
35 40 45
Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser
50 55 60
Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn .Ala
65 70 75 80
Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro
85 90 95
Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val
100 105 110
Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu
115 120 125
Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro
130 135 140
Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr
145 150 155 160
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Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile
165 170 175
Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg
180 185 190
Ile Arg Tyr Asn Arg Arg Ser
195
<210> 6
<211> 189
<212> PRT
<213> Ricinus communis
<400> 6
Met Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly
1 5 10 15
Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg
20 25 30
Leu Thr Val Leu Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe
35 40 45
Ile Leu Val Glu Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala
50 55 60
Leu Asp Val Thr Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser
65 70 75 80
Ala Tyr Phe Phe His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr
85 90 95
His Leu Phe Thr Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly
100 105 110
Asn Tyr Asp Arg Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile
115 120 125
Glu Leu Gly Asn Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr
130 135 140
Tyr Ser Thr Gly Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile
145 150 155 160
Ile Cys Ile Gln Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu
165 170 175
Gly Glu Met Arg Thr Arg Ile Arg Tyr Asn Arg Arg Ser
180 185
<210> 7
<211> 198
<212> PRT
<213> Ricinus communis
<400> 7
Met Val Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
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1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn Arg
35 40 45
Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn
50 55 60
His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala Tyr
65 70 75 80
Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro Asp
85 90 95
Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val Gln
100 105 110
Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu Gln
115 120 125
Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro Leu
130 135 140
Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr Gln
145 150 155 160
Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile Ser
165 170 175
Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile
180 185 190
Arg Tyr Asn Arg Arg Ser
195
<210> 8
<211> 188
<212> PRT
<213> Ricinus communis
<400> 8
Met Val Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Val Leu Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile
35 40 45
Leu Val Glu Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu
50 55 60
Asp Val Thr Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala
65 70 75 80
Tyr Phe Phe His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His
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85 90 95
Leu Phe Thr Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn
100 105 110
Tyr Asp Arg Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu
115 120 125
Leu Gly Asn Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr
130 135 140
Ser Thr Gly Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile
145 150 155 160
Cys Ile Gln Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly
165 170 175
Glu Met Arg Thr Arg Ile Arg Tyr Asn Arg Arg Ser
180 185
<210> 9
<211> 185
<212> PRT
<213> Ricinus communis
<400> 9
Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu
35 40 45
Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr
50 55 60
Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe
65 70 75 80
His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr
85 90 95
Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg
100 105 110
Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn
115 120 125
Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly
130 135 140
Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln
145 150 155 160
Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg
165 170 175
Thr Arg Ile Arg Tyr Asn Arg Arg Ser
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180 185
<210> 10
<211> 200
<212> PRT
<213> Ricinus communis
<400> 10
Met Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly
1 5 10 15
Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg
20 25 30
Leu Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn
35 40 45
Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser
50 55 60
Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala
65 70 75 80
Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro
85 90 95
Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val
100 105 110
Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu
115 120 125
Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro
130 135 140
Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr
145 150 155 160
Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile
165 170 175
Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg
180 185 190
Ile Arg Tyr Asn Arg Arg Ser Ala
195 200
<210> 11
<211> 190
<212> PRT
<213> Ricinus communis
<400> 11
Met Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly
1 5 10 15
Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg
20 25 30
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Leu Thr Val Leu Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe
35 40 45
Ile Leu Val Glu Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala
50 55 60
Leu Asp Val Thr Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser
65 70 75 80
Ala Tyr Phe Phe His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr
85 90 95
His Leu Phe Thr Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly
100 105 110
Asn Tyr Asp Arg Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile
115 120 125
Glu Leu Gly Asn Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr
130 135 140
Tyr Ser Thr Gly Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile
145 150 155 160
Ile Cys Ile Gln Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu
165 170 175
Gly Glu Met Arg Thr Arg Ile Arg Tyr Asn Arg Arg 5er Ala
180 185 190
<210> 12
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: RTA198 primer
for Nde I site.
<400> 12
gaattccata tgatcttccc aaagc 25
<210> 13
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: RTA198 primer
for stop codon and Sal I site.
<400> 13
gtcgacctag gatctacggt tgtatctaat tc 32
<210> 14
<2l1> 40
<212> DNA
<213> Artificial Sequence
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<400> 14
ctgtcagagg tagattgact gtcttgccta acagagttgg 40
<210> 15
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Antisense PCR
oligonucleotide sequence
<400> 15
ccaactctgt taggcaagac agtcaatcta cctctgacag 40
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