Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR ACTIVE VACCINATION
BACKGROUND OF THE INVENTION
This application relates. to an active vaccine approach to the treatment of
cancer and other diseases. The approach is applicable to a number of cancers
and diseases,
although a preferred embodiment provides an active vaccine for treatment of B
cell Non-
Hodgkin's Lymphoma (NHL).
NHL is characterized b~y a clonal proliferation of malignant B cells. The
treatment of NHL across a broad spectrum of patients remains a challenge,
although
numerous therapeutic approaches have been proposed and tried.
The most common therapeutic approach being used today is chemotherapy.
While chemotherapy is effective for some period of time in most patients, a
significant
percentage of patients are not cured and experience a relapse.
Treatments have been proposed based on anti-idiotype therapy. In anti-
idiotype therapy, a cell surface molecule which is expressed by malignant
cells but not by
normal cells is used to create patient-specific antibodies which are then
administered to the
patient. See, Miller, et al., New Engl. J. Med. 306: 517-522 (1982}.
Autologous patient--
derived idiotype proteins have also been conjugated with keyhole limpet
hemocyanin to
produce a vaccine which has demonstrated efficacy and can elicit B and T cell
immune
responses. Kwak et al., New Engl. J. .Med. 327: 1209-1215 (1992). Hybridoma-
derived
idiotype was co-cultured with patient-derived dendritic cells which acted as
antigen
presenters upon re-infusion into the patient and showed clinical efficacy. Hsu
et al., Nature
Medicine 2: 52-58 (1996). Idiotypic vaccines made in lipid-based earners are
disclosed in
International Patent Publication W098/14170.
Treatments have also been proposed using antibodies directed to CD20, a
transmembrane protein that is expressed by both normal and malignant B-cells
during parts of
the B cell development cycle. Using single-dose infusions with anti-CD20
monoclonal
antibodies, partial or minor tumor regressions were observed in 6 of 15
patients in a Phase I
clinical study. Maloney et al., Blood 84: 2457-2466 (1994). In Phase II
studies, 17 of 37
patients showed complete or partial rE;missions. In December 1997, the FDA
approved the
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first antibody-based therapy for NHI~. Rituximab (Ritvaxan, IDEC/Genentech) is
a chimeric
human/murine antibody approved for the treatment of patients with relapsed or
refractory
low-grade or follicular CD20+ B cell NHL. Maloney et al., Blood 90: 2188-2195
(1997).
Combinations of chemotherapy and anti-CD20 therapy have been reported as
having better therapeutic efficacy, with 11 of 11 patients showing complete or
partial
remission. Czuczrnan et al., Abstraca 53, Ann. Oncol. 7, Supp. 1: 56 (1996).
While therapeutic regimens using anti-CD20 concepts are potentially
effective, all of these therapies have the drawback of being passive
therapies, i.e., they do not
directly involve the immune system of the patient. Thus, these therapies may
require the
continued administration of the ther<ipeutic agent for efficacy and do not
provide any long-
terns protection against recurrence. :ln addition, the passive therapy is
monoclonal in nature,
therefore escape is possible. It would therefore be desirable to have an
active therapy, that is
a therapeutic agent which when administered to the patient stimulates an
immune response
against CD20 found in B-cells.
It is an object of the present invention to provide such a therapy. It is a
further
object of the invention to provide an active polyclonal therapy that is
difficult to evade.
SUMMARY OF THE INVENTIOI\f
In accordance with the present invention, NHL is treated, not by
administration of an anti-CD20 monoclonal antibody, but by the administration
of CD20
itself, or an immunogenic fragment of the extracellular portion thereof,
coupled to or
administered with an antigenic carrier moiety such as keyhole limpet
hemocyanin (KLH).
This results in the stimulation of the production of polyclonal antibodies
against CD20 (or an
immunogenic fragment thereof) which has the affect of reducing the number of B-
cells,
including malignant B-cells. Thus, the invention provides an active vaccine.
The same
approach can be used for therapeutics for other diseases and conditions in
which target cells
possess a transmembrane protein, and is particularly applicable to those
diseases and
conditions for which administration of antibodies to transmembrane proteins or
peptides (i.e.,
passive therapy) have been shown to provide therapeutic benefits, and
especially in the
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situations where the target is also capable of transducing or receiving a
signal important for
cell growth or function. This would include, for example, Her2/neu, VEGF
receptor,
epidermal growth factor receptor, the CD 19 molecule, interleukin-2-receptor,
interleukin-4-
receptor, and the P-glycoprotein, also known as the multidrug-resistance
protein.
BRIEF DESCRIPTION OF THE FIGURES
Figs. lA and B show ELISA results for formation of antibodies to human and
mouse CD20 in vaccinated mice;
Figs. 2A and B shoves results for binding of control B 1 antibodies or
antibodies in plasma from a mouse treated with human CD20-KLH conjugate with
Raji B
NHL cells;
Fig. 3 shows CP19+B cell levels in mice treated with human or mouse CD20-
KLH conjugate;
Fig. 4 shows the donnain structure of human Her2;
Fig. 5 shows the domain structure of human EGFR;
Figs. 6A-D shows the cross-reactivity of antibodies generated in response to
human or mouse CD20 fragments;
Figs. 7A-D show thc: importance of carrier protein and adjuvant in generating
an immune response;
Figs. 8A-D shows the immune response generated using different adjuvants;
and
Figs. 9A-I shows CI'19+B cell levels in mice treated with human or mouse
CD20-KLH conjugate.
DETAILED DESCRIPTION OF T'HE INVENTION
The present invention provides an active vaccine therapy which can be used in
the treatment of a variety of cancer's and related conditions in which it is
desirable to bring
about the death of a target group of cells. Conventionally, immunotherapies
targeting cells
have sought to obtain a cellular immune response (T-cells that recognize the
target cells) ,
since a humorai immune response (antibodies that recognize the target cells)
alone is not
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deemed sufficient to achieve the desired result of cell death. The present
invention departs
from this conventional wisdom, and effectively utilizes a humoral immune
response against
the target cells to provide therapeutic benefit. The targets for therapy
include cell surface
proteins that when bound by a ligand signal to the cell. The vaccine induced
antibody
response will mimic ligand binding and cause similar signaling events which
can imitate the
process of programmed cell death (apoptosis) or halt the cell from growing or
change the
cancer cell's sensitivity to chemotherapy.
By way of example, the invention is suitably employed in the treatment of
NHL and other B cell diseases such as chronic lymphocytic leukemia, auto-
immune disorders
and B-cell regulatory disorders. In accordance with this embodiment of the
invention, a
peptide antigen is prepared which contains at least an immunogenic portion of
the
extracellular domain of CD20 coupled to or administered with an antigenic
carrier protein.
The CD20 component of the peptide antigen may be syngeneic or it may be
xenogeneic.
Thus, for example, human patients may be treated with a peptide vaccine
containing a human
or a mouse CD20-fragment. There is evidence that strong immune responses can
be elicited
against xenogeneic proteins. Naftz~;er et al., Proc. Natl. Acad. Sci. (USA)
93: 14809-14814
(1996); International Patent Application PCT/US97/22669, filed December 10
199?,
incorporated herein by reference. A suitable fragment is the 44 amino acid
peptide spanning
amino acids 136 to 179 of the sequf;nce of mouse or human CD20. (Seq. ID Nos.
l and 2)
Other immunogenic fragments derived from the extracellular domain of CD20, or
the entire
CD20 molecule may also be used. Seq. ID. Nos. 3 and 4 shows the nucleic acid
and amino
acid sequences, respectively, of exon VI (the extracellular domain) of human
CD20 as
reported by Tedder et al., J. Immun~ol. 142: 2560-2568 (1989).
As used in the specification and claims hereof, an "immunogenic fragment" is
a molecule which includes at least a portion of the extracellular domain of a
transmembrane
protein to direct and immunologica.l response to that transmembrane protein
when the
immunogenic fragment is coupled to or administered with an antigenic earner
protein
effective to break tolerance and administered with an adjuvant. It is not
required that the
immunogenic fragment alone be effective to stimulate an immune response,
although such
stimulation would not take a given fragment outside the scope of the present
invention.
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A preferred antigenic. carrier protein is keyhole limpet hemocyanin which can
be coupled to peptides using techniques described in Pierce Catalog Protocol.
Other
antigenic carrier proteins which can be used to break tolerance might be used
in the invention
include immunoglobulins, tuberculin, tetanus toxin and others well known in
the art.
The peptide antigen containing the CD20 component and the antigenic carrier
protein is formulated with a pharmaceutically acceptable adjuvant in a liquid
carrier and
administered to a patient suffering from NHL or another B cell disease. The
composition will
generally be administered by injection, for example, intramuscular,
subcutaneous or
intradermal injection, but might also be administered by way of a DNA vaccine
(See US
Patent No. $,580,859, incorporated herein by reference) or a viral vaccine, or
after mixing
with antigen presenting cells (APC';s) such as dendritic cells, ex vivo.
Alternatively, the
antigen may be administered without adjuvant by injection into a host prepared
by prior or
simultaneous injection of an immune adjuvant. Specific amounts to be
administered to a
patient can be determined by monitoring the titer of anti-CD20 antibodies
developed by the
1 S patient, or by an average group of patients using well-known technology.
When a peptide of the extracellular domain of human or mouse CD20 is
coupled to KLH and administered with an adjuvant to mice, antibodies which
react with
CD20 are found in plasma. (Figs. lA and B) These antibodies bind to Raji
cells, a human
lymphoma cell line, indicating the ability to bind to a cell expressing CD20.
(Figs. 2A and
B). Moreover, the number of CDl9t B cells present in mice injected with either
of the two
CD20-KLH conjugates declines substantially (~30% decrease relative to
controls). (Figs. 3
and 9). The assay used to quantitatc; B cell depletion detects CD 19 which is
also expressed
on immature B cells that are CD20-. Thus, the 30% depletion actually
underestimates the
efficacy of the vaccine against CD20' B cells. .
Antibodies generated in mice after vaccination with human or mouse-derived
CD20 fragments are specific for the peptides used, yet are capable of inducing
immunity to
the corresponding peptide from other species (Figs. 6A-D). Studies showed that
in most
instances the peptide, carrier protein and adjuvant are all needed for optimal
response,
although some responses were detected using less than all of the components.
(Figs. 7A-D).
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Several different adjuvants were also tested, and QS21 was found to be the
most effective of
those tested. (Fig. 8A-D).
While not intending to be bound by any particular mechanism, it is believed
that the vaccines of the present invention are effective via at least two
pathways. First, the
S generation of a humoral immune response to CD20 is effective to some extent
to reduce the
numbers of B cells bearing CD20 antigen in a manner consistent with normal
immunological
response to a target antigen. In addiction, however, because CD20 has a
signaling function,
the binding of antibody to the CD2C1 moiety activates this signaling function
to trigger
apoptotic cell death. Such stimulation of apoptosis has been demonstrated to
occur in vitro
following passive treatments with a chimeric anti-CD20 antibody. Maloney et
al., Blood 88
(Supp. 1): 637a (1996).
It is also possible that T cell mediated effector mechanisms are involved in
the
immune response. As evidence of this, we illustrate in Table 1 the mouse and
human peptide
sequences capable of binding to the corresponding mouse and human
histocompatability
1 S antigens. This information was deriived from a search of the NIH
Bioinformatics and
Molecular Analysis Section HLA Binding Predictions database using the mouse
and human
CD20 amino acid sequences. (Parke;r et al., J. Immunol. 152: 163 (1994)).
While the method of the invention is illustrated here using CD20 or CD20-
derived peptides as the antigen to target B cells, the invention is not
limited to this
embodiment. Rather, the inventions encompasses the use of vaccine compositions
comprising an immunogenic portions of the extracellular domain of
transmembrane protein
or peptide, particularly a transmembrane protein or peptide having signaling
function,
coupled to or administered with an antigenic protein and/or adjuvant to break
tolerance.
A non-limiting exarr~ple of another transmembrane protein which can be used
in whole or in part in the method of the invention is Her-2/neu. The Her-2/neu
oncogene is a
receptor-like tyrosine kinase that is expressed on the cell surface of a
significant portion of
solid tumors. It has been shown that patients with early stage breast cancer
have a high titer
of antibodies to Her-2/neu. Discs e1; al., J. Clin. Oncol. 15: 3363-3367
(19967). The amino
acid sequence and domain structure: of human Her-2/neu are shown in Seq. OD.
No. 5 and
Fig. 4, and isolation and expression of the extracellular domain has been
disclosed.
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International Patent Publication No, WO 90/14357, which is incorporated herein
by
reference. There is clinical data showing efficacy of monoclonal antibodies
against Her-2-
neu in the treatment of patients with Her-2/neu+ tumors, and potential
synergism with
chemotherapy. Thus, in accordance; with the present invention, a vaccine
composition
comprising at least an immunogenic; portion of the extracellular domain of Her-
2-neu (amino
acids 22 to 652) coupled to or administered with an antigenic protein or
peptide such a KLH
can be used as a vaccine to provide the same therapeutic benefits using an
active as opposed
to a passive approach.
A further non-limiting example of a transmembrane protein which can be used
in whole or in part in the method of the invention is epidermal growth factor
receptor
(EGFR). The amino acid sequence; and domain structure of human EGFR are shown
in Seq.
ID. No. 6 and Fig. 5. There is significant data showing that antibodies to
EGFR can have
anti-tumor activity in breast and prostate cancer, as well as several head and
neck tumors.
Prewett et al., J. Immunother. Emp~iasis Tumor Humoral 19: 419-27 (1996). The
mechanism
by which antibody therapy against EGFR may be efficacious can be through the
ability to
down-regulate vascular endothelial growth factor production by tumor cells and
thereby
decrease angiogenesis. Petit et al., Am. J. Pathol. 151: 1523-30 (1997). In
accordance with
the present invention, a vaccine composition comprising at least an
immunogenic portion of
the extracellular domain of EGFR I;amino acids 25 to 645) coupled to or
administered with an
antigenic protein or peptide such a KLH can be used as a vaccine to provide
the same
therapeutic benefits using an active: as opposed to a passive approach.
Preferred
immunogenic peptides would be selected from regions not deleted in the various
types of
truncated EGFR mutants associated with some cancers.
A further non-limiting example of a transmembrane protein which can be used
in whole or in part in the method of the invention is VEGF receptor. There are
significant
data showing that antibodies to VE?(JF receptor can inhibit angiogenesis and
thereby halt
tumor progression. In accordance with the present invention, a vaccine
composition
comprising at least an immunogenic portion of the extracellular domain of VEGF
receptor
coupled to or administered with an antigenic protein or peptide such a KLH can
be used as a
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vaccine to provide the same therapeutic benefits using an active as opposed to
a passive
approach.
Still a further non-limiting example of a transmembrane protein which can be
used in whole or in part in the method of the invention is the IL-2 receptor.
The IL-2
receptor is expressed on most T-cells malignancies, and there is a data
showing that
antibodies to the IL-2 receptor can be used in the treatment of T-cell
malignancies and
autoimmune disorders. In the present invention, a composition is made
comprising at least an
immunogenic portion of the extrace:llular domain of the IL-2 receptor (e.g.,
PSS or P7S) ,
coupled to or administered with an antigenic carrier protein or peptide such
as KLH. and
used as a vaccine.
The vaccine compositions of invention can be used alone or in combination
(concurrently or sequentially) with drugs or chemotherapy agents that provide
therapeutic
benefit for the condition being treated. In the case of NHL, suitable
chemotherapy agents
which can be used in combination with the CD20 based vaccine include
alkylating agents,
1 S anthrocyclines, cis-platinum, fludanabine, corticosteroids and vinca
alkaloids. These same
chemotherapy agents which might lie used in combination with other vaccine
compositions
for other forms of cancer.
EXAMPLE 1
44 amino acid fragments of the extracellular domains of humans and murine
CD20 {amino acids 136-179, Seq. l:D Nos. 1 and 2) were synthesized using a
solid-phase
FMOC peptide synthesizer and coupled to KLH using the methodology described in
the
Pierce Catalog Protocol. The peptide coupled to KLH was then prepared for
injection by
formulation with QS-21 adjuvant. Balb/c mice were injected according to one of
the
following protocols on clays 1, 8, 15, 22 and SO of the experiment:
A. Murine CD20 fragment-KLH with QS-21 adjuvant
B. Human CD2.0 fragment-KLH with QS-21 adjuvant
C. KLH with Q~S-21 adjuvant
D. QS-21 adjuvant
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E. P 190 (irrelevant protein) coupled to KLH with QS-21 adjuvant
F. B3A2 (irrele:vant peptide) coupled to KL,H with QS-21 adjuvant.
The animals were sacrificed on day 62 of the experiment.
Serum samples from the mice were diluted 1:200 and evaluated by BSA-
blocked ELISA using goat-anti-mouse antibody conjugated to alkaline
phosphatase for
antibodies which bind to human CD20, mouse CD20 and KLH. As shown in Figs lA
and B,
mice injected with human CD20 coupled to KLH {Fig. lA) or mouse CD20 coupled
to KLH
(Fig. 1B) administration of xenoge;neic antibody produced a significant
polyclonal antibody
response to both human and mouse CD20, while the response following
administration of
syngeneic antibody was principally limited to antibodies to the syngeneic form
of CD20.
Either xenogeneic or syngeneic peptide can therefore be used to generate an
immune
response.
To confirm the ability of the antibodies to bind to B cells, Raji cells (a
form of
human B-cell lymphoma that expresses CD20 on its surface) were blocked with
human IgG,
washed and then incubated for 30 minutes on ice with a 1:10 dilution of plasma
from a mouse
vaccinated with P-190-KLH control or huCD20-KLH. As a positive control, Raji
cells were
incubated with Bl antibody, or IgC~2 as an isotypic negative control. After
washing, the cells
were incubated with goat-anti-mouse antibody, washed and fixed with 1 %
paraformaldehyde.
Flow cytometry analysis was performed in a Becton-Dickinson FACScan. The
results are
shown in Figs 2A and 3B, wherein the shaded data set are the experimental data
set and the
outlined data set is the negative controls. As is apparent, there is a strong
binding of mouse
antibodies and Raji cells, comparable to that observed with B1 antibody .
EXAMPLE 2
To assess the number of B cells present in vaccinated mice, an evaluation was
made of cells expressing CD19, a :standard phenotypic marker for B cells.
Spleens were
harvested from the animals vaccinated in Example 1 and put into a single-cell
suspension.
After counting the total number of cells, the cells were stained with FITC-
labeled anti-mouse
CD19 and the samples were analy~:ed by flow cytometry with a FACScan. 10,000
events
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were collected. The percentage of CD19 positive cells minus the control gate
was multiplied
by the total number of cells to determine the number of CD19 positive cells in
mice treated
with the mouse and human CD20 peptide conjugates, and the P190 irrelevant
peptide
conjugate control.
As shown in Fig. 3, t:he absolute number of CD19 positive cells was
significantly reduced in mice treated with either of the CD20 peptide
conjugates. The level of
CD19 positive cells is a reflection of the number of CD20 positive B cells,
and the number of
immature CD19+, CD20- B cells in the samples. The absolute number of CD19+ B
cells
actually underestimates the therapeutic efficacy of the treatment for
elimination of CD20+ B
cells, however, since CD19 is expressed on B cell progenitor cells before
expression of
CD20.
EXAMPLE 3
Mice were injected five times over two months with one of four treatment
protocols as follows:
human CD20 (44 as fragment)-KLH plus QS 1
human CD20 (44 as fragment)-KLH
human CD20 (44 as fragment) plus QS21
KLH plus QS21
Blood was collected on week 9 for analysis by ELISA. Sera from the vaccinated
mice were
diluted 1:200 and incubated on BSA blocked plates coated with msCD20, huCD20,
P190 or
KLH. Secondary goat anti-mouse antibody conjugated to alkaline phosphatase was
added,
and the color change of p-nitrophenyl phosphate substrate was measured at 405
nm. The
results are summarized in Figs. 7A-D. In most instances the peptide, earner
protein and
adjuvant are all needed for optimal response, although some responses were
detected using
less than all of the components.
EXAMPLE 4
Mice were vaccinated according to the schedule of Example 3 using one of
four treatment protocols: human CL>20 (44 as fragment)-KLH plus QS21 adjuvant,
mouse
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CD20 {44 amino acid fragment)-KLH plus QS21, P190 (irrelevant protein)-KLH
+QS21 and
KLH and QS21 alone. Mouse semm samples were evaluated by ELISA for the
presence of
antibodies reactive with msCD20, huCD20, P190 and KLH. The results are shown
in Figs.
6A-D. Antibodies generated in mice after vaccination with human or mouse-
derived CD20
fragments are specific for the peptides used, yet are capable of inducing
immunity to the
corresponding peptide from other species.
EXAMPLE 5
Mice were vaccinated five times over two months with huCD20 fragment-
KLH conjugate with no adjuvant o~r in combination with one of three adjuvants:
QS'? 1, BCG
or Alum. Serum samples from the vaccinated mice were tested by ELISA. The
results are
summarized in Figs. 8A-D. QS21 was found to be the most effective of those
tested.
EXAMPLE 6
To confirm the observations of Example 2, nucleated spleen cells were
recovered by centrifugation in a dc;nsity gradient from mice vaccinated with a
CD20-KLH
conjugate (human or mouse) in thf: presence of QS21 adjuvant. 1 X 10G cells
from each
mouse were incubated with 2 ,ug o~f rat anti-mouse CD19 FITC-labeled antibody
or with
isotope-matched FITC labeled rat antibody. Cells were washed, fixed and
analyzed with a
Becton Dickinson FACScaliber cytometer. Figs 9A-C, D-F and G-I show the
results for
three exemplary mice of each vaccination group. The decrease in the peak
reflecting levels of
CD19 positive spleen cells in eaclu of the mice is apparent.
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Lcu 'L'yr G1:. ~» ::ys G1~: Val Val Glo Cly ~~~n Leu Gi,: .,eu 1'hr Tyr
SO . 55 GO
Lau pro Thr Asn n~a Ser Leu Ser phe Lw Gln Asp Tle Gln Glu Val
65 70 75 80
Gln Gly Tyr Va::eu Ile Ala His Asn Gln Val Arg G1:: Val Pro Leu
g5 90 95
Gir. i;rg Le;: :~r:, I_.: Val F~rg G~,: Thr G: ~ Leu Phe G:.v Asp Asn Tyr
;Ov i05 ii0
Ala Leu Ala V~:i ~e:: ~lsp Asn Gly Asp Pro Leu Asn As:: Thr Thr Pro
115 120 1 2:i
Val T::r Gly .:la Se: pro Gly Gly Leu Arg Glu Leu G~ : Leu Arc Ser
130 135 110
Leu Thr Glu :~.e Le:: :.ys Gly Gly Val Leu Ile Gln «rg Asn Pro Gin
lg5 150 .155 1G0
Leu Cys Tyr Gi:". :1JJ Thr Ile Leu Trp Ly:, Asp I1 a Phe L:is Lys Asn
_",, ? 7~ 175
Asn G1:~ Leu yla :,~:: Thr Leu Ile Asp Tr Asn Arg Ser Arg Ala Cys
:80 135 190
:Iis Pro Cyis 5:: ?~o !~iet Cys Lys Gly Se: :?rg Cys Try, Gly Glu Ser
SUSSTiTUTE SHEET (RULE 2fi)
CA 02330212 2000-11-08
wo ms~9si pcrius~noo6s
4
195 200 ?v5
Ser Glu Asp C; s Gln Ser Leu Thr tt:~0 Thr Val Cys :,'.~a Gly Gly Cys
210 215 3::0
F,la .~'.~c- Cys Lys Gly Pro Leu Pro ~~~ Asp Cys Cys s Glu Gln Cys
225 . 230 235 240
r.ia aia Gly Cys Thr Gly Pro Ly:, K-s Se_ :~si~ Crr~ -.e4 Ala Cys Leu
245 250 255
His Pile Asn ::is Ser Gl.y Ile Cys G:.',. Leu His Cys ?ro Ala Leu Val
260 2G5 270
Thr Tyr Asn Thr Asp Thr Plie Glu Ser Met: Pro Asrl Pro Glu Gly Arg
275 280 . 285
T~ r Tt:r Phe Gly Ala Ser Cy Val "..._ ::la C,:~~; i~rc Ayr Asn Tyr Leu
J
2~p 295 300
Ser Thr Asp V~1 Gly Ser Cys '!'hr ~~:u Val Cys i~ro Leu ciis Asn Gln
305 310 315 320
Glu Vai Thr :,la Glu rlsp Gly Thr G~:. Ark Cys G1u ~ys Cys Ser Lys
325 330 335
Pro Cys ~11a ~lr~ Val Cys Tyr Gly Lo:: GIy M~:.~ G1;: :iis Leu Arg Glu
3.0 3~i:i 350
~~ai srr Ala Val Thr Ser Ala Asn Iie.Gln Glu Phc .~',la Gly Cys Lys
355 3G0
Lys .le Phe Gly Ser Leu Ala Phe tee;: Pro Glu See ?::e Asp Gly Asp
370 375 380
Pro is Ser As:: Thr Ala Pro Leu G:.:: Pro Glu Gln ~eu Gln Val Phe
390 395 400
385
Glu Thr Leu Glu Glu Ile Thr Gly 'yr Leu 't'yr Ile Se: Ala Trp Pro
415
~i 10
.05
Asp Scr Leu :': o rap Leu Ser Val :'::~~ Gln i,sn Leu G:. : Val Ile Arg
4'2U ~:~~; 430
Giy Arg Ile Leu His Asn Gly Ala ~;~~ Ser Leu '!'hr ~eu Gln Gly Leu
445
940
435
Gyy ;1~ Ser ~.r ~ Leu Gly Leu Arg Si:_ Leu Ar J Glu Leu Cly Ser Giy
SUBSTITUTE SHEET (RULE 28)
CA 02330212 2000-11-08
WO 99/57981 PCTNS99/10065
450 455 4Go
Leu t:la Leu Ile His fiis Asn Thr Ni.~ Leu Cy~ Pl7e V~1 ~iis Thr 'Jal
4G5 470 475 480
PYO '''rp tZ:i7 Gi:l L6'u l~hC: :1'-C: flSn I~YO 1-~l:i Gli7 Alc'1 Lel: Le,::
ftl,i Thr
485 490 495
Ala rsi: ArS Pro Glu Asp G;u Cys Val Gly Glu Gly i:eu Ala Cys His
50C 5i~5 S10
Gln Lcu Cys Air: ark Gly ais Cys 'i'rp Gly Pro Gly Pro Thr Gln Cys
515 530 535
Val Asn Cys See Gln Pi7c Lc:: .:,rr Gly Gln G1u Cys Vai Glu Glu Cys
X30 53~ 540
..rr VG1 Leu Gi : Gly Leu Pro Arg G~.u Tyr Vai Asn A?a yrg ais Cys
545 550 555 5G0
Leu Pro Cy:; i:is Pro Glu Cys Gln Pro Gln r'ssn Cly See Val Thr Cys
5G5 570 575
Phe Gly Pro Glu ?sla Asp Gln Cys Val A1~ Cys Ala His Tyr Lys Asp
580 585 590
Pro Pro Phe CyVal Ala Arc3 Cys Pro Ser Gly Val Lys Pro Asp Leu
595 G00 G05
Se: '.";m P:et Pro Ile Trp Ly~~ Phe Pro lisp Glu Glu Giy ::la Cys Gin
Gi0 G15 G20
Pro Cys Pro Ile an Cys Thr ~:is Ser Cys Val Asp Leu Asp Asp Lys
G25 G30 G35 G40
Giy Cys Pro Ala G;u Gln Arr Ala Ser Pro Leu Thr Sc:r Ile Ile Ser
G45 G50 655
Ala Val Val Gly .le Leu Leu Val Val Val Leu Gly Vai Val Phe Gly
6G0 GG5 G70
.'e Leu T_1~ Lys .-.-'::g Arg Gln Gln Lys Ile Ark Lys Tyr Thr Met Arg
G75 G80 G85
Arn Leu Lcu Glr ~=a Thr G1~ Leu Val Glu Pro Leu T::r Pro Ser Gly
G90 G95 700
ria !".et Pro :a : G:: Aia Gin idea ArJ Ilc Leu Lys Gu :'hr Glu Leu
SUBSTTTUTE SHEET (RULE 26)
CA 02330212 2000-11-08
WO 99/57981 PCTNS99/10065
6
70~ 710 715 720
Arg Lys Val Lya Val Lcu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
725 730 735
Gly Iia Trn ie Pro Asp Gly Glu Flsn Val Lys Ile Pro Val Ala Iie
7d0 '1 ~5 750
Lys Val Leu Arr, Glu Asn Thr Ser Pro Lys Ala Asn :.ys Glu Ile Leu
755 7G0 765
:asp Glu Ala ':yr V4i I~ct Ala Gly Val Cly Ser Pro Tyr Val Ser hrg
770 775 780
Leu Leu Gly ile Cys Leu Thr. Ser Th: Val Gin Leu Val Thr Gln Leu
785 790 795 800
tiet ?~o Tyr G;.l Cys Leu Leu Asp Nis Val elrg Clu Asn Arg Gly Arg
805 810 815
Leu Giy Sir G_:: Asp Leu Leu Asn Tr p Cys Met Gln Ile Ala Lys Gly
820 825 830
Met Ser Tyr LeL Glu Asp Val Arg L::u Val Fiis Arg Asp Leu Ala Ala
835 840 945
Arc3 As:~ Val Leu Val Lys Ser Pro Asn Flis Val Lys Ile Thr Asp Phe
850 B55 860
G1}J I,e;: Ala ?~: ;, Luu Leu Asp Ile Asp Glu 'lhr Glu ':yr His Ala Asp
8G5 870 3'75 880
Gly Gly Lys Vaaro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg
885 890 895
Arg Arg Phe T~° :iis Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val
900 905 910
Trp Glu Leu Ple~ Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala
915 920 925
Arc3 Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro
930 935 940
Pro Ile Cys Th_ plc Asp Val Tyr Met Ile Met Val Lys Cys Trp Met
945 950 955 960
Ile ~:sp Se. Gi;: ~ys :.Yc °ro Ar J Phe ::~~ Giu Leu Val Ser Glu
Phe
SUBSTITUTE SHEET (RULE 26)
CA 02330212 2000-11-08
wo ms~9si pcrius9~noo6s
7
9G5 97G 975
S~r Ar0 Met r-a yr~ rasp Pro Cln r.rJ Phe Vsl Val T_le Gln Asn Glu
985 990
Asp Leu Gly Pro nla Se:r Pro Leu Asp Ser :'::_~ Nhv Tyr Arg Scr Leu
995 1000 1005
Leu Glu t~sp A~:~ Asp Met Gly Asp Leu Val r?sip r'!la Glu Gl.u Tyr Leu
O10 1015 20'0
Val Pro Gln C1: Gly Phe Phe Cys Pro r?sp Pro Ala Pro Gly Ala Gly
1025 1030 1035 1040
Gly 1':~.t VaI ~iis :his Arch I-tis ~Ar 0 Sa: Ser Scr '1'hr Ar J Ser Gly Gly
;OqS 1050 1055
Giy :~sp L4u Thr Leu Gly Leu Glu Pro Svr Glu Glu Git; Ala Pro Arg
106"v lOGS 1070
Ser Pro Leu hia Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly
1075 1080 1085
Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His
1090 1095 1100
A:;p Pro Scr Pro ~cu Cln Ark Tyr Scr Cl.u Asp Pro '!'hr Val Pro Leu
1105 1110 1115 1120
P ro S'° Glu Tr :?sp Gl y Tyr Val T~'ia Pro Lieu '1'hr Cys Ser
E'ro Gln
t_
;125 1130 1135
Pro Glu Tyr Val :>,sn Gln Pro Asp Val ~lrg Pro Cln Pro Pro Ser Pro
114C 1145 1150
Ar0 -Glu Cly Pro Le~,: Pro Ala Ala ArcJ 1'ro Ala Gly Aia '!'hr Leu Glu
1155 1160 1165
Arg Pro Lys Th= :,eu Ser Pro Gly Lys ~~~sn Gly Vai Val Lys Asp Val
y170 1175 1180
Phe :via i~h~ Gly Gly Ala Val Glu As.~ Pro Glu '!'yr Leu Tl:r Pro Gln
1185 1190 1195 1200
Gly Gly Ala r?la Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala
.205 1210 1215
Phe ysa ~s Le:: ':.,~r Tyr Trp Asp Gin Asp Pro Pro Clu Arg Gly Ala
suesn~ sHE~ ~RU~ Zs~
CA 02330212 2000-11-08
WO 99/57981 PCT/US99/10065
8
1320 1225 330
Pro Pro Ser ~'::r Phe Lys GIy Thr Pro Ti:r Ala G1a As : Pro Glu Tyr
1235 1240 1245
Leu Giy Leu As;: Val Pro Val
;250 1255
<210> 6
<211> 1210
<212> PRT
< 213 > : i i3MAN
<220>
<223> '.~.u:aan ~G:'R
<400> 6
Met :erg Pro Scr Gly~ Thr Ala Gl.y Ala i;la Leu Lcu Al.a Leu Leu Ala
1 5 7.0 15
Ala Leu Cys Pro «la Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln
20 25 30
Gly T.::r Ser Asn Lys Leu Thr Gln Leu Giy Thr Phe Glu Asp His Phe
35 40 45
Leu Ser Leu Gln err, Met Phe Asn Asn Cys Glu Val Vai ~eu Gly Asn
50 55 60
Leu Gi:: Iie T'.~.r Tyr Val Gln Arg Asn Tyr Asp Leu Ser ?ne Leu Lys
65 70 75 80
Thr I.e Gln Glu Vai Ala Gly Tyr Val Leu Iie Ala Lei: As:. Thr Val
85 90 95
Glu Arg ire Pro Leu Glu Asn Leu Gln Ile Iie Arg Gly nsn Met Tyr
100 105 .10
'Pyr Giu :~s : Sw °;.~ rlia Leu Ala Val Lcu :cur A::n 'I'yr usp hla Asn
115 120 125
Lys T:r Giy Leu Lys Glu Leu Pro Met rrg Asn Leu Gln Glu Iie Leu
1.3u 13-'i 140
His Giia Val ~=.rs Phe Sor Asn Asn Pro Ala Leu Cys ~lsn Val Glu
145 150 15:i 16G
suesmurE sHE~r ~aut.s zs~
CA 02330212 2000-11-08
wo s9is~9si 9 rcrius~noo6s
Ser Ile Gin Trp Arg Asp Ile Val Ser 5or Asp 1'hc: LoL Ser Asn Met
17O 175
165
Ser Met Asp 1'he Gln Asn l~lis Lcu Gly Scr Cy:. Gln L:.~:: Cys Asp Pro
ly ' 190
130
Sar Cys Pro Asn Gly Ser Cys Trp Gly ::la Gly G1U G1;: Asn Cys Gln
X00 205
195
Lys Leu Thr Ly~ ale Ile Cys Aia Gln Gln Cy~ Ser G1~% Arg Cys Arg
215 220
210
Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Alu aIa Gly Cys
240
230 235
225
Thr Gly Prp Arg Giu SCr A5p CYS L~U Val <:y~ Ar J L}'S PhC Ar C ASp
4~'.5 250 255
Glu Ala Thr Cys Lys App Thr Cys p~~o Pro Leu Met Leu Ty= Asn Pro
270
260
mhr mrr Tyr Gln Met App Val :?~n Pro Glu Gly Ly~ Tyr Scr Phe Gly
280 285
275
Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His
295 300
290
Gly Ser Cys Val ArJ Aia Cys Giy Ala Asp Ser '1'yr G.u MGt Glu Glu
315 320
305 310
asp Gly Vai Arc3 Lys Cys Lys Lys Cys Glu Gly Pro C}'s Arg Lys Val
330 335
325
Cys Asn Gly Ile Gly Ile Gly Glu.Phe Lys Asp Ser Leu Ser Ile Asn
340 345 350
Ala Thr Asn Ile Lys fiis Phe Lys Asn Cys Tar Ser Ile Ser Gly Asp
360 :565
355
Leu His Ile Leu ?ro Val Ala Phe Arc; Gly Asp Ser Phc Thr His Thr
375 380
370
Pro Pro Lcu Asp ?ro Gln Glu L~:u Asp Ile Lcu Lys Thr Val Lys Glu
395 400
385 390
~ c Tr G1~ pt~e :,cu Leu Ile Gln Ala Trp Pro ,Glu easn Arg Thr Asp
410 4I~
~: 0 5
suBSmur~ sHe~ cRU~ is)
CA 02330212 2000-11-08
WO 99157981 1 ~ PCT/US99/10065
Lcu ::is Nla Phe Glu As.~. Leu Glu Ilc .ilc Arr, Gly ~rg Thr Lys Gln
930
~12 5
~.ii:; Gly Cln Phc :per. Lcu ~~ia Va.l Va:. Ser i~eu As:: Ile Thr Ser Leu
. i ~1:i
.l~Ii~
435
Gly Leu ArJ Se: Lcu Lys Glu ale Scr Asp Gly lisp Val Ile Ile Ser
960
955
950
Gly Asn Lys AS.~. ~eu Cys Tyr Ala Ann :'hr Ile Asr. Trp Lys Lys Leu
975 980
4G5 970
Phe GIy Thr See Gly Gln Lys 'fhr Lys I1:Ile Si:r Asn Arg Gly Glu
~.S5 490 495
rlsn Ser Cys Lys .::a Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro
50G 505 510
Giu Giy Cys '::i~ GlY Pro Glu Fro Arg Asp Cys Vai Ser Cys Arc,~ Asn
520 525
515
Val Ser Arg Gly yrg Glu Cys Val Asp Lys Cys Lys Leu Leu Glu Gly
530 535 540
Glu Pro Arg Glu ?he Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro
550 555 SGO
595
Glu Cys Leu Pro Gln Ala Met hsn Ilc Th r Cys Thr Gly Arch Gly Pro
570 575
~G5
r:sp i~sn CyJ T_1V ,:t.~. Cys Ala His Tyr .Lle Asp Gly LLO h1s CyJ Val
58G 585 590
Lys Tl:r Cys Pro :.tea Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp
G00 G05
595
Lys Tyr rla F~sp ::la Gly His Val Cys His Leu Cys H is Pro Asn Cys
G15 G20
G10
Thr 'fyr Gly Cys ~ r Gly Pro Gly Lcu Giu Gly Cys i~:o Thr Asn Gly
G35 G90
625 630
Pro Lys Ile Pro Ser I'_e Ala Thr Gly i4ct Val Gly Ala Leu Leu Leu
G5G G55
.i 5
Leu Leu Val Val :.:.a Lcu Gly Ile Cly Leu Phe Me:t Flrg Arc3 Arg His
GG~ GG5 G70
suesmu~~ sHE~r twin 2s~
CA 02330212 2000-11-08
WO 99/57981 PCI'NS99/10065
11
I lc Val Ar ~ Ly s :,rg Thr Leu Ar g Arc3 Le:u Lou Gln Giu Arg Glu Leu
G80 685
G'7 5
Val Glu Pro Leu ~nr Pro Ser Gly Gla Ala Pro As:. Gln Ala Leu Leu
.. G95 7U0
G9U
Arc3 T_le Leu Lys G:.u Thr Glu Phe Lys Lys Ile Lys Vai Leu Giy Ser
,i5 720
705 710
Gly yla ?he Gly :':~r Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu
730 735
-25
Lys V41 Lye Ile ?wo V41 Ala Ile Lys Glu LW F:rg Giu Ala Thr Ser
74G ' . 745 750
?ro Lys Ala as- Lys Glu Ile Leu Asp Glu T~la Tyr Val Met Aia S~:r
9G0 7G5
755
Val asp Asn Pre is Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser
775 780
770
Thr VaI Gln i~e:: ~lc Thr Gln Leu Met ?ro Phc Gly Cys Leu Leu Asp
795 800
795 790
Tyr Val Arg Gy;: is Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn
810 815
X05
Trp Cys Val G1~ ~'e Ala Lys Gly Met Asn 'fy_ Leu Glu Asp Arg Arg
.. - 825 830
8?~
Leu Val His n.=.' ~~p Leu Ala Ala Arg A:i:: Val Leu Val Ly~ 'f hr Pro
840 845
835
Gln His Val Lys .le Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala
855 860
850
Glu Glu Lys G:.;: ~yr His Ala Glu Gly Gly Lys Val Pro Ile LYs Trp
875 880
8G5 870
Met Ala Leu G'_'.: Sor Ile Leu His Arg Ilc '1'y~r Thr His Gln Ser Asp
385 890 895
Vai Trp Ser Ty_~ ply Val Thr Val 'frp Glu Li:u Met Thr Phe Gly Ser
905 910
9C~.
Lys Pro Tyr Fs~ ~ly -le Pro Ala Sir Glu Ile .'per Ser Ile Leu Glu
920 925
SUBSTITUTE SHEET (RUL.E 26)
CA 02330212 2000-11-08
WO 99/57981 PCT/US99/10065
12
Ly:; G:y Giu r.r~~ Lcu Pro Gln Pro Pro Iie Cy:: 't'hr lie App Val Tyr
9~0 935 940
iret ;~c btc;t Vai Lys Cy~ Trp MeL Ilc: :lip :?ia rl~p S~: Ary Pro Lys
)~t ~ ~.'i0 ~)!~!; 9G0
Phe hrg Giu Leu Ile Ile Glu Phe Ser Ly:: A:i:t .~'~la ::=g Asp Pro Gln
965 970 975
Arg iyr Leu Val le Gln Gly Asp Glu ::r c,~ M4i. l;is Lau Pro Ser Pro
980 985 990
~'hr rs? Ser Asn ?he Tyr Ary Ala Leu :~ic~ flsp Glu G.u Asp Met Asp
995 1000 lOCS
:;sp .',: Vai ?.sp .~'..a Asp Giu :yr Le:u :.;:c L'ro G.in G1:: Gly Phe L'he
iG:~ 1015 1020
Ser Ser Pro Ser ~_':.r Ser Ary Thr Pr o Leu Leu Ser Ser Leu Ser Ala
1025 1030 1035 1040
Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln
1045 1050 1055
Ser Cys Pro Iie Lys Glu Asp Ser Phe Lcu Gln Ary Tyr Ser Ser Asp
lOGO lOGS 1070
Pro T: Giy Aia Leu Thr Glu Asp Ser T_le iap :asp ~r Phe Leu Pro
;075 lOBO iOVS
Val P~c Glu T:~r ~ie Asr. Gln Ser Val Pro Lys :arc _ro Ala Gly Ser
1090 1095 1100
Val Gin Asn Pro Val Tyr !-lis Asn Gln Pro Lcu tan ?ro Ala Pro Ser
1105 1110 1115 1120
Ary :;sp ?ro His ".'yr Gln Asp L'ro I~li:: Si:r Thr t~la V4= Gly Asn Pro
1:25 1130 1135
Glu Ty: Leu As~ '"'.hr Val Gln Pro Thr Cy~ Val t~.sn Scr Thr Phe Asp
1140 1145 1150
Ser Pro .~':la His "'~? Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp
=155 1160 liG:i
Asn Pr o tap ~'yr Gin Gln Asp Phe Phe Pr J Lys Glu hia Lys Pro Asn
1:"; 1175 110
SUBS'TtTtJTE SHEET (RULE 2B)
CA 02330212 2000-11-08
WO 99/57981 PCTNS99/10065
13
Gly Ile Pipe Lys Gly Scr Thr tlla Glu Asn Ala Clu ':fir Leu Arch Val
1185 1190 1195 1200
~~la :~~o Gln Scr Scr Glu ?hc Ile G~y Ala
;~05 i210
SU88TITUTE SHEET (RULE 2B)
