Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN MONOCLONAL ANTIBODIES
Field of the Invention:
This invention relates to novel human monoclonal antibodies (mAbs) and to
the genes encoding same. More specifically, this invention relates to human
monoclonal antibodies specifically reactive with an epitope of the fusion (F)
protein
of Respiratory Syncytial Virus (RSV). Such antibodies are useful for the
therapeutic
and/or prophylactic treatment of RSV infection in human patients, particularly
infants and young children.
Background of the Invention:
Respiratory syncytial virus (RSV) is the major cause of lower respiratory
disease in children, giving rise to predictable annual epidemics of
bronchiolitis and
pneumonia in children worldwide. The virus is highly contagious, and
infections can
occur at any age. Immunity to RS V appears to be short-lived, thus
reinfections are
frequent. Zero to 2 year old infants are the most susceptible and represent
the
primary affected population. In this group, 1 out of 5 will develop lower
respiratory
(below larynx) disease upon infection and this ratio stays the same upon
reinfection.
Depending on age, environment and other associated factors, hospitalization is
required in I-3% of cases of RSV infection and is usually of long duration (up
to 3
weeks). The high morbidity of RSV infection, especially in infancy, has also
been
implicated in the development of respiratory problems later in life. Mortality
is
generally very low in more developed countries, but much higher in less
developed
countries and in certain risk groups such as children with heart/lung disease,
making
prophylactic treatment desirable for these groups of children.
A vaccine for RSV infection is not currently available. Severe safety issues
surrounding an attenuated whole virus vaccine tested in the 1960x, as well as
the
potential of induced immunopathology associated with the newer candidate
subunit
vaccines make the prospects of a vaccine in the near future appear remote. To
date
' 30 one drug therapy, Ribavirin, a broad spectrum antiviral, has been
approved.
Ribavirin has gained only minimal acceptance owing to problems of
administration,
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mild toxicity and questionable efficacy. In the majority of cases,
hospitalized
children receive no drug therapy and receive only intensive supportive care
which is
extremely costly. It is clear that there is a need for a safe, effective and
easily
administered drug for the treatment of RSV infection.
The feasibility of passive antibody treatmentlprotection against RSV has
been well established using animal models. Most of the earlier passive
transfer
studies in animals against infectious agents, including RSV, utilized murine
mABs.
Recently, the FDA has approved for use intravenous gammaglobulins (IVIG)
isolated from pooled human sera. Initial reports from this study had been
encouraging (Groothuis, J. R. et al., Antimicrob. Agents Chemo. 35(7):1469-
1473
( 1991 )). However, generic shortcomings of IVIGs exist and include, without
limitation, the fact that such products are human blood derived and grams of
antibody often need to be administered to achieve an effective dose.
Alternatively, monoclonal antibodies have been employed. The advantages
of such an approach include: a higher concentration of specific antibody can
be
achieved thereby reducing the amount of globulin required to be given; the
reliance
on direct blood products can be eliminated; the levels of antibody in the
preparation
can be more uniformly controlled and the routes of administration can be
extended.
While passive immunotherapy employing monoclonal antibodies from a
heterologous species (e.g., murine) has been suggested (See: PCT Application
PCT/US94/08699, Publication No. WO 95/04081 ), one alternative to reduce the
risk
of an undesirable immune response on the part of the patient directed against
the
foreign antibody is to employ "humanized" antibodies. These antibodies are
substantially of human origin, with only the Complementarity Determining
Regions
(CDRs) being of non-human origin. Particularly useful examples of this
approach
are disclosed in PCT Application PCT/GB91/01554, Publication No. WO 92/04381
and PCT Application PCT/GB93100725, Publication No. W093/20210.
A second and more preferred approach is to employ fully human mAbs.
Unfortunately, there have been few successes in producing human monoclonal
antibodies through classic hybridoma technology. Indeed, acceptable human
fusion
partners have not been identified and murine myeloma fusion partners do not
work
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well with human cells, yielding unstable and low producing hybridoma lines.
However, recent advances in molecular biology and immunology make it now
possible to isolate human mABs, particularly directed against foreign
infectious
agents, as explained in greater detail below.
Comprehensive details concerning RSV infection and its clinical features can
be obtained from excellent recent reviews by McIntosh, K. and R. M. Chanock,
In:
"Respiratory Syncytial Virus", Ch. 38, B.N. Fields ed., Raven Press ( 1990)
and
Hall) C.B., In: "Textbook of Pediatric Disease" Feigin and Cherry, eds., W.B.
Saunders, pgs 1247-1268 ( 1987). RS V, belonging to the family
paramyoxoviridae,
is a negative-strand unsegmented RNA vines with properties similar to those of
the
paramyxoviruses. It has, however been placed in a separate genus Pneumovirus,
based on morphologic differences and lack of hemagglutinin and neuraminidase
activities. RSV is pleomorphic and ranges in size from 150-300 nm in diameter.
The virus matures by budding from the outer membrane of a cell and virions
appear
as membrane-bound particles with short, closely spaced projections or
"spikes". The
RNA genome encodes 10 unique viral polypeptides ranging in size from 9.5 kDa
to
160 kDa (Huang, Y. T. and G. W.Wertz, J. Virol. 43:150-157 ( 1982)). Seven
proteins (F, G, N, P, L, M, M2) are present in RSV virions and at least three
proteins
(F, G, and 5H) are expressed on the surface of infected cells. The F protein
has been
conclusively identified as the protein responsible for cell fusion since
specific
antibodies to this protein inhibit syncytia formation in vitro and cells
infected with
vaccinia virus expressing recombinant F protein form syncytia in the absence
of
other RS V virus proteins. In contrast, antibodies to the G protein do not
block
syncytia formation but prevent attachment of the virus to cells.
RSV can be divided into two antigenically distinct subgroups, (A & B)
(Mufson, M. A. et al., J. Gen'1. Virol. 66:2111-2124 (1985)). This antigenic
---- . dimorphism is linked primarily to the surface attachment (G)
glycoprotein (Johnson,
R. A. et al., Proc. Nat'1. Acad. Sci. USA 84:5625-5629 ( 1987)). Strains of
both group
A and B circulate simultaneously, but the proportion of each may vary
unpredictably
from year to year. An effective therapy must therefore target both subgroups
of the
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virus and this is the reason for the selection of the highly conserved surface
fusion
(F) protein as target antigen for mAb therapy as will be discussed later.
RSV is distributed worldwide. One of the most remarkable features of the
epidemiology of RSV virus, as mentioned above, is the consistent pattern of
infection and disease. Other respiratory viruses cause epidemics at irregular
intervals
or exhibit a mixed endemic/epidemic pattern, but RSV is the only respiratory
viral
pathogen that produces a sizable epidemic every year in large urban centers.
In the
temperate areas of the world, RS V epidemics have occurred primarily in the
late fall,
winter or spring but never during the summer. The occurrence and spread of
infection within a community is characteristic and easily diagnosed) leading
to sharp
rises in cases of bronchiolitis and pediatric pneumonia and the number of
hospital
admissions of young children with acute lower respiratory tract disease. Other
respiratory viral agents that occur in outbreaks are rarely present at the
same time as
RSV . Primary RSV infection occurs in the very young and virtually all
children
have been infected before they have entered school. By 1 year of age, 2_5-50%
of
infants have specific antibodies as a result of natural infection and this is
close to
100% by age 4-5. Age, sex, socioeconomic and environmental factors can all
influence the severity of disease. With current intensive care in the U.S.,
overall
mortality for normal subjects is low (less than 2% of hospitalized subjects)
but can
be much higher in infants with underlying cardiac condition (cyanotic
congenital
heart disease) or respiratory disease (bronchopulmonary dysplasia) where the
progression of symptoms may be rapid. For instance, mortality in infants with
cyanotic congenital heart disease has been reported to be as high as 37%. In
premature infants apneic spells due to RSV infection may occur and, in rare
cases,
cause neurologic or systemic damage. Severe lower respiratory tract illness
(bronchiolitis and pneumonia) is most common in patients under six months of
age.
Infants who have apparently recovered completely from this illness may display
symptomatic respiratory abnormalities for years (recurrent wheezing, decreased
pulmonary function, recurrent cough, asthma, and bronchitis).
The mechanisms by which the immune system protects against RSV
infection and reinfection are not well understood. It is clear, however, that
immunity
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is only partially protective since reinfection is common at all ages, and
sometimes
occurs in infants only weeks after recovery from a primary infection. Both
serum and
secretory antibodies (IgA) have been detected in response to RSV infection in
adults
as well as in very young infants. However, the titers of serum antibodies to
the viral
F or G glycoprotein, as well as of neutralizing antibodies found in infants (
1-8
months of age) are 15-25% of those found in older subjects. These reduced
titers
may contribute to the increased incidence of serious infection in younger
children.
Evidence for the role of serum antibodies in protection against RSV virus has
emerged from epidemiological as well as animal studies. In adults exposed
naturally
to the virus, susceptibility correlated well with low serum antibody level. In
infants,
titers of maternally transmitted antibodies correlate with resistance to
serious disease
(Glezen, W.P. et al., J. Pediatr. 98:708-715 ( 1981 )). Other studies show
that the
incidence and severity of lower respiratory tract involvement is diminished in
the
presence of high serum antibody ( Mclntosh, K. et al., J. Infect. Dis. 138:24-
32
( 1978)) and high titers of passively administered serum neutralizing
antibodies have
been shown to be protective in a cotton rat model, of RS V infection (Prince,
G. A. et
al., Virus Res. 3:193-206 (1985)).
Children lacking cell-mediated immunity are unable to cease their infection
and shed virus for many months in contrast to children with normal immune
systems. Similarly, nude mice infected with RSV virus persistently shed virus.
These mice can be cured by adoptive transfer of primed T cells (Cannon, M. J.
et al.,
Immunolo~y 62:133-138 (1987)).
In summary, it appears that both cellular and humoral immunity are involved
in protection against infection, reinfection and RSV disease and that although
antigenic variation is limited, protective immunity is not complete even after
multiple exposures.
This invention relates to the use of human mABs specific for the F protein of
RSV virus to passively treat or prevent infection. The use of passive antibody
therapy in humans is well documented and is being used to treat other
infectious
diseases such as hepatitis and cytomegalovirus. Clinical trials are also on-
going to
evaluate the efficacy of humanized antibodies for treatment of RSV infection
in
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young children. Studies in animals have clearly demonstrated that polyclonal
and
monoclonal antibody against both F and G glycoprotein can confer passive
protection in RSV virus infection when given prophylactically or
therapeutically
(Prince, et al., s- upra). In these studies, passive transfer of neutralizing
F or G mAbs
to mice, cotton rats or monkeys, significantly reduce or completely prevent
replication of the RSV virus in the lungs.
The induction of neutralizing antibodies to RSV virus appears to be limited
to the F and G surface glycoproteins. Of these two proteins, the F protein is
the
major target for cross-reactive neutralizing antibodies associated with
protection
against different strains of RSV virus. In addition, experimental vaccination
of mice
or cotton rats with F protein also results in cross protection. The antigenic
relatedness of the F protein across strains and subgroups of the virus is
reflected in
its high degree of homology at the amino acid level. In contrast, in the two
subgroups and various strains of RSV, antigenic dimorphism was linked
primarily to
the G glycoprotein. The F protein has a predicted molecular weight of 68-70
kDa; a
signal peptide at its N-terminus; a membrane anchor domain at its C terminus;
and is
cleaved proteolytically in the infected cell prior to virion assembly to yield
disulfide
linked F2 and F 1. Five neutralizing epitopes have been identified within the
F
protein sequence and map to residues 205-225; 259-278; 289-299: 483-488 and
417-438. Studies to determine the frequency of sequence diversion in the F
protein
showed that the majority of the neutralizing epitopes were conserved in all of
the 23
strains of RS V virus isolated in Australia, Europe, and regions of the U.S.
over a
period of thirty years. In another study, seroresponses of forty three infants
and
young children to primary infection with subgroup A or a subgroup B strain
showed
that responses to homologous and heterologous F antigens were not
significantly
different, while the G proteins of the subgroup A and B strains were quite
unrelated.
--._ Moreover, antibody inhibition of virus-mediated cell fusion irt vitro
versus
inhibition of infection correlates best with protection in animal models and
fusion
inhibition is primarily restricted to F protein specific antibodies. Clearly,
the F
protein is the more important target for antibody therapy.
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Fully human mAbs to RSV F.~rotein remain a desirable option for the
treatment of this disease. Although some-success has been reported in
obtaining
fragments of such mAbs ( Barbas, C.F. et al., Proc. Nat'1. Acad. Sci. USA
89:10164-
10168 ( 1992);Crowe, J. E. et al., Proc. Nat'1. Acad. Sci. USA 91: 1386-1390 (
1994)
and PCT application number PCT/US93/08786, published as W094/06448, March
31, 1994)), the achievement of such results is not straight forward and novel
human
mABs as described herein, when and however obtained, are particularly useful
alone
or in combination with existing molecules to form immunotherapeutic
compositions.
This invention relates to one such group of human mAbs.
Brief Descriution of the Invention:
This invention relates to fully human monoclonal.antibodi~s and functional
fragments thereof specifically reactive with an F protein epitope of RSV and
capable
of neutralizing RSV infection.
In a related aspect, the present invention provides modifications to
neutralizing Fab fragments or F(ab')., fragments specific for the F protein of
RSV
produced by random combinatorial cloning of human antibody sequences and
isolated from a filamentous phage Fab display library.
In still another aspect, there is provided a reshaped human antibody
containing human heavy and light chain constant regions from a first human
donor
and heavy and light chain variable regions or the CDRs thereof derived from
human
neutralizing monoclonal antibodies for the F protein of RSV derived from a
second
human donor.
In yet another aspect, the present invention provides a pharmaceutical
composition which contains one (or more) altered antibodies and a
pharmaceutically
acceptable carrier.
In a further aspect, the present invention provides a method for passive
immunotherapy of RSV disease in a human by administering to said human an
effective amount of the pharmaceutical composition of the invention for the
prophylatic or therapeutic treatment of RSV infection.
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In yet another aspect, the present invention provides methods for, and
components useful in, the recombinant production of human and altered
antibodies
(e.g., engineered antibodies, CDRs, Fab or F(ab), fragments, or analogs
thereof)
which are derived from human neutralizing monoclonal antibodies (mAbs) for F
protein of RSV. These components include isolated nucleic acid sequences
encoding same, recombinant plasmids containing the nucleic acid sequences
under
the control of selected regulatory sequences which are capable of directing
the
expression thereof in host cells (preferably mammalian) transfected with the
recombinant plasmids. The production method involves culturing a transfected
host
cell line of the present invention under conditions such that the human or
altered
antibody is expressed in said cells and isolating the expressed product
therefrom.
In yet another aspect of the invention is a method to diagnose the presence of
RSV in a human which comprises contacting a sample of biological fluid with
the
human antibodies and altered antibodies of the instant invention and assaying
for the
occurrence of binding between said human antibody (or altered antibody) and
RSV.
In yet another embodiment of the invention is a pharmaceutical composition
comprising at least one dose of an immunotherapeutically effective amount of
the
monoclonal antibody of this invention in combination with at least one
additional
monoclonal antibody. Especially, when the additional monoclonal antibody is an
anti-RSV antibody distinguished from the subject antibody of by virtue of
being
reactive with a different epitope of the RSV F protein antigen.
Other aspects and advantages of the present invention are described further in
the detailed description and the preferred embodiments thereof.
Brief Descriution of the Drawings:
Figure 1 illustrates the cloning strategy used for the construction of the Hu
19A monoclonal antibody. The heavy chain V region was cloned into the PCD
derivative vector as a XhnI - Bsp 1201 fragment. The entire light chain V and
C
regions were cloned into the PCN derivative vector as a SacI - X(~aI fragment.
Details are described in the hereinbelow.
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Figure 2 provides a comparison of the heavy chain amino acid sequences of
various monoclonal antibodies of this invention. The amino acid sequences of
the
heavy chains for the A, B, C and D constructs are shown (SEQ ID NOS: 5, 6, 7
and
8, respectively). Numbering of the residues is based on the germline (GL) gene
Dp58 (SEQ ID No: 4), beginning at the mature processed amino terminus and
ending at CDR3. The "-" indicates identity to the preceding sequence (eg., C
compared to B). Sequence A has an amino acid insertion between positions 4 and
5
due to the cloning strategy utilized by Barbas et al. ( Proc. Nat'I. Acad.
Sci. (USA)
89, 10164-10168 ( 1992), PCT publication W094/06448). Bold residues correspond
to the leader region, and to CDRs 1-3. The underlined sequence in CDR2
identifies
the N-linked glycosylation site in versions A and B that was mutated in
version C.
Residues P 14 and G 15, marked with an "*" were listed as L and A,
respectively in
the published sequence (Barbas et al., supra).
Figure 3 provides a comparison of the light chain amino acid sequences of
various monoclonal antibodies of this invention. The amino acid sequences of
the
light chains for the A, B, C and D constructs are shown (SEQ ID NOS: 10, 11,
12
and 13). Numbering of the residues in the VK region is based on the germline
(GL)
gene Dpk9 (SEQ ID NO: 9), beginning at the mature processed amino terminus and
ending at CDR3; but for reference to framework 4, the actual numbering is also
shown for Hu 19ALc. As in Fig. 2, the "-" indicates identity to the preceding
sequence. The G at position 97 in framework 4 of Hu 19A, marked with an "*",
was
listed as E in the published sequence (see text). Sequence A has a two amino
acid
deletion at residues 1 and 2 due to the cloning strategy. Bold residues
correspond to
the leader region, and to CDRs 1-3. The tc constant region is shown for
constructs A
and B in comparison to the germline gene. The L mutation near the C-terminus
was
corrected in version C (See; Figure 3, SEQ ID N0:13).
Figure 4 illustrates the DNA sequences of plasmids for the expression of the
Hu 19 mAB heavy and light chains. Figure 4A is the DNA sequence of
Hul9AHcpcd (SEQ ID N0:14). The start of translation, leader peptide, amino-
terminal processing site (SEQ ID NO:15 ), carboxy terminus of the 19A heavy
chain
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(SEQ ID NO: 16} and Eco RI restriction endonuclease cleavage site are shown.
Figure 4B is the DNA sequence of Hul9ALcpcn (SEQ ID NO: 17}, and shows the
corresponding features for the light chain and the Xba I restriction site
following the
end of the coding region for the light chain (SEQ ID NO'S: 18, 19). Figure 4C
is the
DNA sequence of the coding region of the heavy chain of plasmid Hu l9BHcpcd
(SEQ ID NO'S 20,21 }. Figure 4D is the DNA sequence of the coding region for
the
light chain of plasmid Hu l9BLcpcn (SEQ ID N0:22,23 & 24). Figure 4E is the
DNA sequence of the coding region of the heavy chain of the plasmid Hu
l9CHcpcd
(SEQ ID NO'S 25,26}. Figure 4F is the DNA sequence of the coding sequence of
the
heavy chain of plasmid Hu l9DHcpcd (SEQ ID NO:'S 27,28). Figure 4G is the DNA
sequence of the coding region of the light chain of plasmid Hu 19CLcpcn (SEQ
ID
NO'S: 29, 30). In Figures 4C-G, bolded residues indicate_differ_eno~s from the
full
vector sequences for Hu l9AHcpcd and Hu l9ALc shown in Figures 4A and 4B,
respectively. _
Figure 5 illustrates a Coomassie stained SDS-PAGE gel of Hul9B and
Hu 19C under reducing conditions.
Figure 6 illustrates the separation of Hu 19 Glycovarients by anion exchange
chromatography.
Figure 7 illustrates SDS-PAGE analysis of Hu 19B Fab glycovarients.
Detailed Description of the Invention:
This invention provides useful human monoclonal antibodies (and fragments
thereof) reactive with the F protein of RSV, isolated nucleic acids encoding
same
and various means for their recombinant production as well as therapeutic,
prophylactic and diagnostic uses of such antibodies and fragments thereof.
1. Defii2itions.
As used in this specification and the claims, the following terms are defined
as follows:
"Altered antibody" refers to a protein encoded by an altered immunoglobulin
coding region, which may be obtained by expression in a selected host cell.
Such
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altered antibodies are engineered antibodies (e.g., chimeric, humanized, or
reshaped
or immunologically edited human antibodies) or fragments thereof lacking all
or part
of an immunoglobulin constant region, e.g., Fv, Fab, or F(ab')~ and the like.
"Altered immunoglobulin coding region" refers to a nucleic acid sequence
encoding an altered antibody of the invention or a fragment thereof.
"Reshaped human antibody" refers to an altered antibody in which minimally
at least one CDR from a first human monoclonal donor antibody is substituted
for a
CDR in a second human acceptor antibody. Preferrably all six CDRs are
replaced.
More preferrably an entire antigen combining region (e.g., Fv, Fab or F(ab')2
) from
a first human donor monoclonal antibody is substituted for the corresponding
region
in a second human acceptor monoclonal antibody. Most preferrably the Fab
region
from a first human donor is operatively linked to the appropriate constant
regions of
a second human acceptor antibody to form a full length monoclonal antibody.
The
reshaped human monoclonal antibodies designated herein as Hu 19A, Hu 19B,
Hu 19C and Hu 19D are defined as reshaped human antibodies comprising a light
chain amino acid sequence selected from Sequences 19A, 19B, 19C and 19D of
Figure 3 and a heavy chain amino acid sequence selected from Sequences 19A,
19B,
19C and 19D of Figure 2, or functional partial sequences thereof.
"First immunoglobulin partner" refers to a nucleic acid sequence encoding a
human framework or human immunoglobulin variable region in which the native
(or
naturally-occurring) CDR-encoding regions are replaced by the CDR-encoding
regions of a donor human antibody. The human variable region can be an
immunoglobulin heavy chain, a light chain (or both chains), an analog or
functional
fragments thereof. Such CDR regions, located within the variable region of
antibodies (immunoglobulins) can be determined by known methods in the art.
For
example Kabat et al. (Sequences of Proteins of Immunolo~ical Interest, 4th
Ed., U.S.
Department of Health and Human Services, National Institutes of Health ( I
987))
disclose rules for locating CDRs. In addition, computer programs are known
which
are useful for identifying CDR regions/structures.
"Second fusion partner" refers to another nucleotide sequence encoding a
protein or peptide to which the first immunoglobulin partner is fused in frame
or by
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means of an optional conventional linker sequence (i.e., operatively linked).
Preferably the fusion partner is an immunoglobulin gene and when so, it is
referred
to as a "second immunoglobulin partner". The second immunoglobulin partner may
include a nucleic acid sequence encoding the entire constant region for the
same
(i.e., homologous - the first and second altered antibodies are derived from
the same
source) or an additional (i.e., heterologous) antibody of interest. It may be
an
immunoglobulin heavy chain or light chain (or both chains as part of a single
polypeptide). The second immunoglobulin partner is not limited to a particular
immunoglobulin class or isotype. In addition, the second immunoglobulin
partner
may comprise part of an immunoglobulin constant region, such as found in a
Fab, or
F(ab), (i.e., a discrete part of an appropriate human constant region or
framework
region). A second fusion partner may also comprise a sequence encoding an
integral
membrane protein exposed on the outer surface of a host cell, e.g., as part of
a phage
display library, or a sequence encoding a protein for analytical or diagnostic
detection, e.g., horseradish peroxidase, (3-galactosidase) etc.
The terms Fv) Fc, Fd, Fab, or F(ab'), are used with their standard meanings
(see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor
Laboratory, ( 1988)).
As used herein, an "engineered antibody" describes a type of altered
antibody, i.e., a full-length synthetic antibody (e.g., a chimeric, humanized,
reshaped
or immunologically edited human antibody as opposed to an antibody fragment)
in
which a portion of the light and/or heavy chain variable domains of a selected
acceptor antibody are replaced by analogous parts from one or more donor
antibodies which have specificity for the selected epitope. For example, such
molecules may include antibodies characterized by a humanized heavy chain
associated with an unmodified light chain (or chimeric light chain), or vice
versa.
Engineered antibodies may also be characterized by,alteration of the nucleic
acid
sequences encoding the acceptor antibody light and/or heavy variable domain
framework regions in order to retain donor antibody binding specificity. These
30-----antibodies can comprise replacement of one or more CDRs (preferably
all) from the
acceptor antibody with CDRs from a donor antibody described herein.
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A "chimeric antibody" refers to a type of engineered antibody which contains
naturally-occurring variable region (light chain and heavy chains) derived
from a
donor antibody in association with light and heavy chain constant regions
derived
from an acceptor antibody from a heterologous species.
A "humanized antibody" refers to a type of engineered antibody having its
CDRs derived from a non-human donor immunoglobulin, the remaining _..
immunoglobulin-derived parts of the molecule being derived from one (or more)
human immunoglobulin(s). In addition, framework support residues may be
altered
to preserve binding affinity (see, e.g.) Queen et al., Proc. Nat'1. Acad. Sci.
LJSA.
86:10029-10032 ( 1989), Hodgson et al., Bio/Technolo~y, 9:421 ( 1991 )).
An "immunologically edited antibody" refers to a type of engineered
antibody in which changes are made in donor and/or acceptor sequences to edit
regions in respect of cloning artifacts, germ line enhancements, etc. aimed at
reducing the likelihood of an immunological response to the antibody on the
pan of
a patient being treated with the edited antibody.
The term "donor antibody" refers to an antibody (monoclonal, or
recombinant) which contributes the nucleic acid sequences of its variable
regions,
CDRs, or other functional fragments or analogs thereof to a first
immunoglobulin
partner, so as to provide the altered immunoglobulin coding region and
resulting
expressed altered antibody with the antigenic specificity and neutralizing
activity
characteristic of the donor antibody. One donor antibody suitable for use in
this
invention is a Fab fragment of a human neutralizing monoclonal antibody
designated
as Fab Hu 19. Fab Hu 19 is defined as a having the variable light chain DNA
and
amino acid sequences Hu 19A as shown in Figures 2, 3, 4A and 4B.
The term "acceptor antibody" refers to an antibody (monoclonal, or
recombinant) from a source genetically unrelated to the donor antibody, which
contributes all (or any portion, but preferably all) of the nucleic acid
sequences
encoding its heavy and/or light chain framework regions and/or its heavy
and/or light
chain constant regions to the first immunoglobulin partner. Preferably a human
antibody is the acceptor antibody.
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"CDRs" are defined as the complementarity determining region amino acid
sequences of an antibody which are the hypervariable regions of immunoglobulin
heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of
Immunological Interest, 4th Ed., U.S. Department of Health and Human Services,
National Institutes of Health ( 1987). There are three heavy chain and three
light
chain CDRs (or CDR regions) in the variable portion of an immunoglobulin.
Thus,
"CDRs" as used herein refers to all three heavy chain CDRs, or all three light
chain
CDRs (or both all heavy and all light chain CDRs, if appropriate). CDRs
provide
the majority of contact residues for the binding of the antibody to the
antigen or
epitope. CDRs of interest in this invention are derived from donor antibody
variable
heavy and light chain sequences, and include analogs of the naturally
occurring
CDRs, which analogs also share or retain the same antigen binding specificity
and/or
neutralizing ability as the donor antibody from which they were derived.
By "sharing the antigen binding specificity or neutralizing ability" is meant,
for example, that although Fab Hu 19 may be characterized by a certain level
of
antigen affinity, a CDR encoded by a nucleic acid sequence of Fab Hu 19 in an
appropriate structural environment may have a lower, or higher affinity. It is
expected that CDRs of Fab Hu I 9 in such environments will nevertheless
recognize
the same epitope(s) as does the intact Fab Hu 19. A "functional fragment" is a
partial
heavy or light chain variable sequence (e.g., minor deletions at the amino or
carboxy
terminus of the immunoglobulin variable region) which retains the same antigen
binding specificity and/or neutralizing ability as the antibody from which the
fragment was derived.
An "analog" is an amino acid sequence modified by at least one amino acid,
wherein said modification can be chemical or a substitution or a rearrangement
of a
few amino acids (i.e., no more than 10), which modification permits the amino
acid
sequence to retain the biological characteristics, e.g., antigen specificity
and high
affinity, of the unmodified sequence. For example, (silent) mutations can be
constructed, via substitutions, when certain endonuclease restriction sites
are created
within or surrounding CDR-encoding regions.
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Analogs may also arise as allelic variations. An "allelic variation or
modification" is an alteration in the nucleic acid sequence encoding the amino
acid
or peptide sequences of the invention. Such variations or modifications may be
due
to degeneracy in the genetic code or may be deliberately engineered to provide
desired characteristics. These variations or modifications may or may not
result in
alterations in any encoded amino acid sequence.
The term "effcctor agents" refers to non-protein carrier molecules to which
the altered antibodies, and/or natural or synthetic light or heavy chains of
the donor
antibody or other fragments of the donor antibody may be associated by
conventional means. Such non-protein carriers can include conventional
carriers
used in the diagnostic field, e.g., polystyrene or other plastic beads,
polysaccharides,
e.g., as used in the BIAcore (Pharmacia) system, or other non-protein
substances
useful in the medical field and safe for administration to humans and animals.
Other
effector agents may include a macrocycle, for chelating a heavy metal atom, or
radioisotopes. Such effector agents may also be useful to increase the half-
life of the
altered antibodies, e.g., polyethylene glycol.
Il. Combinatorial Cloning:
As mentioned above, a number of problems have hampered the direct
application of the hybridoma technology of G. Kohler and C. Milstein (Nature
256:
495-497 ( 1975)) to the generation and isolation of human monoclonal
antibodies.
Among these are a lack of suitable fusion partner myeloma cell lines used to
form
hybridoma cell lines as well as the poor stability of such hybridomas even
when
formed. These shortcomings are further exacerbated in the case of RSV because
of
the paucity of viral specific B cells in the perpherial circulation.
Therefore, the
molecular biological approach of combinatorial cloning is preferred.
---- Combinatorial cloning is disclosed generally in PCT Publication No.
W090/ 14430. Simply stated, the goal of combinatorial cloning is to transfer
to a
population of bacterial cells the immunological genetic capacity of a human
cell,
tissue or organ. It is preferred to employ cells, tissues or organs which are
immunocompetent. Particularly useful sources include, without limitation,
spleen,
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thymus, lymph nodes, bone marrow, tonsil and perpherial blood lymphocytes. The
cells may be optionally RS V stimulated in vitro, or selected from donors
which are
known to have produced an immune response or donors who are HIV+ but
asymptomatic.
The genetic information isolated from the donor cells can be in the form of
DNA or RNA and is conveniently amplified by Polymerase Chain Reaction (PCR)
or similar techniques. When isolated as RNA the genetic information is
preferably
converted into cDNA by reverse transcription prior to amplification. The
amplification can be generalized or more specifically tailored. For example,
by a
careful selection of PCR primer sequences, selective amplification of
immunoglobulin genes or subsets within that class of genes can be achieved.
Once the component gene sequences are obtained, in this case the genes
encoding the variable regions of the various heavy and light antibody chains,
the
light and heavy chain genes are associated in random combinations to form a
random combinatorial library. Various recombinant DNA vector systems have been
described to facilitate combinatorial cloning (see: PCT Publication No.
W090/ 14430 s-upra, Scott and Smith, Science 249:386-406 ( 1990) or U. S.
Patent
5,223,409). Having generated the combinatorial library, the products can,
after
expression, be conveniently screened by biopanning with RSV F protein or, if
necessary, by epitope blocked biopanning as described in more detail below.
Initially it is generally preferred to use Fab fragments of mAbs for
combinatorial cloning and screening and then to convert the Fabs to full
length
mAbs after selection of the desired candidate molecules. However, single chain
antibodies can also be used for cloning and screening.
lll. Antibody Fragments
The present invention contemplates the use of Fab fragments or F(ab')
fragments to derive full-length mAbs directed against the F protein of RSV.
Although these fragments may be independently useful as protective and
therapeutic
agents ift vivo against RSV-mediated conditions or in vitro as part of an RSV
diagnostic, they are employed herein as a component of a reshaped human
antibody.
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A Fab fragment contains the entire light chain and amino terminal portion of
the
heavy chain; and an F(ab'), fragment is the fragment formed by two Fab
fragments
bound by additional disulfide bonds. RS V binding monoclonal antibodies
provide
sources of Fab fragments and F(ab')~ fragments and can be obtained via
combinatorial phage library (see, e.g., Winter et al., Ann. Rev. Immunol.,
12:433-
455 ( 1994) or Barbas et al. ( Proc. Nat'1. Acad. Sci-. (USA) 89, 10164-10168
( 1992))
which are both hereby incorporated by reference in their entirety).
IV. Anti-RSV Antibody Amino Acid and Nucleotide Sequences of Interest
The Fab Hu 19 or other antibodies described herein may contribute
sequences, such as variable heavy and/or light chain peptide sequences,
framework
sequences, CDR sequences, functional fragments, and analogs thereof, and the
nucleic acid sequences encoding them, useful in designing and obtaining
various
altered antibodies which are characterized by the antigen binding specificity
of the
donor antibody.
As one example, the present invention thus provides variable light chain and
variable heavy chain sequences from the RS V human Fab Hu 19A-D and sequences
derived therefrom.
The nucleic acid sequences of this invention, or fragments thereof, encoding
the variable light chain and heavy chain peptide sequences are also useful for
mutagenic introduction of specific changes within the nucleic acid sequences
encoding the CDRs or framework regions, and for incorporation of the resulting
modified or fusion nucleic acid sequence into a plasmid for expression. For
example, silent substitutions in the nucleotide sequence of the framework and
CDR-
encoding regions can be used to create restriction enzyme sites which would
facilitate insertion of mutagenized CDR (and/or framework) regions. These CDR-
encoding regions may be used in the construction of reshaped human antibodies
of
this invention.
Taking into account the degeneracy of the genetic code, various coding
sequences may be constructed which encode the variable heavy and light chain
amino acid sequences, and CDR sequences of the invention as well as functional
17
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WO 98/19704 PCT/US97/19203
fragments and analogs thereof which share the antigen specificity of the donor
antibody. The isolated nucleic acid sequences of this invention, or fragments
thereof, encoding the variable chain peptide sequences or CDRs can be used to
produce altered antibodies, e.g., chimeric or humanized antibodies, or other
engineered antibodies of this invention when operatively combined with a
second
immunoglobulin partner.
It should be noted that in addition to isolated nucleic acid sequences
encoding portions of the altered antibody and antibodies described herein,
other such
nucleic acid sequences are encompassed by the present invention, such as those
complementary to the native CDR-encoding sequences or complementary to the
modified human framework regions surrounding the CDR-encoding regions. Such
sequences include all nucleic acid sequences which by virtue of the redundancy
of
the genetic code are capable of encoding the same amino acid sequence as given
in
Figures 2 and 3. Other useful DNA sequences encompassed by this invention
include those sequences which hybridize under stringent hybridization
conditions
(See: T. Maniatis et al., Molecular Cloning (A Laboratory Manual), Cold Spring
Harbor Laboratory ( 1982), pages 387 to 389) to the DNA sequences encoding the
antibodies of Figures 2 and 3 and which retain the antigen binding properties
of
those antibodies. An example of one such stringent hybridization condition is
hybridization at 4XSSC at 65°C, followed by a washing in O.1XSSC at
65°C for an
hour. Alternatively an exemplary stringent hybridization condition is in 50%
formamide, 4XSSC at 42°C. Preferably, these hybridizing DNA sequences
are at
least about 18 nucleotides in length, i.e., about the size of a CDR.
V. Altered ImnaunnRlobulin Coding Regions and Altered Antibodies
Altered immunoglobulin coding regions encode altered antibodies which
include engineered antibodies such as chimcric antibodies, humanized) reshaped
and
immunologically edited human antibodies. A desired altered immunoglobulin
coding region contains CDR-encoding regions in the form of Fab regions that
encode peptides having the antigen specificity of an RS V antibody, preferably
a high
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affinity antibody such as provided by the present invention, inserted into an
acceptor
immunoglobulin partner. --
When the acceptor is an immunoglobulin partner, as defined above, it
includes a sequence encoding a second antibody region of interest, for example
an
Fc region. Immunoglobulin partners may also include sequences encoding another
immunoglobulin to which the light or heavy chain constant region is fused in
frame
or by means of a linker sequence. Engineered antibodies directed against
functional
fragments or analogs of RSV may be designed to elicit enhanced binding with
the
same antibody.
The immunoglobulin partner may also be associated with effector agents as
defined above, including non-protein carrier molecules, to which the
immunoglobulin partner may be operatively linked by conventional means.
Fusion or linkage between the immunoglobulin partners, e.g., antibody
sequences, and the effector agent may be by any suitable means, e.g., by
conventional covalent or ionic bonds, protein fusions, or hetero-bifunctional
cross-
linkers, e.g., carbodiimide, glutaraldehyde, and the like. Such techniques are
known
in the art and readily described in conventional chemistry and biochemistry
texts.
Additionally, conventional linker sequences which simply provide for a
desired amount of space between the second immunoglobulin partner and the
effector agent may also be constructed into the altered immunoglobulin coding
region. The design of such linkers is well known to those of skill in the art.
In addition, signal sequences for the molecules of the invention may be
modified to enhance expression. For example the reshaped human antibody having
the signal sequence and CDRs derived from the Fab Hu 19 heavy chain sequence,
may have the original signal peptide replaced with another signal sequence
such as
the Campath leader sequence {Page, M. J. et al., BioTechnolo~y 9:64-68{ 1991
)).
An exemplary altered antibody, a reshaped human antibody, contains a
variable heavy and the entire light chain peptide or protein sequence having
the
antigen specificity of Fab Hu 19, fused to the constant heavy regions CI-1_ 1-
CH-3
derived from a second human antibody.
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In still a further embodiment, the engineered antibody of the invention may
have attached to it an additional agent. For example, the procedure of
recombinant
DNA technology may be used to produce an engineered antibody of the invention
in
which the Fc fragment or CH2 CH3 domain of a complete antibody molecule has
been replaced by an enzyme or other detectable molecule (i.e., a polypeptide
effector
or reporter molecule).
Another desirable protein of this invention may comprise a complete
antibody molecule, having full length heavy and light chains, or any discrete
fragment thereof, such as the Fab or F(ab')~ fragments, a heavy chain dimes,
or any
minimal recombinant fragments thereof such as an F~, or a single-chain
antibody
(SCA) or any other molecule with the same specificity as the selected donor
Fab
Hu 19. Such protein may be used in the form of an altered antibody, or may be
used
in its unfused form.
Whenever the immunoglobulin partner is derived from an antibody different
from the donor antibody, e.g., any isotype or class of immunoglobulin
framework or
constant regions, an engineered antibody results. Engineered antibodies can
comprise immunoglobulin (Ig) constant regions and variable framework regions
from one source, e.g., the acceptor antibody, and one or more (preferably all)
CDRs
from the donor antibody, e.g., the anti-RSV antibody described herein. In
addition,
alterations, e.g., deletions, substitutions, or additions, of the acceptor mAb
light
and/or heavy variable domain framework region at the nucleic acid or amino
acid
levels, or the donor CDR regions may be made in order to retain donor antibody
antigen binding specificity or to reduce potential immunogenicity. In the
present
invention, a preferred mutation is the alteration of the consensus N-linked
glycosylation site in CDR2 of the Hu 19A and Hu 19B heavy chain, as
exemplified in
the heavy chains of Hul9C and Hul9D (Fig. 2) (SEQ ID NO'S 7 and 8).
Such engineered antibodies arc designed to employ one (or both) of the
variable heavy and/or light chains of the RSV mAb (optionally modified as
described) or one or more of the below-identified heavy or light chain CDRs.
The
engineered antibodies of the invention are neutralizing, i.e., they desirably
inhibit
virus growth in vitro and in vivo in animal models of RSV infection.
CA 02270288 1999-04-30
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Such engineered antibodies may include a reshaped human antibody
containing the human heavy and light chain constant regions fused to the RS V
antibody functional fragments. A suitable human (or other animal) acceptor __
antibody may be one selected from a conventional database, e.g., the KABAT't
database, Los Alamos database, and Swiss Protein database, by homology to the
nucleotide and amino acid sequences of the donor antibody. A human antibody
characterized by a homology to the framework regions of the donor antibody (on
an
amino acid basis) may be suitable to provide a heavy chain constant region
and/or a
heavy chain variable framework region for insertion of the donor CDRs. A
suitable
acceptor antibody capable of donating light chain constant or variable
framework
regions may be selected in a similar manner. It should be noted that the
acceptor
antibody heavy and light chains are not required to originate from the same
acceptor
antibody.
Desirably the heterologous framework and constant regions are selected from
human immunoglobulin classes and isotypes, such as IgG (subtypes 1 through 4),
IgM, IgA and IgE. The Fc domains are not limited to native sequences, but
include
mutant variants known in the art that alter function. For example, mutations
have
been described in the Fc domains of certain IgG antibodies that reduce Fc-
mediated
complement and Fc receptor binding (see, e.g., A. R. Duncan et al., Nature
332:563-
564 ( 1988); A. R. Duncan and G. Winter, Nature 332:738-740 ( 1988); M.-L.
Alegre
et al., J. Immunol. 148:3461-3468 (1992); M.-H. Tao et al., J. Exp. Med.
178:661-
667 ( 1993); V. Xu et al. J. Biol. Chem., 269:3469-2374 ( 1994)), alter
clearance rate
(J.-K. Kim et al., Eur. J. Immunol. 24:542-548 ( 1994), and reduce structural
heterogeneity (S. Angal et al., Mol. Immunol. 30:105-108 (1993)). Also, other
modifications are possible such as oligomerization of the antibody by addition
of the
tailpiece segment of IgM and other mutations (R. I. F. Smith and S. L.
Morrison,
Biotechnolo~y 12:683-688 ( 1994); R. I. F. Smith et al., J. lmmunol., 154:
2226-2236
( 1995)) or addition of the tailpiece segment of IgA (l. Kariv et al., J.
Immunol. 157:
29-38 ( 1996). However, the acceptor antibody need not comprise only human
immunoglobulin protein sequences. For instance a gene may be constructed in
which a DNA sequence encoding part of a human immunoglobulin chain is fused to
21
CA 02270288 1999-04-30
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a DNA sequence encoding a non-immunoglobulin amino acid sequence such as a
polypeptide effector or reporter molecule.-
The altered antibody thus preferably has the structure of a natural human
antibody or a fragment thereof, and possesses the combination of properties
required
for effective therapeutic use, e.g., treatment of RSV mediated diseases in
man, or for
diagnostic uses.
It will be understood by those skilled in the art that an altered antibody may
be further modified by changes in variable domain amino acids without
necessarily
affecting the specificity and high affinity of the donor antibody (i.e., an
analog). It is
anticipated that heavy and light chain amino acids may be substituted by other
amino
acids either in the variable domain frameworks or CDRs or both. Particularly
preferred is the immunological editing of such reconstructed sequences as
illustrated
in the examples herein.
In addition, the variable or constant region may be altered to enhance or
decrease selective properties of the molecules of the instant invention, as
described
above. For example, dimerization, binding to Fc receptors, or the ability to
bind and
activate complement (see, e.g., Angal et al., Mol. Immunol, 30:105-108 (1993),
Xu
et al., J. Biol. Chem, 269:3469-3474 ( 1994), Winter et al., EP 307,434-B).
Such antibodies are useful in the prevention and treatment of RS V mediated
disorders, as discussed below.
Vl. Prodacction of Altered antibodies and Engineered Antibodies
The resulting reshaped human antibodies of this invention can be expressed
in recombinant host cells, e.g., COS, CHO or myeloma cells. A conventional
expression vector or recombinant plasmid is produced by placing these coding
sequences for the altered antibody in operative association with conventional
regulatory control sequences capable of controlling the replication and
expression in,
and/or secretion from, a host cell. Regulatory sequences include promoter
sequences, e.g., CMV promoter, and signal sequences, which can be derived from
other known antibodies. Similarly, a second expression vector can be produced
having a DNA sequence which encodes a complementary antibody light or heavy
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chain. Preferably this second expression vector is identical to the first
except insofar
as the coding sequences and selectable markers are concerned, so to ensure as
far as
possible that each polypeptide chain is functionally expressed. Alternatively,
the
heavy and light chain coding sequences for the altered antibody may reside on
a
single vector.
A selected host cell is co-transfected by conventional techniques with both
the first and second vectors (or simply transfected by a single vector) to
create the
transfected host cell of the invention comprising both the recombinant or
synthetic
light and heavy chains. The transfected cell is then cultured by conventional
techniques to produce the engineered antibody of the invention. The production
of
the antibody which includes the association of both the recombinant heavy
chain and
light chain is measured in the culture by an appropriate assay, such as ELISA
or
RIA. Similar conventional techniques may be employed to construct other
altered
antibodies and molecules of this invention.
Suitable vectors for the cloning and subcloning steps employed in the
methods and construction of the compositions of this invention may be selected
by
one of skill in the art. For example, the conventional pUC series of cloning
vectors,
may be used. One vector used is pUC 19, which is commercially available from
supply houses, such as Amersham (Buckinghamshire, United Kingdom) or
Pharmacia (Uppsala, Sweden). Additionally, any vector which iS capable of
replicating readily, has an abundance of cloning sites and selectable genes
(e.g.,
antibiotic resistance), and is easily manipulated may be used for cloning.
Thus, the
selection of the cloning vector is not a limiting factor in this invention.
Similarly, the vectors employed for expression of the engineered antibodies
according to this invention may be selected by one of skill in the art from
any
conventional vectors. Preferred vectors include for example plasmids pCD or
pCN.
The vectors also contain selected regulatory sequences (such as CMV promoters)
which direct the replication and expression of heterologous DNA sequences in
selected host cells. These vectors contain the above described DNA sequences
-....._ vsrhich code for the engineered antibody or altered immunoglobulin
coding region.
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In addition, the vectors may incorporate the selected immunoglobulin sequences
modified by the insertion of desirable restriction sites for ready
manipulation.
The expression vectors may also be characterized by genes suitable for .
amplifying expression of the heterologous DNA sequences, e.g., the mammalian
dihydrofolate reductase gene (DHFR). Other preferable vector sequences include
a
poly A signal sequence, such as from bovine growth hormone (BGH) and the
betaglobin promoter sequence (betaglopro). The expression vectors useful
herein
may be synthesized by techniques well known to those skilled in this art.
The components of such vectors, e.g. replicons, selection genes, enhancers,
promoters, signal sequences and the like, may be obtained from commercial or
natural sources or synthesized by known procedures for use in directing the
expression and/or secretion of the product of the recombinant DNA in a
selected
host. Other appropriate expression vectors of which numerous types are known
in
the art for mammalian, bacterial, insect, yeast, and fungal expression may
also be
selected for this purpose.
The present invention also encompasses a cell line transfected with a
recombinant plasmid containing the coding sequences of the engineered
antibodies
or altered immunoglobulin molecules thereof. Host cells useful for the cloning
and
other manipulations of these cloning vectors are also conventional. However,
most
desirably, cells from various strains of E. cnli are used for replication of
the cloning
vectors and other steps in the construction of altered antibodies of this
invention.
Suitable host cells or cell lines for the expression of the engineered
antibody
or altered antibody of the invention are preferably mammalian cells such as
CHO,
COS, a fibroblast cell (e.g., 3T3), and myeloid cells, and more preferably a
CHO or a
myeloid cell. Human cells may be used, thus enabling the molecule to be
modified
with human glycosylation patterns. Alternatively, other eukaryotic cell lines
may be
employed. The selection of suitable mammalian host cells and methods for
transformation) culture) amplification, screening and product production and
purification are known in the art. See, e.g., Sambrook et al., cited above.
Bacterial cells may prove useful as host cells suitable for the expression of
the recombinant Fabs of the present invention (see, e.g., Pliickthun, A.,
Immunol.
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CA 02270288 1999-04-30
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Rev., 130:151-188 (1992)). The tendency of proteins expressed in bacterial
cells to
be in an unfolded or improperly folded form or in a non-glycosylated form does
not
pose as great a concern as Fabs are not normally glycosylated and can be
engineered
for exported expression thereby reducing the high concentration that
facilitates
misfolding. Nevertheless, any recombinant Fab produced in a bacterial cell
would
have to be screened for retention of antigen binding ability. If the molecule
expressed by the bacterial cell was produced and exported in a properly folded
form,
that bacterial cell would be a desirable host. For example, various strains of
E. coli
used for expression are well-known as host cells in the field of
biotechnology.
Various strains of B. subtihs, Streptomyces, other bacilli and the like may
also be
employed in this method.
Where desired, strains of yeast cells known to those skilled in the art are
also
available as host cells, as well as insect cells, e.g. Drosophila and
Lepidoptera and
viral expression systems. See, e.g. Miller et al., Genetic En~ineerina, 8:277-
298,
Plenum Press ( 1986) and references cited therein.
The general methods by which the vectors of the invention may be
constructed, the transfection methods required to produce the host cells of
the
invention, and culture methods necessary to produce the altered antibody of
the
invention from such host cell are all conventional techniques. Likewise, once
produced, the altered antibodies of the invention may be purified from the
cell
culture contents according to standard procedures of the art, including
ammonium
sulfate precipitation, affinity columns, column chromatography, gel
electrophoresis
and the like. Such techniques are within the skill of the art and do not limit
this
invention.
Yet another method of expression of resphaped antibodies may utilize
expression in a transgenic animal, such as described in U. S. Patent No.
4,873,316.
This relates to an expression system using the animal's casein promoter which
when
transgenically incorporated into a mammal permits the female to produce the
desired
recombinant protein in its milk.
Once expressed by the desired method, the engineered antibody is then
examined for in vitro activity by use of an appropriate assay. Presently
conventional
CA 02270288 1999-04-30
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ELISA assay formats are employed to assess qualitative and quantitative
binding of
the altered antibody to RSV. Additionally, other in vitro assays and in vivo
animal
models may also be used to verify neutralizing efficacy prior to subsequent
human
clinical studies performed to evaluate the persistence of the altered antibody
in the
body despite the usual clearance mechanisms.
Vll. TherapeuticlProphytactic Uses
This invention also relates to a method of treating humans experiencing
RSV-related symptoms which comprises administering an effective dose of
antibodies including one or more of the altered antibodies described herein or
fragments thereof.
The therapeutic response induced by the use of the molecules of this
invention is produced by the binding to RSV and thus subsequently blocking RSV
propagation. Thus, the molecules of the present invention, when in
preparations and
formulations appropriate for therapeutic use, are highly desirable for those
persons
experiencing RSV infection. For example, longer treatments may be desirable
when
treating seasonal episodes or the like. The dose and duration of treatment
relates to
the relative duration of the molecules of the present invention in the human
circulation, and can be adjusted by one of skill in the art depending upon the
condition being treated and the general health of the patient.
The altered antibodies, antibodies and fragments thereof of this invention
may also be used alone or in conjunction with other antibodies, particularly
human
or humanized tnAbs reactive with other epitopes on the F protein or other RS V
target antigens as prophylatic agents.
The mode of administration of the therapeutic and prophylatic agents of the
invention may be any suitable route which delivers the agent to the host. The
altered
antibodies, antibodies, engineered antibodies, and fragments thereof, and
pharmaceutical compositions of the invention are particularly useful for
parenteral
administration, i.e., subcutaneously, intramuscularly, intravenously, or
intranasally.
30------ Therapeutic and prophylacticagents of the invention may be prepared
as
pharmaceutical compositions containing an effective amount of the altered
antibody
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WO 98/19704 PCT/iJS97119203
of the invention as an active ingredient in a pharmaceutically acceptable
carrier. An
aqueous suspension or solution containing the antibody, preferably buffered at
physiological pH, in a form ready for injection is preferred. The compositions
for
parenteral administration will commonly comprise a solution of the engineered
antibody of the invention or a cocktail thereof dissolved in an
pharmaceutically
acceptable carrier, preferably an aqueous carrier. A variety of aqueous
carriers may
be employed, e.g., 0.4% saline, 0.3% glycine, and the like. These solutions
are
- sterile and generally free of particulate matter. These solutions may be
sterilized by
conventional, well known sterilization techniques (e.g., filtration). The
compositions may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH adjusting and
buffering
agents, etc. The concentration of the antibody of the invention in such
pharmaceutical formulation can vary widely, i.e., from less than about 0.5%,
usually
at or at least about 1 % to as much as 15 or 20°~o by weight and will
be selected
primarily based on fluid volumes, viscosities, etc., according to the
particular mode
of administration selected.
Thus, a pharmaceutical composition of the invention for intramuscular
injection could be prepared to contain 1 mL sterile buffered water, and
between
about 1 ng to about 100 mg, e.g. about 50 ng to about 80 mg or more
preferably,
about 5 mg to about 75 mg of an engineered antibody of the invention.
Similarly, a
pharmaceutical composition of the invention for intravenous infusion could be
made
up to contain about 250 ml of sterile Ringer's solution, and about 1 to about
75 and
preferably 5 to about 50 mg/ml of an engineered antibody of the invention.
Actual
methods for preparing parenterally administrable compositions are well known
or
will be apparent to those skilled in the art and are described in more detail
in, for
example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing
Company,
--.. . Easton, Pennsylvania.
It is preferred that the therapeutic and prophylactic agents of the invention,
when in a pharmaceutical preparation, be present in unit dose forms. The
appropriate therapeutically effective dose can be determined readily by those
of skill
in the art. To effectively treat an inflammatory disorder in a human or other
animal,
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CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
one dose of approximately 0. I mg to approximately 20 mg per 70 kg body weight
of
a protein or an antibody of this invention should be administered
parenterally,
preferably i.v. or i.m. (intramuscularly). Such dose may, if necessary, be
repeated at
appropriate time intervals selected as appropriate by a physician.
The altered antibodies and engineered antibodies of this invention may also
be used in diagnostic regimens, such as for the determination of RSV mediated
disorders or tracking progress of treatment of such disorders. As diagnostic
reagents, these altered antibodies may be conventionally labeled for use in
ELISAs
and other conventional assay formats for the measurement of RSV levels in
serum,
plasma or other appropriate tissue, or the release by human cells in culture.
The
nature of the assay in which the altered antibodies are used are conventional
and do
not limit this disclosure.
The antibodies, altered antibodies or fragments thereof described herein can
be lyophilized for storage and reconstituted in a suitable carrier prior to
use. This
technique has been shown to be effective with conventional immunoglobulins and
art-known lyophilization and reconstitution techniques can be employed.
The following examples illustrate various aspects of this invention including
the construction of exemplary engineered antibodies and expression thereof in
suitable vectors and host cells, and are not to be construed as limiting the
scope of
this invention. All amino acids are identified by conventional three letter or
single
letter codes. All necessary restriction enzymes, plasmids, and other reagents
and
materials were obtained from commercial sources unless otherwise indicated.
All
general cloning ligation and other recombinant DNA methodology were as
performed in T. Maniatis et al., cited above, or the second edition thereof (
1989),
eds. Sambrook et al., by the same publisher ("Sambrook et al.").
Example A
Conversion of Hul9 Fab to mAb Version A: Direct Cloning
For expression in mammalian cells, the heavy chain variable region and the
light chain variable and constant regions from the Fab clone 19 plasmid(C.
Barbas
III et al., Proc. Nat'1. Acad. Sci. (USA) 89, 10164-10168 ( 1992) and PCT
application
28
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Publication No. WO 94/06448, Application No. PCT/US93/08786 Cell Line CIone
19 referenced therein as ATCC Accession No. 69072) herein designated Hu 19
Fab,
were cloned into derivatives of plasmid PCDN (Nambi, A. et.al., Molecular and
Cellular Biochemistry 131:75-86 ( 1994), in which the expression of the
antibody
chain is driven by the CMV promoter. Plasmid PCD-HC68B is used for cloning and
expressing full length heavy chains and plasmid PCN-HuLC, for cloning and
expressing full length light chains (Figure 1 shows the strategy for cloning
of version
A of the Hu 19 mAb).
In the initial constructs, the changes in the sequence at the amino terminus,
introduced by the PCR primers used for cloning, were not altered. For the
heavy
chain, the variable region was extracted from the Hu 19 Fab plasmid (C. Barbas
III et
al., Proc. Nat'1. Acad. Sci. (USA) 89, 10164-10168 (1992)) as an Xlio1-Bsp120I
fragment and introduced into the same sites in PCD-HC68B. The Xho 1 site was
introduced at the amino terminus by the PCR primer and, when cloned into PCD-
HC68B at the same site is preceded in frame by the Campath leader sequence
(Page,
J.M. et al., Biotechnology 9:64-68 ( 1991 ). The Bsp 120I site is a naturally
occurring,
highly conserved sequence at the beginning of the CH 1 domain, and when cloned
into PCD-HC68B at the same site is in frame with the remaining sequence for
the
CH 1 through CH3 regions of human IgG 1 (Figure 1 ). In the resulting
construct,
Hu 19AHcpcd, the amino acids immediately following the Campath leader are
EVQLLEE (Fig. 2 SEQ ID NO 5, AMINO ACIDS 20 - 26), where the residues LE
are encoded by the nucleotide sequence for the Xho 1 cloning site. The
complete
nucleotide sequence for the plasmid Hu l9AHcpcd is shown in Fig. 4A (SEQ ID NO
14).
Of note, sequence analysis revealed base differences from the published
sequence (C. Barbas III et al., Proc. Nat'1. Acad. Sci. (USA) 89, 10164-10168
( 1992), PCT publication W094/06448) within the heavy chain region from the Hu
19
Fab plasmid. The changes encode amino acid differences at positions 15 and 16
{ 14
and 15 according to consensus numbering of Kabat et al (Sequences of Proteins
of
Immunological Interest, fifth edition, NIH Publication No. 91-3242, 1991 ):
29
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PG in the Hu 19AHepcd vector versus LA in the published sequence (see Fig 2 of
this application and Fig. 4 of W094/06448)). This discrepancy must represent
an
error in the original published sequence. PG at these positions is the
consensus
sequence in the closest homologues among published human antibodies (Kabat et
al., Sequences of Proteins of Immunological Interest, fifth edition, NIH
Publication
No. 91-3242, 1991 ) and in the likely germline parent sequence (see below,
version
B ). In addition, sequences derived from 3 independent clonings initiated with
the
_ Hu 19 Fab plasmid all encoded PG at these positions.
For the light chain, the variable and constant regions of the Hu 19 Fab
plasmid were cloned as a ScrcllXhal fragment into the same sites in the pCN-
HuLcvector. Both restriction sites correspond to restriction sites introduced
by the
primers used in the PCR amplification. The Sac 1 site is introduced at the
amino
terminus by the PCR primer and, when cloned into pCN-HuLC at the same site, is
preceded in frame by the Campath leader sequence (Page, J.M. et al.,
Biotechnology
9:64-68 ( 1991 ). The first 2 amino acids of the mature light chain are
therefore
deleted. In the resulting construct, Hu 19ALcpcn, the first 2 amino acids
immediately following the leader are EL (Fig. 3, part A), where the residues
EL are
encoded by the nucleotide sequence for the Sac 1 cloning site . The PCR primer
used
at the carboxy terminus of the constant region introduces a nucleotide
substitution
which changes the amino acid at position 202 of the mature light chain, from a
serine to a leucine (Fig 3, part B). The Xba 1 restriction site, introduced by
the same
PCR primer, lies outside the coding region and has no effect on the final
amino acid
sequence of the mature light chain. The complete nucleotide sequence of the
plasmid Hu 19Apcn is shown in Fig. 4B.
As for the heavy chain above, there was a sequence discrepancy for the light
chain between the published sequence (C. Barbas III et al., Proc. Nat'l. Acad.
Sci.
-_ U( SA) 89: 10164-10168 ( 1992), PCT publication W094/06448) and the
sequence
obtained in the Hu I 9ALepen vector. A single base change resulted in glycine
in
Hu 19ALepcn in place of glutamic acid at position 97 (also consensus position
97 in
Kabat et al (Sequences of Proteins of Immunological Interest, fifth edition,
NIH
Publication No. 91-3242, 1991)) in framework 4 (see Fig. 3 of this application
and
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Fig. 4 of W094/06448). Glycine, but not glutamic acid, is encoded at this
position
in a human J germline J mini-gene and glutamic acid was not observed among a
large collection of human antibody sequences (Kabat et al., "Sequences of
Proteins
of Immunological Interest", fifth edition, NIH Publication No. 91-3242, 1991
). Also
as for the heavy chain, the glycine encoding sequence was observed for 3
separate
clonings from the original Fab 19 vector (Barbas et al., Proc. Nat'1. Acad.
Sci.
U( SA) 89, 10164-10168 (1992), PCT publication W094/06448). These results
demonstrate that the originally published sequence for Fab 19 light chain is
in error.
The Hu l9AHcpcd and Hu l9ALcpcn set of vectors were used to produce
antibody Hu 19A in COS cells and in CHO cells.
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Example B
Version B :Cloning Of The Edited Fab Hul9 Heavy and Light Chains
In cloning the variable region of the Fab 19 heavy chain, non-consensus
amino acid changes relative to the predicted germline sequence were introduced
at
the amino terminus by the PCR primer (C. Barbas III et al., Proc. Nat'1. Acad.
Sci.
U( SA) 89, 10164-10168 (1992)). To determine the likely amino terminus of the
heavy chain, the peptide sequence of the variable region of the Fab 19 heavy
chain
was aligned with all known human germline heavy chain sequences. Based on the
results of this alignment, the germline amino terminus is predicted to be
either
QVQLVE or EVQLVE rather than the sequence EVQLLEE present in version A.
To correct the N-terminus, the original Fab clone 19 heavy chain peptide was
aligned with human heavy chain sequences previously cloned at SmithKline
Beecham. A clone designated 97B27, which was obtained via PCR amplification
from the beginning of its leader sequence, had the acceptable N-terminus of
QVQLVE and was used to replace this region in the Fabl9 heavy chain.
Specifically, the Fabl9 heavy chain in the Hul9 Fab plasmid was PCR amplified
using a constant region primer which spanned the naturally occurring Bspl20 I
site
at the beginning of CH 1, and a variable region primer which created a PvuII
site
(corresponding to the site naturally occurring in clone 97B27) at amino acids
3 and 4
of the mature protein. This primer also introduced changes in the coding
sequence at
the amino terminus of the Fab I 9 heavy chain, coding for the amino acid
sequence of
QLVE for amino acids 3-6 instead of QLLEE, as in the version A construct. The
PCR fragment was cut with restriction enzymes PvccII and Bsp 120I, and,
through a
series of cloning steps, was combined with 97B27 at its PvuII site. The
resulting
clone, designated Hu l9BHcpcd, contained the leader and first 3 amino acids of
the
variable region of clone 97B27 and coded for the consensus sequence QVQI_VE at
its amino terminus (Fig. 2). The nucleotide sequence of Hul9BHepcd is shown in
Fig. 4C (SEQ ID NO: 20) for the region encoding the heavy chain. Sequences
differing from Hu 19AHcpcd are bolded.
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In cloning the variable region of the Fab clone 19 light chain, changes were
introduced at the amino terminus for cloning purposes, by the PCR primer, such
that
the first 4 amino acids of the Fab 19 light chain are EIEL. To determine the
likely
amino terminus of the light chain, the peptide sequence of the variable region
of the
Fab 19 light chain was aligned with all known human germline kappa chain
sequences. Based on the results of this alignment, the germline amino terminus
is
predicted to be DIQM. To convert the amino terminus of Fab 19 Lc to the
predicted
germline sequence, Fab 19 light chain was aligned with human kappa chain
sequences previously cloned at SB. A Clone designated AG I-37, which is the
kappa
chain obtained from cell line AG 1-37 obtained by PCR amplification from the
middle of its leader sequence, had the desired N-terminus and was used to
introduce
the corrections into the Fab 19 light chain. The N-terminal portion of the
leader
sequence was provided by the expression vector and was the consensus sequence
for
this family of leader regions. For this construct, the light chain coding
region was
excised from the Hu 19 Fab vector (Fig. 1 ) as a HirZfIlXba I fragment. Hinfl
recognizes a site which spans amino acids 18 an 19 of the mature protein and
is also
present in clone AG1-37. Through a series of cloning steps) the Hinf'1/Xbal
fragment of the Fab 19 light chain was ligated to the Hinfl site in clone AG I-
37.
The final construct consisted of the leader and first 18 amino acids of the AG
1-37
variable region linked to the variable and constant regions of the Fab 19
light chain,
beginning at amino acid 19 of the V-region. The resulting clone, designated
Hu l9BLcpcn, is altered only in the region encoding the first four amino acids
of the
variable region, coding for the consensus sequence DIQM (SEQ ID NO: 1 I ,
AMINO
ACIDS 21 - 24) instead of EIEL present in version A (Fig. 3A). The nucleotide
sequence for plasmid Hu l9BLcpen is shown in Fig. 4D (SEQ ID NO: 22) for the
region encoding the light chain. Sequences differing from Hu 19ALepcn are
bolded.
The vector set of Hu 19BHcpcd and Hu l9BLcpcn was used to produce
antibody Hu 19B in COS cells and in CHO cells.
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example C
Versions C & D: Mutation Of CDR2 Of Hul9B Heavy Chain To Eliminate a
Glycosvlation Site
An N-linked glycosylation site is encoded within the CDR2 loop of the heavy
chain. This glycosylation adds the potential for heterogeneity in the mAb
produced
in eucaryotic cells and for interference in binding antigen. To eliminate this
glycosylation site, mutations were introduced separately at two different
residues via
PCR overlap technology. For the first mutation the serine at position 61 of
the
mature Hu I 9B heavy chain was substituted with alanine, to create Hu 19C
heavy
chain. For the second substitution, the asparagine at position 59 was changed
to
glutamine, to create Hu I 9D heavy chain.
SITGGSNGINYADSVKR S61A Substitution (SEQ ID NO: 1)
SITGGSNGINYSDSVKR Original HuB CDR2 (SEQ ID NO: 2)
SITGGSNGIQYSDSVKR N59Q Substitution (SEQ ID NO: 3)
Specifically, the mutations were introduced via the PCR overlap technique
using one set
of primers encoding the mutation and a second set of primers annealing to
sequences
within the CMV promoter and the CH2 constant region in plasmid Hu l9Bpcd, as
the
outside 5' and 3' primers, respectfully. The final PCR product was digested
with
restriction enzymes, EcoR 1 and Bsp I 20I, and cloned into the Hu l9BHcpcd
vector at the
same sites to create Hu l9CHcpcd (Ser to Ala mutation) and Hu l9DLcpcd (Asp to
Gln
mutation ) (Fig. 2). The final constructs were sequenced to verify that the
mutations were
present. The nucleotide sequences of the heavy chain regions in Hu 19CHcpcd
and
Hul9DHcped are shown in Figs. 4E and 4F (SEQ ID NO'S 2S AND 27). Differences
from Hu 19Hcpcd are bolded.
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Example D
Version C :Cloning Of The Edited Constant Region
In the original cloning the of the Fab 19 light chain, a change was purposely
introduced at the caboxy terminus by the PCR primer to eliminate a naturally
occurring Sac 1 site (Barbas et al, supra). Consequently, the amino acid at
position
202 of the Fab 19 light chain was changed from a serine to a leucine. This
change
was corrected as follows. Plasmid Hu 19BLcpcn was cut with EcoR 1 and Bbs 1, a
naturally occurring restriction site near the amino terminus of human kappa
constant
region and a 405 by fragment, containing the nucleotide sequence coding for
the
leader, variable region, and first 5 amino acids of the kappa constant region,
was
isolated. Plasmid Lcvector4, a puc 18 derivative containing a normal human
kappa
constant region with a XbaI site just distal to the coding region, was cut
with Bbs 1
and Xba 1 and a 321 by fragment containing the nucleotide sequence coding for
the
entire kappa constant region beginning at amino acid 6 was isolated. This
fragment
contains the naturally occurring Sac 1 site near the end of the carboxy
terminus and
codes for a serine at position 202. Plasmid Hu l9BLcpcn was also cut with EcoR
1
and Xba 1 and a 4947 by fragment, containing the remainder of the vector
sequence
from plasmid Hu l9BLcpcn, was isolated. The three fragments were ligated
together to create Hu l9CLcpcn. The amino acid sequence of the Hu 19C light
chain
is shown in Figs. 3A and 3B (SEQ ID NO'S 11 and 12) and the nucleotide
sequence
of the light chain region is shown in Fig 4G (SEQ ID NO: 29). Differences from
Hu l9ALcpen are bolded. The vector Hu l9CLcpcn, was used with Hu l9CHcpcd or
Hu l9DHcpcd to produce antibody Hu 19C and Hu 19D, respectively, in COS cells
and in CHO cells.
Example E
Production of Hul9 Mabs in mammalian cells:
For initial characterization, the mAb constructs for each version, Hu 19A
heavy and light chain, Hu 19B heavy and light chain, Hu 19C heavy and light
chain,
30---and Hu 19D heavy with Hu 19C light chain, were expressed in COS cells
essentially
as described in Current Protocols in Molecular Biology (edited by F. M.
Ausubel et
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al. 1988, John Wiley & Sons, vol. 1 ( section 9.1 ). On day 1 after the
transfection,
the culture growth medium was replaced with a serum-free medium which was
changed on day 3. The serum-free medium was a proprietary formulation but _
satisfactory results are obtained using DMEM supplemented with ITSTM Premix
(insulin, transferrin, selenium mixture - Collaborative Research, Bedford, MA)
and 1
mg/ml BSA. The mAb was prepared from the day 3 + day 5 conditioned medium by
standard protein A affinity chromatography methods (e.g., as described in
Protocols
in Molecular Biology) using, for example, Prosep A affinity resin
(Bioprocessing
Ltd., UK).
To produce larger quantities of the Hu 19 mAb ( 100-200 mgs), the vectors
were introduced into a proprietary CHO cell system. However, similar results
will
be obtained using dhfu CHO cells as previously described (P. Hensley et al.,
J.
Biological Chemistry 269:23949-23958 ( 1994)). Briefly, a total of 30ug of
linearized plasmid DNA ( l5ug each of the A, B, C or D/C set of heavy chain
and
light chain vectors) was electroporated into 1 x 10~ cells. The cells were
initially
selected in nucleoside-free medium in 96 well plates. After three to four
weeks,
media from growth positive wells was screened for human immunoglobulin using
an
ELISA assay. The highest expressing colonies were expanded and selected in
increasing concentrations of methotrexate for amplification of the transfected
vectors. The antibody was purified from conditioned medium by standard
procedures using protein A affinity chromatography (Protein A sepharose,
Pharmacia) followed by size exclusion chromatography (Superdex 200,
Pharmacia).
The concentration and the antigen binding activity of the eluted antibody are
measured by ELISA. The antibody containing fractions are pooled and further
purified by size exclusion chromatography. As expected for any such antibody,
by
SDS-PAGE, the predominant protein product migrated at approximately 150 kDa
under non-reducing conditions and as 2 bands of 50 and 25 kDa under reducing
conditions. For antibody produced in CHO cells, the purity was > 90%, as
judged by
SDS-PAGE, and the concentration was accurately determined by amino acid
analysis.
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Example F
Preparation of Fab from Hul9B mAb: Samples with and without ~Iycosvlation
in heavy chain CDR2
Purification of MAbs
Each mAb was purified using an essentially similar purification procedure
that is detailed here for mAb 19B. Conditioned media (2L) from a 6 day culture
was
harvested, sterile filtered and applied to a 2.5 X S.lcm Protein A (Pharmacia,
fast
flow) equilibrated in 20mM sodium phosphate, I SOmM sodium chloride, pH 7
(PBS) at a linear flow rate of 98cm/h. The column was washed with
equilibration
buffer and eluted with 100mM glycine pH 2.5. Elution fractions containing the
mAb
were immediately adjusted to pH 5.0 with 1 M sodium hydroxide and applied at a
concentration of 4.2 mg/mL to a Superdex 200 size exclusion column (2.6 x 70
cm}
1 S equilibrated in 20 mM sodium phosphate buffer containing 150 mM NaCI,
pH7Ø
Monomeric mAb that was retained by the column at an apparent molecular weight
of
around 150 kDa was pooled and concentrated by ultrafiltration to Smg/mL, and
stored at 4°C after sterile filtration.
Electrophoretic analysis of MAb 19B and MAb 19C
By reduced SDS-PAGE, mAb 19B resolved as 2 major bands at 52 kDa and 28kDa
corresponding to the heavy and light chains of IgG respectively, with an
additional
band at 59 kDa representing about 7% of the total protein (Fig. 5). LC/mass
spectrometry analysis of the two heavy chains following excision from an SDS-
PAGE and proteolytic digestion (see below), confirmed that the 59 kDa species
represented an additional glycoform of mAb 19B that contained carbohydrate at
the
predicted VFl glycosylation site. In contrast, reduced SDS-PAGE analysis of
mAb
19C (Fig. 5), in which this VH glycosylation site is removed, showed that this
mAb
contains only the lower molecular weight (52 kDa) heavy chain species, as
expected.
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Carbohydrate Analysis of mAb 19B
The Hu 19B construct contains an additional consensus sequence for N-linked
glycosylation in the variable region of the heavy chain, -Asn59-Tyr-Ser-, in
addition
to the normal glycosylation site in the C,~2 domain of the heavy chain, -Asn'-
99-Ser-
Thr-. Analysis of both heavy chain bands by liquid chromatography,
electrospray
mass spectrometry (LC-ELMS) following reduction, alkylation, and tryptic
digestion
revealed that the 59 kDa band contains a variant that is glycosylated at Asn59
in
addition to being glycosylated at Asn299. The carbohydrate at AsnS~ is
predominantly biantennary, core fucosylated carbohydrates having two sialic
acid
residues. This is a common carbohydrate structure found in CHO-expressed
glycoproteins (such as sCR-1 and sCD4), but it differs from the carbohydrate
found
at the Asn299 site which lacks sialic acid altogether.
Purification of mAb 19B Glycovariant
Mab 19B {2 mg) was dialyzed against 20 mM Tris, pH 8.5 and applied to a 0.5 x
Scm Mono Q column (Pharmacia) equilibrated in the same buffer at a linear flow
rate of 300cm/h. The column was washed with equilibration buffer and eluted
with
a 20 column volume gradient from 0 mM to 50 mM NaCI in the same buffer (Fig.
6). Fractions containing the glycovariant, as determined by SDS-PAGE, were
pooled, dialyzed against PBS, sterile filtered and stored at 4°C.
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Preparation of Fabs ~ ProteolYtic Digestion
mAbl9B (48mg) was removed and the pH adjusted to 7.0 with dilute sodium
hydroxide. 2.Sm1 of 100mM sodium phosphate buffer containing IOmM EDTA, pH
7.3; l.3ml of 100mM cysteine in IOmM sodium phosphate buffer containing 1mM
EDTA; and 20u1 of crystalline papain (Boehringer, lOmg/ml) were added. The
sample was incubated at 37°C for 20h and the digest applied to a 1.5 x
2.6cm
Protein G column equilibrated in PBS at a linear flow rate of 67cm/h. The
column
was washed with PBS and the nonbound fraction containing the Fab was collected
and concentrated to Sml in an Amicon ultrafiltration cell fitted with a 10,000
molecular weight cut-off membrane and applied to a 2.6 X 70cm Superdex 200
(Pharmacia) size exclusion column equilibrated in PBS at a linear flow rate of
23cm/h. FAb (total yield, l2mg) eluted as a monomer on the size exclusion
column
and analysis by non-reduced SDS-PAGE revealed a major band at 45kDa and the
glycoform at 47kDa.
Separation of Fab Glycovariant
The mixture of glycosylated and unglycosylated Fab from cleaved mAb 19B was
dialyzed against 20 mM sodium acetate, pH 4.5 and applied (4mg) to a 0.5 X Scm
Mono S column (Pharmacia) at 300cm/h equilibrated with 20mM sodium acetate
buffer, pH 4.5. The column was then washed with equilibration buffer and
eluted
isocratically with the equilibration buffer containing 100 mM NaCI.
Glycosylated
Fab eluted after 5 column volumes whereas the unglycosylated FAb was retained
longer, eluting after 6 column volumes. Fractions that contained only
glycosylated
Fab, as judged by SDS-PAGE, were pooled, diluted 1:1 with starting buffer and
reapplied to a 0.16 X Scm Mono S column at 300cm/h. The Fab was once again
eluted with 100 mM NaCI and fractions most enriched for glycosylated Fab were
pooled, dialyzed against PBS pH 7.0, and sterile filtered. By SDS-PAGE
analysis
this fraction was enriched >90% with the glycosylated species (Fig. 7). The
process
yielded 3.3 mg of unglycosylated Fab and 0.16 mg of glycosylated Fab,
respectively.
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Example G
Binding of Hul9 mAb and Fab clone 19 proteins to recombinant F urotein
Binding of the various antibody constructs to recombinant F protein was
measured in a standard solid phase ELISA. Antigen diluted in PBS pH 7.0 was
adsorbed onto polystyrene round-bottom microplates (Dynatech, Immunolon II)
for
18 hours. Wells were then aspirated and blocked with 0.5% boiled casein (BC)
in
PBS containing 1 % Tween 20 (PBS/0.05% BC) for 2 hours. Antibodies (50
~l/well)
were diluted to varying concentrations in PBS/0.5% BC containing 0.025% Tween
20 and incubated in antigen coated wells for one hour. Plates were washed
three
times with PBS containing 0.05% Tween 20, using a Titertek 320 microplate
washer, followed by addition of HRP-labelled protein A/G (50 p,l) diluted
1:5000.
After washing three times, TMBIue substrate (TSI, #TM 102) was added and
plates
were incubated an additional 15 minutes. The reaction was stopped by addition
of 1
NH2S04 and absorbance read at 450 nm using a Biotek ELISA reader.
The antigen binding epitope of Fab 19 and mAb construct 19B were
examined in a competition ELISA. The test antibody construct was mixed with
increasing concentrations of RSMU 19 or B4 and added to F protein-coated
wells.
The epitope regions recognized by mAbs RSMU 19 and B4 have been previously
described in Arbiza et al., J. Gen'1 Virol. 73:2225-34 ( 1992). The
concentration of
Fab 19 or mAb 19B used in competition studies was determined previously to
give
90% maximal binding to F antigen. Binding of Fab 19 or mAb 19B in the presence
of other mAbs was detected using HRP-labelled goat anti-human IgG. The
reaction
was developed as stated above.
Fab 19 and amAb constructs 19A or 19B, demonstrated equivalent binding to
rF protein based on molar concentrations. Binding of Fabl9 or mAb 19B to rF
(recombinant F) protein was inhibited by mAb B4 but not by RSMU 19 indicating
that the epitope region recognized by these constructs is localized to region
as 255-
275 of the F protein (Table 1 ).
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Table 1: Viral F Protein Epitope Recognized by mAb 19B
Construct Bindin to Com etition Bindin to rF
rF
ECSp RSMU19 mAb B4 mAb
(M) (aa 429)* (aa 268, 272,
275)
_ Fab 19 10-9 -
mAb 19A 10-9 not tested not tested
- mAb 19B 10-9 - -1-
* amino acid residues critical for antigen recognition
The mAb 19B also showed specific binding to RSV infected cells indicating
recognition of the F protein as displayed in its native form. VERO cells
infected
with approximately 50 TCID50 RS Long virus were fixed in 90% methanol when
CPE reached > 90% and were used as antigen in the ELISA format described
above.
Binding of biotinylated mAb 19B was detected with HRP-labelled -Streptavidin.
In
this assay, the EC50 for mAb 19B was 34 +/- ng/ml.
Example H
In vitro antiviral activity of the Hul9 Antibodies
The ability of Fab fragments to inhibit virus-induced cell fusion was
determined using a modification of the in vitro microneutralization assay
described
by Beeler et al (J. of Virology 63: 2941-2950 ( 1989)}. In this assay, 50 ul
of RS
Long strain virus (approximately 100 TCIDSp/well; American Type Culture
Collection ATCC VR-26) were mixed with 0.1 ml VERO cells (5 x 103/well; ATCC
CCL-81 ) in Minimum Essential Media (MEM) containing 2% FCS, for 4 hours at
37°C, 5% CO2. Serial two-fold dilution (in duplicate) of test samples
(50 ul) were
then added to wells containing virus-infected cells. Control cultures
contained cells
incubated with virus only (positive virus control) or cells incubated with
media
alone. Cultures were incubated at 37°C in 5% C02 for 6 days at which
time
cytopathic effects (CPE) in virus control wells were >_ 90%. Neutralization
assays
41
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were performed as described above except that serial dilutions of test samples
were
mixed with 100 TCID50 of RS virus (50 ul each) for 2 hours at 37°C in
5% C02
before the addition of VERO cells (5 x 103).
Microscopic examination for cytopathic effects were confrimed by ELISA.
Media was aspirated from cultures and replaced with 50 ul of 90% methanol/0.6%
H202. After 10 minutes, fixative was aspirated and plates were air dried
overnight.
Viral antigen was detected in the fixed cultures using biotinylated
human/bovine
chimeric derivative of mAb B4 (RSCHB4; 1 ug/ml), followed by HRP-labelled
streptavidin (Boehringer-Mannheim) diluted I :10,000 (each lot was titrated to
determine the optimal concentration). The reaction was developed using TMBIue
and stopped by addition of 1 N H2S04. Absorbance was measured at 450nm
(O.D.450)~
Fusion-inhibition or neutralization titers were defined as the reciprocal
dilution of test sample, or concentration of antibody, which caused a 50%
reduction
in ELISA signal (ED50) as compared to virus controls. Based on the curve
generated in the ELISA by the standard virus titration, a 50% reduction in
O.D.450
in wells corresponded to >_ 90% reduction in virus titer. To determine the
ED50,
mean absorbance for replicate cultures (per dilution of test sample) was
plotted
against dilution of sample. Calculation of the 50% point, defined as (mean
absorbance virus-infected cells + mean absorbance uninfected cells)/2, was
based on
regression analysis of the dose titration.
SB 209763 is a humanized derivative of RSMU19 as described in P. R.
Tempest et al., Biotechnology 9, 266-271 ( 1991 ). To determine the effects of
coadministration of mAb 19B and SB 209763 on in vitro fusion-inhibition, the
antibodies were titrated alone and in combination. Antibody interactions were
analyzed using MacSynergy T"" II software.
The in vitro antiviral titers of the mAb constructs generated either by direct
cloning (version A) or after introduction of various sequence modfications
(versions
B-D) demonstrated potent neutralization and fusion-inhibition activity against
a
prototype RSV Long strain (Table 2). mAb 19B was also shown to neutralize
clinical isolates representing multiple antigenic variants of RS V collected
over the
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1993/1994 season in the PhiladelphiapA area (Table 3). When mAb 19B was co-
administered with a second antibody directed to a different F protein epitope
(SB
209763, critical residue as 429), the effect on inhibition of virus growth in
infected
cell cultures was additive (data not shown).
The antiviral titers of the mAb constructs were approximately 5 to 10-fold
lower than the titers obtained with the corresponding Fab constructs - Fab 19,
Fab 19A or B (Table 2). Fab 19 is the original Fab protein produced directly
in E.
coli from the clone 19 plasmid,whereas Fab 19A and Fab 19B were derived by
papain
cleavage from the coresponding full length mAbs. Removal of the N-linked
glycoslation site encoded within the CDR2 loop of the heavy chain by cloning
had
no effect on the overall antiviral activity of the mAb (Table 2; construct C
compared
to A and B). In addition, enrichment of the mAbl9B Construct for normally
glycosylated antibody did not significantly alter the in vitro fusion-
inhibition titer
(Table 4). However, enrichment for the glycovariant Fab fragment resulted in_a
2 to
10-fold reduction in in vitro antiviral activity compared to normally
glycosylated Fab
fragment (Table 4).
Table 2: Antiviral Activity of 19A, 19B, 19C, and 19D Constructs Against RS
Long strain virus
Construct Neutralization Fusion-Inhibition
EC a ml EC
(u ml) (nM)
Fab 19 0.34 + 0.25 * 0.22 4.4
Fab 19A not tested 0.16 3
Fab 19B not tested 0.12 + 0.06 2.4
mAb 19A 2.2 2.8 + 1.9 18.9
mAb 19B 1.5 2.3 + 1.9 15.3
mAb 19C not tested 2.4 16
mAb 19D not tested 2.6 17.3
* mean + standard deviation
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Table 3: Fusion-Inhibition Activity of mAb 19B against Clinical Isolates of
RSV
Virus Isolate Fusion-Inhibition
Titer
ECSO a ml
mAbl9B SB 209763
RS Lon ( rotot a A 2.3 + 1.9 1.3 + 0.8
1 )
RS 9320 (prototype 0.59 2.5 + 1.1
B 1 )
A 1 - V 1763 2.79 1.95
A2 - 847 0.89 0.27
A2 - 626 0.35 0.36
A3 - 7062 2.65 1.67
A4 - 6652 2.1 1.52
B 1 - 6973 1.77 2.22
B2 - 6556 1.49 2.05
B3 - 447 1.78 1.7
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Table 4: Antiviral Activity of 19B Glycovariants
Construct % Fusion-Inhibition
glycovariant*Titer
ECSp
(u ml)
mAb 19B 40% 2.5 + 1.5
Fraction < 5% 1.8 + 0.8
A
Fraction 40% 3.8 + 0.9
B
Fab 19B < 10% 0.12 + 0.06
Fraction 1 % 0.89
A
Fraction 94% 1.5 + 0.2
B
Fraction 99% 3.7
C
* mAb or Fab fragments were untreated or run on MonoQ (Mab) or MonoS (Fab)
columns to separate glycosylated versus minimally glycosylated forms in the
varible
region.
Example I
In vivo Activity of mAb 19B; Prophylaxis and Therapy in Balb/c Mouse
Model.
Balb/c mice (5/group) were inoculated intraperitoneally with doses ranging
from 0.06 mglkg to 5 mg/kg of mAb 19B either 24 hours prior (prophylaxis) or 4
days after (therapy) intranasal infection with 105 PFU of the A2 strain of
human
RSV. Mice were sacrificed 5 days after infection. Sera was obtained to
determine
antibody levels and lungs were homogenized to determine virus titers. Virus
was
undetectable in the lungs of mice treated prophylactically with > 1.25 ug mAb
19B,
and corresponding serum concentrations of > 5 ug/ml (Table 5). Higher doses of
mAb 19B were required for complete viral clearance when mAb was administered
therapeutically (5 mg/kg).
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Table 5: mAb 19B Prophylaxis and Therapy in Balb/c Mice
Pro h taxis Thera
TreatmentDose Lune Virus Serum Lung Virus Serum
Titer Titer
(mg/kgto lp/ tun Concentration~loglp/ iun Concentration
a ml a ml
mAb 19B 5 <1.7 15.6 <1.7 13.2
1.25 < 1.7 5.0 2.5 + 0.4 2.1
0.31 3.2+0.3 0.79 3.8+0.2 0.61
0.06 3.8+0.6 0.17 4.5+0.1 0.08
PBS - 5.2 + 0.1 <0.02 4.7 + 0.3 <0.036
The results of examples G through I establish that the Hu 19 antibodies have
potent antiviral activity in vitro against a broad range of native RSV
isolates of both
type A and B, and show prophylactic and therapeutic efficacy in vivo in animal
models. Thus, the Hu 19 antibodies, most preferably Hu 19C or Hu 19D, are
candidates for therapeutic, prophylactic, and diagnostic application in man.
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SEQUENCE LISTING
(1) GENERAL INFORMATION
S (i) APPLICANT: Deen, Keith C.
Dillon, Susan B.
Porter, Terence C.
Sweet) Raymond A.
1~ (ii) TITLE OF THE INVENTION: Human Monoclonal Antibodies
(iii) NUMBER OF SEQUENCES: 30
(iv) CORRESPONDENCE ADDRESS:
IS (A) ADDRESSEE: SmithKline Beecham Corporation
(B) STREET: 709 Swedeland Road
(C) CITY: King of Prussia
' (D) STATE: PA
(E) COUNTRY: U.S.A.
(F) ZIP: 19046
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
ZS (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi} CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
3O (B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/030,149
3S ---w (B) FILING DATE: 01-NOV-1997
47
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(viii) ATTORNEY/AGENT INFORMATION: _
{A) NAME: Geiger, Kathleen
S (B) REGISTRATION NUMBER: 35,880
(C) REFERENCE/DOCKET NUMBER: P50504
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5968
(B) TELEFAX: 610-270-5090
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ser Ile Thr Gly Gly Ser Asn Gly Ile Asn Tyr Ala Asp Ser Val Lys
1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
_ S (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Ile Thr Gly Gly Ser Asn Gly Ile Asn Tyr Ser Asp Ser Val Lys
1 5 10 15
Arg
1~
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
IS (A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Ser Ile Thr Gly Gly Ser Asn Gly Ile Gln Tyr Ser Asp Ser Val Lys
2S 1 5 10 15
Arg
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
3S {D) TOPOLOGY: linear
49
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg
2O (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 139 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
3O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr A1a Thr Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu Val Glu Ser Gly Gly Gly Leu
35--- 20 25 30
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Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr
35 40 45
Leu Ser Gly Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
50 55 60
Leu Glu Trp Val Ser Ser Ile Thr Gly Gly Ser Asn Phe Ile Asn Tyr
65 70 75 80
Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
85 90 95
Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala
100 105 110
Val Tyr Tyr Cys Ala Thr Ala Pro Ile Ala Pro Pro Tyr Phe Asp His
115 120 125
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
2~ (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
1 5 10 15
Val Gln Cys Gln Val Gln Leu Val Val Glu Ser Gly Gly Gly Leu Arg
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Leu
40 45
Ser Gly Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
3S 50 55 60
51
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Glu Trp Val Ser Ser Ile Thr Gly Gly Ser Asn Phe Ile Asn Tyr Ser
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
85 90 95
Ser Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Thr Ala Pro Ile Ala Pro Pro Tyr Phe Asp His Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
1 5 10 15
Val Gln CysGln ValGlnLeu ValValGlu SerGlyGly GlyLeuArg
20 25 30
Pro Gly GlySer LeuArgLeu SerCysAla AlaSerGly ThrThrLeu
35 40 45
Ser Gly TyrThr MetHisTrp ValArgGln AlaProGly LysGlyLeu
50 55 60
Glu Trp ValSer SerIleThr GlyGlySer AsnPheIle AsnTyrAla
65 70 75 80
Asp Ser ValLys GlyArgPhe ThrIleSer ArgAspAsn AlaLysAsn
3S 85 90 95
52
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Ser Leu Tyr Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val
100 105 110
Tyr Tyr Cys Ala Thr Ala Pro Ile Ala Pro Pro Tyr Phe Asp His Trp
115 120 125
S Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135
(2) INFORMATION FOR SEQ ID N0:8:
IO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
ZO Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
1 5 10 15
Val GlnCysGln ValGlnLeu ValValGlu SerGlyGly GlyLeuArg
20 25 30
Pro GlyGlySer LeuArgLeu SerCysAla AlaSerGly ThrThrLeu
35 40 45
Ser GlyTyrThr MetHisTrp ValArgGln AlaProGly LysGlyLeu
50 55 60
Giu TrpValSer SerIleThr GlyGlySer AsnPheIle G1nTyrSer
65 70 75 gp
3O Asp SerValLys GlyArgPhe ThrIleSer ArgAspAsn AlaLysAsn
-___ 85 90 95
Ser LeuTyrLeu GlnMetAsn SerLeuThr AlaGluAsp ThrAlaVal
100 105 110
Tyr TyrCysAla ThrAlaPro IleAlaPro ProTyrPhe AspHisTrp
115 120 125
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Gly Gln Gly Thr Leu Val Thr Val Ser Ser
130 135
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 88 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
In (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 124 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Met Gly TrpSerCys IleIleLeu PheLeuVal AlaThrAla ThrGly
1 5 10 15
Va1 His SerGluLeu ThrGlnSer ProSerSer LeuSerAla SerVal
20 25 30
Gly Asp ArgValThr IleThrCys ArgAlaThr GlnSerVal SerAsn
35 40 45
Phe Leu AsnTrpTyr GlnGlnLys ProGlyGlu AlaProThr LeuLeu
50 55 60
Ile Tyr AspAlaSer ThrSerGln SerGlyVal ProSerArg PheSer
65 70 75 80
IS Gly Ser GlySerGly MetAspPhe SerLeuThr IleSerSer LeuGln
85 90 95
Pro Glu AspLeuAla MetTyrTyr CysGlnAla SerIleAsn ThrPro
100 105 110
Leu Phe GlyGlyGly ThrArgIle AspMetArg Arg
2~ 115 120
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
25 (A) LENGTH: 101 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
3~ (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp Leu Arg
35 1 5 10 15
SS
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Gly Ala Arg Cys Asp Ile Gln Met Asn Phe Leu Asn Trp Tyr Gln Gln
20 25 30
Lys Pro Gly Glu Ala Pro Thr Leu Leu Ile Tyr Asp Ala Ser Thr Ser
35 40 45
S Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Met Asp
50 55 60
Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Met Tyr
65 70 75 80
Tyr Cys Gln Ala Ser Ile Asn Thr Pro Leu Phe G1y Gly Gly Thr Arg
]() 85 90 95
Ile Asp Met Arg Arg
100
(2) INFORMATION FOR SEQ ID N0:12:
IS
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 106 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
20 25 30
3U Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
40 45
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
35 65 70 75 80
S6
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His Lys Val Tyr Ala Cys Glu ~lal Thr His Gln Gly Leu Ser Ser Pro
85 90 95
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 105
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 106 amino acids
1~ (B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
1 5 10 15
ZO Leu LysSer GlyThrAla SerValVal CysLeu LeuAsnAsn PheTyr
20 25 30
Pro ArgGlu AlaLysVal GlnTrpLys ValAsp AsnAlaLeu GlnSer
35 40 45
Gly AsnSer GlnGluSer ValThrGlu GlnAsp SerLysAsp SerThr
50 55 60
Tyr SerLeu SerSerThr LeuThrLeu SerLys AlaAspTyr GluLys
65 70 75 80
His LysVal TyrAlaCys GluValThr HisGln GlyLeuSer LeuPro
85 90 95
Val ThrLys SerPheAsn ArgGlyGlu Cys
100 105
(2) INFORMATION FOR SEQ ID N0:14:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6284 base pairs
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GACGTCGCGGCCGCTCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAG60
AGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGCATGGGGC120
IO GGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACT180
ATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGG240
GACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCT300
GGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCGG360
GGATCGATCCGTCGACGTACGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT420
IS CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA480
CCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA540
ATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCA600
GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG660
CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC720
ZO TACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT780
GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT840
TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG900
ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTG960
AACCGTCAGATCGCCTGGAGACGCCATCGAATTCTGAGCACACAGGACCTCACCATGGGA1020
ZS TGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGAGGTCCAA1080
CTGCTCGAGGAGTCTGGGGGAGGCCTGGTCAGGCCTGGCGGGTCCCTAAGACTCTCGTGT1140
GCAGCCTCTGGAACCACCCTCAGTGGCTATACCATGCACTGGGTCCGCCAGGCTCCAGGG1200
AAGGGGCTGGAGTGGGTCTCATCCATTACTGGAGGTAGCAACTTCATAAACTACTCAGAC1260
TCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTTTATCTGCAA1320
3O ATGAACAGCCTGACAGCCGAGGACACGGCTGTCTATTATTGTGCGACCGCCCCTATAGCA1380
CCGCCCTACTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACC1440
AAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG1500
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACCGTGTCGTGGAACTCA1560
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC1620
3S TCCCTCAGCAGCGTGGTGACTGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC1680
AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT1740
5g
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GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTC1800
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA1860
-- TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC1920
GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC1980
S CGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG2040
TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA2100
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG2160
AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAG2220
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC2280
IO GACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG2340
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC?.400
CTCTCCCTGTCTCCGGGTAAATGATAGATATCTACGTATGATCAGCCTCGACTGTGCCTT2460
CTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG2520
CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGT2580
IS GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA2640
ATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCGACAGCGCT2700
GGATCTCCCGATCCCCAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAAAAAAGG2760
AAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAAAGGATG2820
CTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGTACCCAG2880
ZO AGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCTTGTCATC2940
ACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACACAGGATA3000
GAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCCTCACAT3060
TTGCTTCTGACATAGTTGTGTTGGGAGCTTGGATAGCTTGGACAGCTCAGGGCTGCGATT3120
TCGCGCCAAACTTGACGGCAATCCTAGCGTGAAGGCTGGTAGGATTTTATCCCCGCTGCC3180
ZS ATCATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCAAAATATGGGGATTGGCAAG3240
AACGGAGACCTACCCTGGCCTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATGACC3300
ACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGTGATTATGGGTAGGAAAACCTGGTTC3360
TCCATTCCTGAGAAGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTCAGTAGAGAA3420
CTCAAAGAACCACCACGAGGAGCTCATTTTCTTGCCAAAAGTTTGGATGATGCCTTAAGA3480
3O CTTATTGAACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGGATAGTCGGAGGCAGT3540
TCTGTTTACCAGGAAGCCATGAATCAACCAGGCCACCTTAGACTCTTTGTGACAAGGATC3600
ATGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATTGATTTGGGGAAATATAAACTT3660
CTCCCAGAATACCCAGGCGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGTATAAG3720
TTTGAAGTCTACGAGAAGAAAGACTAACAGGAAGATGCTTTCAAGTTCTCTGCTCCCCTC3780
~S-- CTAAAGCTATGCATTTTTATAAGACCATGGGACTTTTGCTGGCTTTAGATCAGCCTCGAC3840
TGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT3900
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CA 02270288 1999-04-30
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GGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCT3960
GAGTAGGTGT CATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG4020
GGAAGACAAT AGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAGCTGGGGCTCG4080
ATCGAGTGTA TGACTGCGGCCGCGATCCCGTCGAGAGCTTGGCGTAATCATGGTCATAGC4140
S TGTTTCCTGT GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA4200
TAAAGTGTAA AGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCT4260
CACTGCCCGC TTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAAC4320
GCGCGGGGAG AGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGC4380
TGCGCTCGGT CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGT4440
IO TATCCACAGA ATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG4500
CCAGGAACCG TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG4560
AGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT4620
ACCAGGCGTT TCCCCCTGGAAGCTCCCTCGTGCGC'PCTCCTGTTCCGACCCTGCCGCTTA4680
CCGGATACCT GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCT4740
IS GTAGGTATCT CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC4800
CCGTTCAGCC CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAA4860
GACACGACTT ATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATG4920
TAGGCGGTGC TACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAG4980
TATTTGGTAT CTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTT5040
ZO GATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA5100
CGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC5160
AGTGGAACGA AAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCA5220
CCTAGATCCT TTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAA5280
CTTGGTCTGA CAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTAT5340
ZS TTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCT5400
TACCATCTGG CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT5460
TATCAGCAAT AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT5520
CCGCCTCCAT CCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTA5580
ATAGTTTGCG CAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTG5640
3O GTATGGCTTC ATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT5700
TGTGCAAAAA AGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCG5760
CAGTGTTATC ACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCG5820
TAAGATGCTT TTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC5880
GGCGACCGAG TTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAA5940
3S CTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTAC6000
CGCTGTTGAG ATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTT6060
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAGGCAAAATGCC GCAAAAAAGG6120
GAATAAGGGC GACACGGAAA TGTTGAATAC TCATACTCTTCCTTTTTCAA TATTATTGAA6180
GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATTTGAATGTATT TAGAAAAATA6240
AACAAATAGG GGTTCCGCGC ACATTTCCCC GAAAAGTGCCACCT 6284
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Gly Trp Ser Cys Ile Ile Leu Phe Ala Thr Ala Thr
Leu Val Gly
1 5 10 15
Val His Ser Glu Val Gln Leu Leu Glu
Val
20 25
(2) INFORMATION FOR SEQ ID N0:16:
ZS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
{B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Ser Pro Gly Lys
1
61
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
$ (A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly
1S 1 5 10 15
Val His Ser Glu Leu Thr Gln Ser Pro
25
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5681 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
GACGTCGCGGCCGCTCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAG 60
AGGCCGAGGC GGCCTCGGCCTCTGCATAAAThAAAAAA.ATTAGTCAGCCATGCATGGGGC 120
GGAGAATGGG CGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACT 180
ATGGTTGCTG ACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGG 240
GACTTTCCAC ACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCT 300
3S----- GGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGAATTAATTCCCGG 360
GGATCGATCC GTCGACGTACGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT 420
62
CA 02270288 1999-04-30
WO 98/19704 PCT/US9?/19203
CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA480
CCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCA540
ATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCA600
GTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGG660
S CCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC720
TACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGT780
GGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGT840
TTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG900
ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGGTACGTG960
IO AACCGTCAGATCGCCTGGAGACGCCATCGAATTCTGAGCACACAGGACCTCACCATGGGA1020
TGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGAGCTCACC1080
CAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA1140
ACTCAGAGTGTTAGTAACTTTTTAAATTGGTATCAGCAGAAGCCAGGGGAAGCCCCTACG1200
CTCCTGATCTATGATGCATCCACTTCGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGT1260
IS GGATCTGGGATGGATTTCAGTCTCACCATCAGCAGTCTGCAGCCTGAAGATCTTGCAATG1320
TATTACTGTCAAGCGAGTATCAATACCCCGCTTTTCGGCGGAGGGACCAGAATAGATATG1380
AGACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAA1440
TCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTA1500
CAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG1560
ZO GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTAC1620
GAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTTGCCCGTCACA1680
AAGAGCTTCAACAGGGGAGAGTGTTAGTGAGATGATCCTCTAGAGTCATCTACGTATGAT1740
CAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTT1800
CCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT1860
~S CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG1920
GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAACCAG1980
CTGGGGCTCGACAGCTCGAGCTAGCTTTGCTTCTCAATTTCTTATTTGCATAATGAGAAA2040
AAAAGGAAAATTAATTTTAACACCAATTCAGTAGTTGATTGAGCAAATGCGTTGCCAAAA2100
AGGATGCTTTAGAGACAGTGTTCTCTGCACAGATAAGGACAAACATTATTCAGAGGGAGT2160
3O ACCCAGAGCTGAGACTCCTAAGCCAGTGAGTGGCACAGCATTCTAGGGAGAAATATGCT~'2220
GTCATCACCGAAGCCTGATTCCGTAGAGCCACACCTTGGTAAGGGCCAATCTGCTCACAC2280
AGGATAGAGAGGGCAGGAGCCAGGGCAGAGCATATAAGGTGAGGTAGGATCAGTTGCTCC2340
TCACATTTGCTTCTGACATAGTTGTGTTGGGA'GCTTGGATCGATCCACCATGGTTGAACA2400
AGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG2460
3S GGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCG2520
CCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGC2580
63
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
AGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGT264
CACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTC27C0
ATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCA2760
TACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGC282
S ACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGG288
GCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCT2940
CGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTC300~~
TGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGC3060
TACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTA3120
IO CGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTT3180
CTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGA324;;
GATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGAC330
GCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAAC336
TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAAT3420
IS AAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAT3480
CATGTCTGGATCGCGGCCGCGATCCCGTCGAGAGCTTGGCGTAATCATGGTCATAGCTGT3540
TTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAA3600
AGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCAC3660
TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCG3720
ZO CGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGC3780
GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT3840
CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA3900
GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGC3960
ATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC4020
ZS AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG4080
GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTA4140
GGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCG420
TTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC4260
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAG4320
3O GCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTA~_'4380
TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT4440
CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGC45CJ
GCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT456
GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCT46~~
3S AGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATAT:AGTAAACTT468:;
GGTCTGACAGTTACCAATGC:'TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC474
64
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
GTTCATCCAT AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTAC4800
CATCTGGCCC CAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTAT4860
CAGCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG4920
CCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATA4980
S GTTTGCGCAA CGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA5040
TGGCTTCATT CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT5100
GCAAAAAAGC GGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAG5160
TGTTATCACT CATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA5220
GATGCTTTTC TGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC5280
IO GACCGAGTTG CTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT5340
TAAAAGTGCT CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGC5400
TGTTGAGATC CAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTA5460
CTTTCACCAG CGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA5520
TAAGGGCGAC ACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCA5580
IS TTTATCAGGG TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAAC5640
AAATAGGGGT TCCGCGCACATTTCCCCGAAAAGTGCCACCT 5681
(2) INFORMATION FOR SEQ ID N0:19:
2O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
2S
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
3O Leu Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
1 5 10
(2) INFORMATION FOR SEQ ID N0:20:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1427 base pairs
6S
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
GAATTCGGTA CCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTGGCTCTTTTAAGAGGT60
GTCCAGTGTC AGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAGGCCTGGCGGGTCC120
IO CTAAGACTCT CGTGTGCAGCCTCTGGAACCACCCTCAGTG'GCTATACCATGCACTGGGTC180
CGCCAGGCTC CAGGGAAGGGGCTGGAGTGGGTCTCATCCATTACTGGAGGTAGCAACTTC240
ATAAACTACT CAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC300
TCACTTTATC TGCAAATGAACAGCCTGACAGCCGAGGACACGGCTGTCTATTATTGTGCG360
ACCGCCCCTA TAGCACCGCCCTACTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTC420
IS TCCTCAGCCT CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC480
TCTGGGGGCA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACC540
GTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG600
TCCTCAGGAC TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC660
CAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT720
ZO GAGCCCAAAT CTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG780
GGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG840
ACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC900
AACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG960
TACAACAGCA CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT1020
~S GGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC1080
ATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG1140
GATGAGCTGA CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC1200
GACATCGCCG TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT1260
CCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC1320
3O AGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC1380
TACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAAATGATAGATATC 1427
(2) INFORMATION FOR SEQ ID N0:21:
3S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
66
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
$ (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly
1 5 10 15
Val Gln Cys Gln Val Gln Leu Val
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 732 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
ZS GAATTCCATGGACATGAGGGTCCCCGCTCAGCTCCTAGGGCTCCTGCTGCTCTGGCTCCG60
AGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG220
AGACAGAGTCACCATCACTTGCCGGGCAACTCAGAGTGTTAGTAACTTTTTAAATTGGTA180
TCAGCAGAAGCCAGGGGAAGCCCCTACGCTCCTGATCTATGATGCATCCACTTCGCAAAG240
TGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGATGGATTTCAGTCTCACCATCAG300
CAGTCTGCAGCCTGAAGATCTTGCAATGTATTACTGTCAAGCGAGTATCAATACCCCGCT360
TTTCGGCGGAGGGACCAGAATAGATATGAGACGAACTGTGGCTGCACCATCTGTCTTCAT420
CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA480
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGG540
TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG600
3S CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC660
CCATCAGGGCCTGAGCTTGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGAGA720
67
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
TGATCCTCTA 732
GA
(2) INFORMATION FOR SEQ N0:23:
ID
S (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
- (D) TOPOLOGY: linear
IU
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: ID N0:23:
SEQ
IS Met Arg Val Pro Ala Gln Leu Leu Leu Leu Leu Trp Leu Arg
Leu Gly
1 5 10 15
Gly Ala Arg Cys Asp Ile Gln
Met Thr
2 0 ?. 5
ZO (2) INFORMATION FOR SEQ N0:24:
ID
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
ZS (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:24:
Leu Pro Val Thr Lys Ser Phe Gly Glu Cys
Asn Arg
1 5 10
3S (2) INFORMATION FOR SEQ N0:25:
ID
68
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(i) SEQUENCE CHARACTERSSTICS:
(A) LENGTH: 1427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
S (D}- TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
IO GAATTCGGTACCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTGGCTCTTTTAAGAGGT60
GTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAGGCCTGGCGGGTCC120
CTAAGACTCTCGTGTGCAGCCTCTGGAACCACCCTCAGTGGCTATACCATGCACTGGGTC180
CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTACTGGAGGTAGCAACTTC240
ATAAACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTC~AGAGACAACGCCAAGAAC300
IS TCACTTTATCTGCAAATGAACAGCCTGACAGCCGAGGACACGGCTGTCTATTATTGTGCG360
ACCGCCCCTATAGCACCGCCCTACTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTC420
TCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC480
TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACC540
GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG600
2O TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCRGCTTGGGCACC660
CAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT720
GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG780
GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG840
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC900
ZS AACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG960
TACAACAGCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT1020
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC1080
ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG1140
GATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC1200
3O GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT1260
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC1320
AGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC1380
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGATAGATATC 1427
3S (2) INFORMATION FOR SEQ ID N0:26:
69
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein.
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Ser Asn Phe Ile Asn Tyr Ala
1 5
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1427 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
2,SGAATTCGGTA CCATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTGGCTCTTTTAAGAGGT60
GTCCAGTGTC AGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAGGCCTGGCGGGTCC120
CTAAGACTCT CGTGTGCAGCCTCTGGAACCACCCTCAGTGGCTATACCATGCACTGGGTC180
CGCCAGGCTC CAGGGAAGGGGCTGGAGTGGGTCTCATCCATTACTGGAGGTAGCAACTTC240
ATACAATACT CAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAC300
3O TCACTTTATC TGCAAATGAACAGCCTGACAGCCGAGGACACGGCTGTCTATTATTGTGCG360
ACCGCCCCTA TAGCACCGCCCTACTTTGACCACTGGGGCCAGGGAACCCTGGTCACCGTC420
TCCTCAGCCT CCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC480
TCTGGGGGCA CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACC540
GTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG600
3S TCCTCAGGAC TCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACC660
CAGACCTACA TCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTT720
n
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
GAGCCCAAAT CTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG780
GGGGGACCGT CAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG840
ACCCCTGAGG TCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTC900
AACTGGTACG TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG960
S TACAACAGCA CGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT1020
GGCAAGGAGT ACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC1080
ATCTCCAAAG CCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG1140
GATGAGCTGA CCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC2200
GACATCGCCG TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT1260
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGC1320
AGGTGGCAGC AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC1380
TACACGCAGA AGAGCCTCTCCCTGTCTCCGGGTAAATGATAGATATC 1427
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
Ser Asn Phe Ile Gln Tyr Ser
1 5
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 762 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
71
CA 02270288 1999-04-30
WO 98/19704 PCT/US97/19203
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GAATTCCATGGACATGAGGGTCCCCGCTCAGCTCCTAGGGCTCCTGCTGCTCTGGCTCCG 6C
S AGGTGCCAGA-TGTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGG 120
AGACAGAGTCACCATCACTTGCCGGGCAACTCAGAGTGTTAGTAACTTTTTAAATTGGTA 18C
TCAGCAGAAGCCAGGGGAAGCCCCTACGCTCCTGATCTATGATGCATCCACTTCGCAAAG 240
TGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGATGGATTTCAGTCTCACCATCAG 300
CAGTCTGCAGCCTGAAGATCTTGCAATGTATTACTGTCAAGCGAGTATCAATACCCCGCT 360
IO TTTCGGCGGAGGGACCAGAATAGATATGAGACGAACTGTGGCTGCACCATCTGTCTTCAT 420
CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAA 480
TAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGG 540
TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG 600
CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCAC 660
IS CCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTGAGA 720
TGATCCTCTAGATCTACGTATGATCAGCCTCGACTGTGCCTT _ 762
(2) INFORMATION FOR SEQ ID N0:30:
2O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
Ser Pro Val Thr Lys Ser Phe Thr Arg Gly Gln Cys
1 5 10
72
