Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN B-CELL ANTIGENS; RELATED REAGENTS
FIELD OF THE INVENTION
5The present invention relates to various biological
reagents which are useful in modulating a human immune
response. More particularly, it is directed towards
compositions and methods useful, e.g., in B and T cell
interaction.
BACKGROUND OF THE INVENTION
Leukemias, lymphomas, carcinom~, and other
malignancies are described in, e.g., Wilson, et al.
(eds.) Harrison's Princi~les of Internal Medicine,
McGraw-Hill, New York, pp. 1599-1612). As one example of
such diseases, malignant lymp~om~ are neoplastic
transformed cells that reside pre~om~n~ntly in lymphoid
tissues (see, e.g., Nadler, L.M. in Harrison's). Ninety
percent of non-Hodgkins lymphomas, of which approximately
30,000 new cases occur each year in the U.S., are B cell
ly~homA~. Less than 25~ of these cases are cured.
Another example is leukemia, of which approximately
30,000 new cases occur ~nn~ ly in the U.S. Thus, a need
exists for a more effective treatment for B and T cell
lymphomas, leukem~ , carcinomas, and other malignancies.
Growth of normal resting B cells (also referred to
as "L lymphocytes") involves two distinct steps. First,
the resting cells are activated to pass from the Go to G
phase of the cell cycle. See, e.g., Alberts, et al.
(eds. 1989) Molecular Biolooy of the Cell Garland Publ.,
NY; and Darnell, et al. (1990) Molecular Cell Bioloov
Freeman, NY. Next, the activated cells are induced to
proliferate. See, e.g., Paul, ed. (1989) Fundamental
Tmmlmoloov, 2nd ed., Raven Press, NY; and the third
edition. Several factors have been identified that
induce growth of B cells, including interleukin-l (IL-l),
IL-2, IL-4, IL-10, and IL-13. In addition, antibodies
against certain B cell surface molecules have been
demonstrated to promote B cell proliferation. T cells
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(also referred to as "T lymphocytes") are also induced to
proliferate by certain factors, which include
phytohemagglutinin, anti-T cell receptor monoclonal
antibodies, anti-CD3 monoclonal antibodies, and other
agents.
Numerous studies implicate the CD40 molecule as a
growth factor receptor and an important regulator of
human B cell proliferation and development. B
lymphocytes are activated by the interaction of CD40
molecules on the surface of the B lymphocytes with a
ligand that is transiently expressed on activated helper
T cells.
The CD40 is a membrane-associated glycoprotein
expressed on normal B lymphocytes and B cell
malignancies, interdigitating cells, follicular dendritic
cells, thymic epithelial cells, and some carc;nom~.
~uman CD40 was first identified in 1985 as the epitope of
a monoclonal antibody that is expressed almost
exclusively on B lymphocytes, and therefore is a useful
marker for B cells. The human CD40 antigen is a 45-50 Kd
glycoprotein.
Anti-CD40 antibodies provide a potent co-stimulatory
signal for B cell proliferation induced by either phorbol
myristic acetate (PMA), anti-CD20 antibodies, or
anti-im~lmoglobulin antibodies. The addition of
anti-human CD40 antibodies plus IL-4 to activated B cells
also causes numerous effects, including short-term
replication, induction of IgE synthesis, and long-term
proliferation when cultures are further supplemented with
CD32 transfected L cells.
B7 (CD80) and B70 (CD86) are the second "group~ of
molecules which strongly mediate B and T cell
interaction. These molecules, on B cells, interact with
their ligands CD28 and CTLA-4 on T cells. These
interactions are major co-stimulatory signals for
activation of both B and T cells.
During the last 15 years, it has become apparent
that these two pairs of surface molecules play
flln~Am~ntal functions in T cell and B cell activation
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Numerous in vitro and in vivo experiments have
o~trated that these two pairs of molecules represent
important targets for ;mmnnosuppression. See, e.g.,
Banchereau, et al. (1994) Ann. Rev. Tm~llnol. 12:881-922;
van Kooten, et al. (1996) Adv. Immunol. 61:1-77; Linsley
and Ledbetter (1993) Ann. Rev. T ~llrlol. 11:191-212).
Immunosuppression represents a very important
;~llnological intervention to prevent and cure auto;mmllne
diseases and graft rejection during transplantation.
TmmllnQsuppression can be achieved by a) anti-
proliferative drugs (cyclophosphamide, 15-
deoxyspergualin, et al.); b) glucocorticosteroids; c)
inhibitors of intracellular signaling processes (e.g.,
cyclosporine); d) ;mmllnosuppressive cytokines (e.g., TGF-
~ -10); e) specific tolerance induction by antigens;
and f) inhibition of cell surface molecules involved in T
and B lymphocyte activation, such as the CD40/CD40 ligand
and B7/B70 with CD28/CTLA-4.
In 1995, another molecule called RP105 was cloned
from mouse splenic cells. See Miyake, et al (1995)
~RP105, a novel B cell surface molecule implicated in B
cell activation, is a member of the leucine-rich repeat
protein family" J. Tmmlln~l . 154:3333-3340. Monoclonal
antibody against RP105 induces strong proliferation of
mouse B cells and protects mouse B cells from
irradiation-induced apoptosis in a S;m; 1 ~r fashion to
anti-CD40 antibody or CD40-ligand. See Miyake, et al.
(1994) "Murine B cell proliferation and protection from
apoptosis with an antibody against a 105 kDa molecule:
Unresponsiveness of X-linked ;m~lmodeficient B cells" J.
EXD. Med. 180:1217-1224.
The RP105 and its putative ligand may be a third
pair of molecules that play key roles in the activation
of T cells and B cells. However, until now, the human
sequence has not been available, and its structure and
activities undetermined. The present invention provides
this and many other useful advances.
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SU~ RY OF THE IN~3~TION
The present invention provides a composition
selected from the group consisting of: an isolated or
recombinant nucleic acid encoding human BAS-l protein or
fragment thereof; a substantially pure BAS-l protein or
peptide thereof; a fusion protein comprising BAS-l
protein se~uence; and an antibody raised to a recombinant
or purified primate BAS-l protein.
In embodiments of the isolated or recombinant
nucleic acid, the nucleic acid may comprise a sequence
defined in SEQ ID NO: 1.
In protein embodiments, the protein may be a
substantially pure BAS-l protein or peptide thereof; be
selected from the group consisting of: a protein or
peptide from a primate, including a human; a protein or
peptide comprising at least one polypeptide segment
defined in SEQ ID NO: 2; a protein or peptide which
exhibits a post-translational modification pattern
distinct from a natural BAS-l protein; and a protein
lacking the intracellular ~or-;n of BAS-l; and may be a
composition comprising the protein and a ph~rm~ceutically
acceptable carrier.
Antibody embo~;m~nts include those where the BAS-l
protein is a primate protein, including one from human;
the antibody is raised against a peptide sequence defined
in SEQ ID NO: 2; the antibody exhibits a Kd of at least
about 10 ~M; the antibody is a monoclonal antibody; or
the antibody is labeled. Further embodiments include
where the antibody induces strong proliferation of B
cells; and/or protects B cells from irradiation- or
steroid hormone-induced apoptosis.
The present invention also embraces kits, e.g.,
which comprise, e.g., a nucleic acid encoding a BAS-l
protein or peptide; a substantially pure BAS-l protein or
fragment; or an antibody or receptor which specifically
binds a BAS-l protein. A kit which includes the nucleic
acid may comprise a coding sequence defined in SEQ ID NO:
1. For a kit which includes a protein or fragment, often
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the polypeptide is selected from the group consisting of:
a protein or peptide from a primate, including a human; a
protein or peptide comprising at least one polypeptide
segment defined in SEQ ID NO: 2; and a protein or peptide
which exhibits a post-translational modification pattern
distinct from a natural BAS-1 protein.
Another kit may include an antibody or receptor,
wherein: the BAS-1 protein is a primate protein,
including one from a human; the antibody is raised
against a peptide sequence defined in SEQ ID NO: 2; the
antibody ~xh;h;ts a Kd of at least about 10 ~M; the
antibody is a monoclonal antibody; or the antibody is
labeled.
The invention also provides a method of screening a
sample for a b;n~;ng partner for BAS-1 comprising the
steps of producing a purified or reCo~hin~nt BAS-1
protein, and screening in the sample for a specific
bin~;ng of the b;n~;ng partner to the BAS-1 protein. In
certain embodiments, the sample comprises proteins
derived from a T cell, a dendritic cell, including a
follicular dendritic cell, or a stromal cell, including a
fibroblast cell, an endothelial cell, and an epithelial
cell. In other embodiments, the binding partner is an
antibody, and the sample is a hybridoma supernatant.
Other aspects of the present invention include a
method of modulating physiology or development of a cell
comprising contacting the cell with an agonist or
antagonist of a BAS-1 protein. Typically, the physiology
is selected from~ nosuppression; activation of
cytotoxic killing; modulation of cytokine production; or
growth of a lymphoma. The antagonist often will be an
antibody AgA;nct a primate BAS-1 protein. And the method
may include such in cnmh~nAtion with a mediator of a
signal through the antigen receptor, the CD40:CD40 ligand
pathway, or the CD28/CTLA-4:B7/B70 pathway, e.g., anti-
CD3, anti-CD40, anti-CD40 ligand, anti-CD28, anti-CTLA-4,
anti-B7, anti-B70, or soluble portions of the appropriate
surface signaling molecule.
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DETAILED DESCRIPTION OF THE lNv~N~l~loN
I. General
The present invention provides the amino acid
sequence and DNA sequence of a human protein ~hich
exhibits properties of activation antigens. mis protein
is designated B cell Activation and Survival antigen-1
(BAS-1). The primate sequence described herein was
obtained after a mouse gene was initially described. See
Miyake, et al. (1995) J. Immunol. 154:3333-3340. Similar
sequences for proteins in other primate species should
also be available. The descriptions below are directed,
for exemplary purposes, to the human BAS-1 natural allele
described, but are likewise applicable to allelic and/or
polymorphic variants, e.g., from other individuals, as
well as splicing variants, e.g., natural forms.
These genes will allow isolation of other primate
genes encoding proteins related to this, further
ext~n~;ng the family beyond the specific embodiment
described. The procedure is broadly set forth below.
Human B cell Activation and Survival Ag-1 (BAS-1),
so named because of its biological activity. Monoclonal
antibody ~g~;nct a mouse homolog, RP105, induces strong
proliferation of mouse B cells and protects mouse B cells
from irradiation-induced apoptosis in a similar fashion
to anti-CD40 antibody or CD40-ligand.
The human BAS-1 cDNA was isolated using nucleic acid
sequences based upon sequences from the mouse RP105.
Analysis of the correspo~;ng encoded protein indicates
that the human BAS-1 is a member of the family of
proteins which contain leucine-rich motifs. Other
members of the family include the CD14-LPS receptor,
~SH/LH/TSH receptors, and neurotropin receptors. These
receptors appear to be involved in signal transduction.
Human BAS-1 cDNA was deduced using sequences from
various regions of mouse RP105. A homology comparison
between the mouse and human BAS-1 open re~;n~ frames
exhibit a~oximately 67% DNA sequence identity and
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approximately 73% amino acid sequence identity with the
mouse RP105.
The human BAS-1 may also have functional roles
outside the immune system, e.g., in developmental
regulation in other cell types. See, e.g., Gilbert
(1991) Develo~mental Biolo~v (3d ed.), Sinauer
Associates, Sunderland, MA; Browder, et al. (1991)
Develo~mental Bioloov (3d ed.), Saunders, Philadelphia,
PA.; Russo, et al. ~1992) DeveloT~ment: The Molecular
Genetic A~proach, Springer-Verlag, New York, N.Y.; and
Wilkins (1993) Genetic Analvsis of ~imA1 Develo~ment (2d
ed.) Wiley-Liss, New York, N.Y. Identification of the
ligand for human BAS-1 may help to address some of the
questions raised by these observations.
II. Purified human BAS-1
SEQ ID NO: 1 discloses the nucleotide seguence of
the cDNA and the correspo~;n~ amino acid sequence. The
amino acid sequence is also set forth in SEQ ID NO: 2.
The signal sequence appears to run from met (1) to val
(20); an amino flanking region runs from cys (28) to leu
(58), which contains two potential N-linked glycosylation
sites on asparagine (residues 34, 53); the tandem repeats
of leucine-rich motif run from thr (55) to tyr (592),
which contain 9 potential sites for N-linked
glycosylation on asparagine (residues 70, 78, 201, 234,
244, 394, 402, 451, and 573); the carboxy flanking region
runs from leu (574) to cys (625); the trAncm~ e
region runs from ala (629) to ~al (650); the
intracellular region runs from lys (651) to phe (661),
which contains two potential phosphorylation sites on tyr
(652 and 658).
The following table shows the alignment of the
leucine-rich repeats of the human BAS-1 protein shown in
SEQ ID NO: 2.
Table 1
C I
consensus: TXl~T.~T.lrXNXT.~XT.xxxxxxxl.XX
1: 55-78 TEFLEFSFNFL~ll~NKlr'SR~MN
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2: 79-102 _TFLD_TR~ Q N~ QSHHQ
3: 103-126 LST_VLTG_PLIFMAETSLNGPKS
4: 127-150 _KH_FLIQTGISNLEFIPVHN_EN
5: 151-174 LES_YLGS_HISSlK~K~ARN
5 6: 175-198 kKV_DFQN_A_HYISREDMRSLEQ
7: 199-223 AINLS_NFNGNN-VKGIELGAFDSTV
8: 224-246 FQSLNFGGTPNL-SVIFNGLQNST
9: 247-275 TQS_W_GTFEDIDDEDISSAM_KGLCEMS
10: 276-299 VES_N_QEHRFSDISSTTFQCFTQ
10 11: 300-322 LQE_DLTATH_XGLPSGMKG-~NL
12: 323-346 LKKLVLSVNHFDQ_CQISAANFPS
13: 347-371 _TH_Y_RG_-VXX_HLGVGC -T.T~RT .~'.N
14: 372-397 LQTT~nT~R-nIEAsDccsLQ--xNLsH
15: 398-421 LQT_N_~N~LGLQSQAFXECPQ
15 16: 422-446 T.F~T.--nJ~z~FTR-HINApQ~
17: 447-470 LQVkN_TYCFLDTSNQF~.T.~G~PV
18: 471-497 LRH_N~KG_HFQD~lllKlN~LQTVGS
19: 498-521 _EVLILSSCGLLS_DQQAFHSLGK
20: 522-544 MSHVDLSH_SLTCDSTn~T.. ~R~,~
20 21: 545-568 GIY_N_AA_SINIISPRLLPILSQ
22: 569-592 QSTIN_SH_PLDCTCSNTRF-LTWY
As used herein, the term "human BAS-1" shall refer,
when used in a protein context, to a protein having the
amino acid sequence shown in SEQ ID NO: 2. The present
invention also encompasses proteins comprising a
substantial fragment thereof, e.g., mutants and
polymorphic variants, along with a human derived
polypeptide which exhibits the same biological function
or interacts with human BAS-1 specific b;n~;ng
components. These b;n~;n~ components typically bind to a
human BAS-1 with high affinity, e.g., at least about 100
nM, usually better than about 30 nM, preferably better
than about 10 nM, and more preferably at better than
about 3 nM. Homologous proteins are found in species
other than hl~m~n~, e.g., primates.
The term "polypeptide" as used herein includes a
fragment or segment, and encompasses a stretch of amino
acid residues of at least about 8 amino acids, generally
at least 10 amino acids, more generally at least 12 amino
acids, often at least 14 amino acids, more often at least
16 amino acids, typically at least about 18 amino acids,
more typically at least about 20 amino acids, usually at
least about 22 amino acids, more usually at least about
24 amino acids, preferably at least about 26 amino acids,
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more preferably at least about 28 amino acids, and, in
particularly preferred embodiments, at least about 30 or
more amino acids, e.g., 33, 37, 41, 45, 49, 53, 57, etc.
The term ~ligand" or "b; n~; n~ composition" refers to
molecules that bind with specificity to BAS-1, e.g., in
an antibody-antigen type fashion. Other interactions
include, e.g., ligand-receptor type, or with compounds
which associate with BAS-1, e.g., in a protein-protein
interaction, either covalent or non-covalent. The
molecule may be a polymer, or chemical reagent. No
implication as to whether BAS-1 is either the ligand or
the receptor of a ligand-receptor interaction is
represented, other than the interaction exhibit specific
affinity. A functional analog may be a ligand with
structural modifications, or may be a wholly unrelated
molecule which has a molecular shape which interacts with
the ~ o~riate ligand b;n~;n~ det~rm;nAnts. The ligands
may serve as agonists or antagonists, see, e.g., Goodman,
et al. (eds.) (1990) Goodman & Gilman's: The
Pharmacoloaical Bases of Thera~eutics (8th ed.), Pely~l-ol.
Press.
Solubility of a polypeptide or fragment depends upon
the environment and the polypeptide. Many parameters
affect polypeptide solubility, including temperature,
electrolyte environment, size and molecular
characteristics of the polypeptide, and nature of the
solvent. Typically, the temperature at which the
polypeptide is used ranges from about 4- C to about 65-
C. Usually the temperature at use is greater than about
18- C and more usually greater than about 22- C. For
~;A~nostic purposes, the temperature will usually be
about room temperature or warmer, but less than the
denaturation temperature of components in the assay. For
therapeutic purposes, the temperature will usuall~ be
body temperature, typically about 37- C for hnmAnc,
though under certain situations the temperature may be
raised or lowered in situ or in vitro.
The electrolytes will usually a~Lo~imate in situ
physiological conditions, but may be modified to higher
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or lower ionic strength where advantageous. The actual
ions may be modified to conform to stAn~Ard buffers used
in physiological or analytical contexts.
The size and structure of the polypeptide should
generally be in a substantially stable state, and usually
not in a denatured state. The polypeptide may be
associated with other polypeptides in a quaternary
structure, e.g., to confer solubility, or associated with
lipids or detergents in a ~nner which a~oximates
natural lipid bilayer interactions. In particular, it is
~elieved that the natural protein is often linked to
lipid via a PI l;nkAge.
The solvent will usually be a biologically
compatible buffer, of a type used for preservation of
biological activities, and will usually a~Loximate a
physiological solvent. Usually the solvent will have a
neutral pH, typically between about 5 and 10, and
preferably about 7.5. On some occasions, a detergent
will be added, typically a mild non-denaturing one, e.g.,
CHS (cholesteryl hemisuccinate) or CHAPS (3-([3-
cholamidopropyl]dimethylammonio)-1-propane sulfonate), or
in a low enough detergent concentration to not disrupt
the tertiary structure of the protein.
Solubility is reflected by sedimentation measured in
Svedberg units, which are a measure of the s~;mentation
velocity of a molecule under particular conditions. The
det~rm;nAtion of the sç~;m~ntation velocity was
classically performed in an analytical ultracentrifuge,
but is typically now performed in a stAn~Ard
ultracentrifuge. See, Freifelder (1982) Phvsical
siock~mistrv (2d ed.), W.~. Freeman; and Cantor and
S~h;m~l (1980) Bio~hvsical Chemistrv, parts 1-3, W.H.
Freeman & Co., San Francisco. As a crude det~rm;n~tion,
a sample contA; n; n~ a putatively soluble polypeptide is
spun in a st~n~Ard full sized ultracentrifuge at about
- 50K rpm for about 10 minutes, and soluble molecules will
remain in the supernatant. A soluble particle or
polypeptide will typically be less than about 30S, more
typically less than about 15S, usually less than about
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10S, more usually less than about 6S, and, in particular
embodiments, preferably less than about 4S, and more
preferably less than about 3S.
5 III. Physical Variants
This invention also encompasses proteins or peptides
having substantial amino acid sequence identity with the
amino acid sequence of the human BAS-1. It will embrace,
e.g., 1-fold, 2-fold, and 3-fold conservative
10 substitutions. Preferably the substitutions will be away
from the conserved cysteines, and often will be in the
regions away from the helical structural ~om~;n~. Such
variants may be useful to produce specific antibodies,
and often will share many or all biological properties.
Amino acid sequence identity is determined by
optimizing residue matches. This changes when
considering conservative substitutions as matches.
Conservative substitutions typically include
substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid,
glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. Similar
amino acid sequences are intended to include natural
allelic variations in each respective protein sequence.
Typical homologous proteins or peptides will have from
85-100% identity (if gaps can be introduced), to 90-100%
identity (if conservative substitutions are included)
with the amino acid sequence of the human BAS-1 Identity
measures will be at least about 85%, generally at least
about 87%, often at least about 89%, typically at least
about 91%, usually at least about 93%, more usually at
least about 95%, preferably at least about 9796, and more
preferably at least about 98%, and in particularly
preferred embodiments, at least about 99% or more. See
also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;
Sankoff, et al. (1983) Chapter One in Time War~s, Strincr
Edits, and Macromolecules: The Theorv and Practice of
Sec~uence Com~arison Addison-Wesley, Re~-l;ng, MA; and
software packages from IntelliGenetics, Mountain View,
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1~
CA; and the University of Wisconsin Genetics Computer
Group, Madison, WI.
The isolated human BAS-1 DNA can be readily modified
by nucleotide substitutions, nucleotide deletions,
nucleotide insertions, and inversions of nucleotide
stretches. These modifications will result in novel DNA
sequences which encode useful antigens, their
derivatives, or proteins having similar or antagonist
activity. These modified sequences can be used to
produce mutant antigens or to ~nh~nce expression.
Enhanced expression may involve gene amplification,
increased transcription, increased translation, and other
mech~n; ~m~. Such mutant BAS-1 derivatives include
predetermined or site-specific mutations of the
respective protein or its fragments. "Mutant BAS-1"
encompasses a polypeptide otherwise sharing important
features of the human BAS-1 as set forth above, but
having an amino acid sequence which differs from that of
BAS-1 as found in nature, whether by way of deletion,
substitution, or insertion. In particular, "site
specific mutant BAS-1" is defined as having homology with
an antigen defined in SEQ ID NO: 2, and as sharing
various biological activities with those antigens.
Similar concepts apply to different BAS-1 proteins,
particularly those found in various other primates. As
stated before, it is P~rh~ized that descriptions are
generally meant to encompass additional BAS-1 proteins,
not limited solely to the human embodiment specifically
discussed.
Although site specific mutation sites are
predetermined, mutants need not be site specific. Human
BAS-1 mutagenesis can be conducted by making amino acid
insertions or deletions. Substitutions, deletions,
insertions, or any combinations may be generated to
arrive at a final construct. Insertions include amino-
or carboxy- t~rm; n~ 1 fusions. ~n~ mutagenesis can be
conducted at a target codon and the expressed mutants can
then be screened for the desired activity. Methods for
making substitution mutations at predetermined sites in
CA 022~4843 1998-11-13
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13
DNA having a known sequence are well known in the art,
e.g., by M13 primer mutagenesis. See also Sambrook, et
al. (1989) and Ausubel, et al. (1987 and Supplements).
The mutations in the DNA normally should not place
coding sequences out of reading frames and preferably
will not create complementary regions that could
hybridize to produce secondary mRNA structure such as
loops or hairpins.
The present invention also provides recombinant
proteins, e.g., heterologous fusion proteins using
segments from these proteins. A heterologous fusion
protein is a fusion of proteins or segments which are
naturally not normally fused in the same manner. Thus,
the fusion product of an ;n~ noglobulin with a BAS-l
polypeptide is a continuous protein molecule having
sequences fused in a typical peptide linkage, typically
made as a single translation product and exhibiting
properties derived from each source peptide. A similar
concept applies to heterologous nucleic acid sequences.
In addition, new constructs may be made from
combining similar functional domains from other proteins.
For example, ligand-binding or other segments may be
"swapped" between different new fusion polypeptides or
fragments. See, e.g., Cunningham, et al. (1989~ Science
243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem.
263:15985-15992. Thus, new ch;m~ric polypeptides
exhibiting new combinations of specificities will result
from the functional linkage of ligand-b; n~; ng
specificities and other functional ~n~;n~,
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will
produce suitable synthetic DNA fragments. A double
stranded fragment will often be obt~;ne~ either by
synthesizing the complementary strand and ~nne~ling the
strand together under appropriate conditions or by ~;ng
the complementary strand using DNA polymerase with an
appropriate primer sequence.
IV. Functional Variants
.. ...
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1~
The blocking of physiological response to BAS-l
antigens may result from the inhibition of binding of the
ligand to the BAS-l, likely through competitive
inhibition. Thus, in vitro assays of the present
invention will often use isolated protein, membranes from
cells expressing a recombinant BAS-l, soluble fragments
comprising ligand binding segments of these antigens, or
fragments attached to solid phase substrates. These
assays will also allow for the diagnostic det~rm;n~tion
of the effects of either binding segment mutations and
modifications, or ligand mutations and modifications,
e.g., ligand analogs.
This invention also contemplates the use of
competitive drug screening assays, e.g., where
neutralizing antibodies to the antigen or antigen
fragments compete with a test compound for b;n~;ng to the
protein. In this manner, the antibodies can be used to
detect the presence of any polypeptide which shares one
or more b; n~; n~ sites of the antigen and can also be used
to occupy b; n~; n~ sites on the protein that might
otherwise be occupied by a ligand.
Additionally, neutralizing antibodies against the
BAS-l and soluble fragments of the BAS-l which contain a
high affinity ligand binding site, can be used to inhibit
ligand function in tissues, e.g., tissues experiencing
abnormal physiology.
"Derivatives" of the BAS-l antigens include amino
acid sequence mutants, glycosylation variants, and
covalent or aggregate conjugates with other chemical
moieties. Covalent derivatives can be prepared by
linkage of functionalities to groups which are found in
the BAS-l antigen amino acid side ~h~;n~ or at the N- or
C- termini, by means which are well known in the art.
These derivatives can include, without limitation,
aliphatic esters or amides of the carboxyl tPrm;n~ or
of residues cont~;n;ng carboxyl side rh~;n~, O-acyl
derivatives of hydroxyl group-contA;n;ng residues, and N-
acyl derivatives of the amino term;n~l amino acid or
amino-group cont~;n;n~ residues, e.g., lysine or
CA 022~4843 1998-11-13
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I j
arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby
forming alkanoyl aroyl species.
In particular, glycosylation alterations are
included, e.g., made by modifying the glycosylation
patterns of a polypeptide during its synthesis and
processing, or in further processing steps. Particularly
preferred means for accomplishing this are by exposing
the polypeptide to glycosylating enzymes derived from
cells which normally provide such processing, e.g., human
glycosylation enzymes. Deglycosylation enzymes are also
contemplated. Also embraced are versions of the same
primary amino acid sequence which have other minor
modifications, including phosphorylated amino acid
residues, e.g., phosphotyrosine, phosphoserine, or
phosphothreonine.
A major group of derivatives are covalent conjugates
of the BAS-1 antigens or fragments thereof with other
proteins of polypeptides. These derivatives can be
synthesized in recombinant culture such as N- or C-
terminal fusions or by the use of agents known in the art
for their usefulness in cross-linking proteins through
reactive side groups. Preferred ligand derivatization
sites with cross-linking agents are at free amino groups,
carbohydrate moieties, and cysteine residues.
Fusion polypeptides between the BAS-1 antigens and
other homologous or heterologous proteins are also
provided. Homologous polypeptides may be fusions between
different surface markers, resulting in, for instance, a
hybrid protein exhibiting ligand specificity of one or
more marker proteins. Likewise, heterologous fusions may
be constructed which would exhibit a combination of
properties or activities of the derivative proteins.
Typical examples are fusions of a reporter polypeptide,
e.g., luciferase, with a segment or ~om~in of an antigen,
e.g., a ligand-b; n~; ng segment, so that the presence or
location of a desired ligand may be easily determined.
See, e.g., Dull, et al., U.S. Patent No. 4,859,609, which
is hereby incorporated herein by reference. Other gene
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648
1~
fusion partners include bacterial g-galactosidase, trpE,
Protein A, ~-lactamase, alpha amylase, alcohol
dehydrogenase, and yeast alpha mating factor. See, e.g.,
Godowski, et al. (1988) Science 241:812-816.
The phosphoramidite method described by Beaucage and
Carruthers (1981) Tetra. Letts. 22:1859-1862, will
produce suitable synthetic DNA fragments. A double
stranded fragment will often be obtained either by
synthesizing the complementary strand and annealing the
strand together under a~o~riate conditions or by ~; ng
the complementary strand using DNA polymerase with an
appropriate primer sequence.
Such polypeptides may also have amino acid residues
which have been chemically modified by phosphorylation,
sulfonation, biotinylation, or the addition or removal of
other moieties, particularly those which have molecular
shapes similar to phosphate groups. In some embodiments,
the modifications will be useful labeling reagents, or
serve as purification targets, e.g., affinity ligands.
Fusion proteins will typically be made by either
recombinant nucleic acid methods or by synthetic
polypeptide methods. Techniques for nucleic acid
manipulation and expression are described generally, for
example, in Sambrook, et al. (1989) Molecular Clonina: A
LaboratorY Manual (2d ed.), Vols. 1-3, Cold Spring Harbor
Laboratory. Techniques for synthesis of polypeptides are
described, for example, in Merrifield (1963) J. Amer.
Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232:
341-347; and Atherton, et al. (1989) Solid -Phase Peptide
Svnthesis: A Practical An~roach, IRL Press, Oxford.
This invention also contemplates the use of
derivatives of the BAS-1 antigens other than variations
in amino acid sequence or glycosylation. Such
derivatives may involve covalent or aggregative
association with chemical moieties. These derivatives
generally fall into three classes: (1) salts, (2) side
chain and t~rm;n~l residue covalent modifications, and
(3) adsorption complexes, for example with cell
membranes. Such covalent or aggregative derivatives are
CA 022~4843 1998-11-13
W097/~4S2 PCT~S97/07648
l7
useful as immllnogens, as reagents in immunoassays, or in
purification methods such as for affinity purification of
ligands or other binding ligands. For example, a BAS-l
antigen can be immobilized by covalent bonding to a solid
support such as cyanogen bromide-activated Sepharose, by
methods which are well known in the art, or adsorbed onto
polyolefin surfaces, with or without glutaraldehyde
cross-linking, for use in the assay or purification of
anti-BAS-l antibodies or its ligAnd. The BAS-l antigens
can also be labeled with a detectable group, for example
radioiodinated by the chloramine T procedure, covalently
bound to rare earth chelates, or conjugated to another
fluorescent moiety for use in diagnostic assays.
A solubilized BAS-l antigen of this invention can be
used as an immunogen for the production of antisera or
antibodies specific for the antigen or any fragments
thereof. The purified antigens can be used to screen
monoclonal antibodies or antigen-b; n~; ng fragments
prepared by ;mml]n;zation with various forms of impure
preparations contA;n;n~ the protein. In particular, the
term "antibodies" also encompasses antigen b;n~;ng
fragments of natural antibodies. The purified BAS-l can
also be used as a reagent to detect antibodies generated
in response to the presence of elevated levels of BAS-l
or cell fragments contA;n;n~ the antigen, both of which
may be ~;Agnostic of an abnormal or specific
physiological or disease condition. Additionally, BAS-l
fragments may also serve as immunogens to produce the
antibodies of the present invention, as described
immediately below. For example, this invention
contemplates antibodies raised AgA; n~t amino acid
sequences of, or encoded by nucleotide se~lenc~s shown in
SEQ ID NO: 1 or fragments thereof. In particular, this
invention contemplates antibodies having b;n~;n~ affinity
to or being raised against specific fragments which are
predicted to lie outside of the lipid bilayer.
Additionally, various constructs may be produced from
fusion of a membrane associating segment to the otherwise
extracellular exposed portion of the molecule.
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648-
The present invention contemplates the isolation of
additional closely related variants. It is high~y li~ely
that allelic variations exist in different individuals
exhibiting, e.g., better than 90-97% identity to the
embodiment described herein.
The invention also provides means to isolate a group
of related antigens displaying both distinctness and
similarities in structure, expression, and function.
Elucidation of many of the physiological effects of the
antigens will be greatly accelerated by the isolation and
characterization of distinct species counterparts of the
antigens. In particular, the present invention provides
useful probes for identifying additional homologous
genetic entities in different species.
The isolated genes will allow transformation of
cells lacking expression of BAS-l, e.g., either species
types or cells which lack correspo~; ng antigens and
exhibit negative background activity. Expression of
transformed genes will allow isolation of antigenically
pure cell lines, with defined or single specie variants.
This approach will allow for more sensitive detection and
discrimination of the physiological effects of any
ligands. Subcellular fragments, e.g., cytoplasts or
membrane fragments, can be isolated and used.
Dissection of the critical structural elements which
effect the various differentiation functions provided by
ligands is possible using stAn~Ard techniques of modern
molecular biology, particularly in comparing members of
the related class. See, e.g., the homolog-scAnn;ng
mutagenesis technique described in Cunningham, et al.
(1989) Science 243:1339-1336; and approAche~ used in
O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992; and
Lechleiter, et al. (1990) EMBO J. 9:4381-4390.
In particular, ligand b;n~;ng segments can be
substituted between species variants to determine what
structural features are important in both ligand b;n~;ng
affinity and specificity. An array of different BAS-l
variants will be used to screen for ligands exhibiting
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07~8--
1~
combined properties of interaction with different species
variants.
Intracellular functions would probably involve
segments of the antigen which are normally accessible to
the cytosol. However, antigen internalization may occur
under certain circumstances, and interaction between
intracellular components and the designated
"extracellular" segments may occur. The specific
segments of interaction of BAS-l with other intracellular
components may be identified by mutagenesis or direct
biochemical means, e.g., cross-lin~ing or affinity
methods. Structural analysis by crystallographic or
other physical methods will also be applicable. Further
investigation of the mech~n;sm of signal transduction
will include study of associated components which may be
isolatable by affinity methods.
Further study of the expression and control of BAS-l
antigens will be pursued. The controlling elements
associated with the antigens exhibit differential
developmental, tissue specific, or other expression
patterns. Upstream or downstream genetic regions, e.g.,
control elements, are of interest.
Structural studies of the BAS-l antigens will lead
to design of new ligands, particularly analogs exhibiting
agonist or antagonist properties. This can be combined
with previously described screening methods to isolate
ligands exhibiting desired spectra of activities.
Expression in other cell types will often result in
glycosylation differences in a particular antigen.
Various species variants may exhibit distinct functions
based upon structural differences other than amino acid
sequence. Differential modifications may be responsible
for differential function, and elucidation of the effects
are now made possible.
Thus, the present invention provides important
developmental antigens and reagents developed from them.
Although the foregoing description has focused primarily
upon the human BAS-l, those of skill in the art will
;mmeA;~tely recognize that the invention encompasses
~ , . .
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other BAS-1 antigens, e.g., primate and other mammalian
species variants.
V. Antibodies
Antibodies can be raised to the various allelic
variants of BAS-1 antigens and fragments thereof, both in
their naturally occurring forms and in their recombinant
forms. Additionally, antibodies can be raised to BAS-1
in either their active forms or in their inactive forms.
Anti-idiotypic antibodies are also contemplated.
Antibodies, including b; n~; ng fragments and single
chain versions, against predetermined fragments of BAS-1
can be raised by ;mm~n; zation of ~n;m~l S with conjugates
of the fragments with immunogenic proteins. Monoclonal
antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding
to normal or defective BAS-1, or screened for agonistic
or antagonistic ligand activity. These monoclonal
antibodies will usually bind with at least a KD of better
than about 1 mM, more usually better than about 300 ~M,
typically better than about 10 ~M, more typically better
than about 30 ~M, preferably better than about 10 ~M, and
more preferably better than about 3 ~M, e.g., 1 ~M, 300
nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 300 pM, 100 pM, 30
pM, etc.
The antibodies, including antigen b; n~; ng fragments,
of this invention can have significant diagnostic or
therapeutic value. They can be potent antagonists that
bind to BAS-1 and inhibit ligand b;n~;ng or inhibit the
ability of a ligand to elicit a biological response.
They also can be useful as non-neutralizing antibodies
and can be coupled to toxins or radionuclides so that
when the antibody binds to the antigen, the cell itself
is killed. Further, these antibodies can be conjugated
to drugs or other therapeutic agents, either directly or
indirectly by means of a linker.
The antibodies of this invention can also be useful
in ~;~gnostic applications. As capture or non-
neutralizing antibodies, they can bind to the BAS-1
CA 022~4843 1998-11-13
W097/444S2 PCT~S97/07648 .
~1
without inhibiting ligand b;n~;n~. As neutralizing
antibodies, they can be useful in competitive b;n~; ng
assays. They will also be useful in detecting or
quantifying BAS-1 or its ligands.
BAS-1 fragments may be joined to other materials,
particularly polypeptides, as fused or covalently joined
polypeptides to be used as ;m~llnogens. A BAS-1 and its
fragments may be fused or covalently linked to a variety
of immunogens, such as keyhole limpet hemocyanin, bovine
serum albumin, tetanus toxoid, etc. See Microbiolooy,
Hoeber Medical Division, Harper and Row, 1969;
Landsteiner (1962) S~ecificitv of Seroloaical Reactions,
Dover Publications, New York, and Williams, et al. (1967)
Methods in ImmunolooY and ImmunochemistrY, Vol. 1,
Academic Press, New York, for descriptions of methods of
preparing polyclonal antisera. A typical method involves
hyper;mmlln;zation of an An;m~l with an antigen. The
blood of the An;mAl is then collected shortly after the
repeated ;mmlm;zations and the gamma globulin is
isolated. Alternatively, cells may be collected for
producing hybridomas.
In some instances, it is desirable to prepare
monoclonal antibodies from various ~mmAlian hosts, such
as mice, rodents, primates, hl]m~ns, etc. Description of
techniques for preparing such monoclonal antibodies may
be found in, e.g., Stites, et al. (eds.) Basic and
Clinical Immunologv (4th ed.), Lange Medical
Publications, Los Altos, CA, and references cited
therein; Harlow and ~ane (1988) Antibodies: A Laboratory
Manual, CSH Press; Goding (1986) Monoclonal Antibodies:
Princi~les and Practice (2d ed.) AcA~m~c Press, New
York; and particularly in Kohler and Milstein (1975) in
Natl~re 256:495-497, which discusses one method of
generating monoclonal antibodies. Summarized brie~ly,
this method involves injecting an An;~l with an
;mmllnogen. The An;m-l is then sacrificed and cells taken
from its spleen, which are then fused with myeloma cells.
The result is a hybrid cell or "hybridoma" that is
capable of reproducing in vitro. The population of
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a~
hybridomas is then screened to isolate individual clones,
each of which secrete a single antibody species to the
immunogen. In this manner, the individual antibody
species obtained are the products of immortalized and
cloned single B cells from the immune ~n;m~l generated in
response to a specific site recognized on the ;m~l~nogenic
substance.
Other suitable techniques involve in vitro exposure
of lymphocytes to the antigenic polypeptides or
alternatively to selection of libraries of antibodies in
phage or similar vectors. See, Huse, et al. (1989)
"Generation of a Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage T.Amh~ Science
246:1275-1281; and Ward, et al. (1989) Nature 341:544-
546. The polypeptides and antibodies of the presentinvention may be used with or without modification,
including ch;m~ric or hl~m~n;zed antibodies. Frequently,
the polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance
which provides for a detectable signal. A wide variety
of labels and conjugation techniques are known and are
reported extensively in both the scientific and patent
literature. Suitable labels include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent
moieties, chemiluminescent moieties, magnetic particles,
and the like. Patents, te~ch; ng the use of such labels
include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also,
recombinant immunoglobulins may be produced, see Cabilly,
U.S. Patent No. 4,816,567.
The antibodies of this invention can also be used
for affinity chromatography in isolating the protein.
Columns can be prepared where the antibodies are linked
to a solid support, e.g., particles, such as agarose,
SEPHADEX, or the like, where a cell lysate may be passed
through the column, the column w~he~, followed by
increasing concentrations of a mild denaturant, whereby
the purified BAS-l protein will be released.
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2 3
The antibodies may also be used to screen expression
libraries for particular expression products. Usually
the antibodies used in such a procedure will be labeled
with a moiety allowing easy detection of presence of
antigen by antibody binding.
Antibodies raised against a BAS-1 antigen will also
be used to raise anti-idiotypic antibodies. These will
be useful in detecting or diagnosing various
immunological conditions related to expression of the
respective antigens.
VI. Nucleic Acids
The human BAS-1 probe, or fragments thereof, will be
used to identify or isolate nucleic acids encoding
homologous proteins from other species, or other related
proteins in the same or another species.
This invention contemplates use of isolated DNA or
fragments to encode a biologically active corresponding
BAS-1 polypeptide. In addition, this invention covers
isolated or recombinant DNA which encodes a biologically
active protein or polypeptide which is capable of
hybridizing under appropriate conditions with the DNA
sequences described herein. Said biologically active
protein or polypeptide can be an intact BAS-1, or
fragment, and have an amino acid sequence encoded by a
nucleic acid shown in SEQ ID NO: 1. Further, this
invention covers the use of isolated or recombinant DNA,
or fragments thereof, which encodes a protein which is
homologous to a BAS-1 or which was isolated using cDNA
encoding human BAS-1 as a probe. The isolated DNA can
have the respective regulatory sequences in the 5' and 3'
flanks, e.g., promoters, ~nh~ncers, poly-A addition
signals, and others.
An '~isolated" nucleic acid is a nucleic acid, e.g.,
an RNA, DNA, or a mixed polymer, which is substantially
separated from other components which naturally accompany
a na~ive sequence, e.g., ribosomes, polymerases, and
flanking genomic seguences from the originating species.
The invention embraces a nucleic acid sequence which has
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W097/444S2 PCT~S97/07648 -
~ '1
been removed from its naturally occurring environment,
and includes recombinant or cloned DNA isolates and
chemically synthesized analogs or analogs biologically
synthesized by heterologous systems. A substantially
pure molecule includes isolated forms of the molecule.
An isolated nucleic acid will generally be a
homogeneous composition of molecules, but will, in some
embodiments, contain minor heterogeneity. This
heterogeneity is typically found at the polymer ends or
portions not critical to a desired biological function or
activity.
A '~recombinant" nucleic acid is defined either by
its method of production or its structure. In reference
to its method of production, e.g., a product made by a
process, the process is use of recombinant nucleic acid
techniques, e.g., involving human intervention in the
nucleotide sequence. Alte~rnatively, it can be a nucleic
acid made by generating a sequence comprising fusion of
two fragments which are not naturally contiguous to each
other, but is meant to exclude products of nature, e.g.,
naturally occurring mutants. Thus, for example, products
made by transforming cells with any unnaturally occurring
vector is encompassed, as are nucleic acids comprising
sequence derived using any synthetic oligonucleotide
process. Such is often done to replace a codon with a
re~-ln~nt codon encoding the same or a conservative amino
acid, while typically introducing or removing , e.g., a
restriction or sequence recognition site. Alternatively,
it is performed to join together nucleic acid segments of
desired functions to generate a single genetic entity
comprising a desired combination of functions not found
in the commonly available natural forms. Restriction
enzyme recognition sites are often the target of such
artificial manipulations, but other site specific
3~ targets, e.g., promoters, DNA replication sites,
regulation sequences, control sequences, or other useful
features may be incorporated by design. A s;m;l~r
concept is intended for a rec~mh;n~nt, e.g., fusion,
polypeptide. Specifically included are synthetic nucleic
CA 022~4843 1998-11-13
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~ LS
acids which, by genetic code re~l~n~ncy, encode similar
polypeptides to fragments of these antigens, and fusions
of sequences from various different species variants.
A "fragment" in a nucleic acid context is a
contiguous segment of at least about 17 nucleotides,
generally at least 20 nucleotides, more generally at
least about 23 nucleotides, ordinarily at least about 26
nucleotides, more ordinarily at least about 29
nucleotides, often at least about 32 nucleotides, more
often at least about 35 nucleotides, typically at least
about 38 nucleotides, more typically at least about 41
nucleotides, usually at least about 44 nucleotides, more
usually at least about 47 nucleotides, preferably at
least about 50 nucleotides, more preferably at least
about 53 nucleotides, and in particularly preferred
embodiments will be at least about 56 or more
nucleotides, e.g., ~0, 75, 100, 150, 200, 250, 300, etc.
A DNA which codes for a BAS-l protein will be
particularly useful to identify genes, mRNA, and cDNA
species which code for related or homologous antigens, as
well as DNAs which code for homologous proteins from
different species. Various BAS-l proteins should be
similar in sequence and are encompassed herein. However,
even proteins that have a more distant evolutionary
relation~h;p to the BAS-l can readily be isolated using
these sequences if they exhibit sufficient similarity.
Primate BAS-l proteins are of particular interest.
This invention further encompasses recombinant DNA
molecules and fragments having a DNA sequence identical
to or highly homologous to the isolated DNAs set forth
herein. In particular, the se~uences will often be
operably linked to DNA segments which control
transcription, translation, and DNA replication.
Alternatively, recombinant clones derived from the
genomic sequences, e.g., cont~;n;ng introns, will be
useful for transgenic studies, including, e.g.,
transgenic cells and org~n;~m~, and for gene therapy.
See, e.g., Goodnow (1992) "Transgenic ~n;m~ in Roitt
(ed.) Encvclopedia of Tmmllnoloqv Ac~m'c Press, San
CA 022~4843 1998-11-13
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~ 2~
Diego, pp. 1502-1504; Travis (1992) Science 256:1392-
1394; Kuhn, et al. ~1991) Science 254:707-710; Capecchi
(1989) Science 244:1288; Robertson (1987)(ed.)
Teratocarcinomas and Embrvonic Stem Cells: A Practical
A~roach IRL Press, Oxford; and Rosenberg ~1992) J.
Clinical OncoloqY 10:180-199.
Homologous nucleic acid sequences, when compared,
exhibit significant sequence similarity. The st~n~rds
for homology in nucleic acids are either measures for
homology generally used in the art by sequence comparison
or based upon hybridization conditions. The
hybridization conditions are described in greater detail
below.
Substantial identity in the nucleic acid sequence
comparison context means either that the segments, or
their complementary strands, when compared, are identical
when optimally aligned, with appropriate nucleotide
insertions or deletions, in at least about 50% of the
nucleotides, generally at least about 56%, more generally
at least about 59%, ordinarily at least about 62%, more
ordinarily at least about 65%, often at least about 68%,
more often at least about 71%, typically at least about
74%, more typically at least about 77%, usually at least
about 80%, more usually at least about 85%, preferably at
least about 90%, more preferably at least about 95 to 98%
or more, and in particular embodiments, as high at about
99% or more of the nucleotides. Alternatively,
substantial identity exists when the segments will
hybridize under selective hybridization conditions, to a
strand, or its complement, typically using a sequence
derived from SEQ ID NO: 1. Typically, selective
hybridization will occur when there is at least about 55%
homology over a stretch of at least about 14 nucleotides,
preferably at least about 65%, more preferably at least
about 75%, and most preferably at least about 90%. See,
Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length
of homology comparison, as described, may be over longer
stretches, and in certain embodiments will be over a
stretch of at least about 17 nucleotides, usually at
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least about 20 nucleotides, more usually at least about
24 nucleotides, typically at least about 28 nucleotides,
more typically at least about 40 nucleotides, preferably
at least about 50 nucleotides, and more preferably at
least about 75 to 100 or more nucleotides, e.g., 125,
150, 200, 250, 300, etc.
Stringent conditions, in referring to identity in
the hybridization context, will be stringent combined
conditions of salt, temperature, organic solvents, and
other parameters typically controlled in hybridization
reactions. Stringent temperature conditions will usually
include temperatures in excess of about 30 C, more
usually in excess of about 37- C, typically in excess of
about 45- C, more typically in excess of about ~5- C,
preferably in excess of about 65- C, and more preferably
in excess of about 70 C. Stringent salt conditions will
ordinarily be less than about 500 mM, usually less than
about 350 mM, more usually less than about 200 mM,
typically less than about 150 mM, preferably less than
about 100 mM, and more preferably less than about 50 mM.
However, the combination of parameters is much more
important than the measure of any single parameter. See,
e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-
370.
BAS-1 from other human subjects can be cloned and
isolated by hybridization or PCR. Alternatively,
preparation of an antibody preparation which exhibits
less allelic specificity may be useful in expression
cloning approaches. Allelic variants may be
characterized using, e.g., a comh;nAtion of re~-m~Ant PCR
and seguence analysis, e.g., using defined primers,
thereby providing information on allelic variation in a
human population.
VII. Making BAS-1; Mimetics
DNA which encodes the BAS-1 antigen or fragments
thereof can be obtA;ne~ by chemical synthesis, screening
cDNA libraries, or by screening genomic libraries
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prepared from a wide variety of cell lines or tissue
samples.
This DNA can be expressed in a wide variety of host
cells for the synthesis of a full-length antigen or
fragments which can in turn, e.g., be used to generate
polyclonal or monoclonal antibodies; for binding studies;
for construction and expression of modified molecules;
and for structure/function studies. Each antigen or its
fragments can be expressed in host cells that are
transformed or transfected with appropriate expression
vectors. These molecules can be substantially purified
to be free of protein or cellular contAm;n~nts, e.g.,
those derived from the recombinant host, and therefore
are particularly useful in pharmaceutical compositions
when combined with a pharmaceutically acceptable carrier
and/or diluent. The antigen, or portions thereof, may be
expressed as fusions with other proteins.
Expression vectors are typically self-replicating
DNA or RNA constructs cont~;n;ng the desired antigen gene
or its fragments, usually operably linked to suitable
genetic control elements that are recognized in a
suitable host cell. These control elements are capable
of effecting expression within a suitable host. The
specific type of control elements necessary to effect
expression will depend upon the eventual host cell used.
Generally, the genetic control elements can include a
prokaryotic promoter system or a eukaryotic promoter
expression control system, and typically include a
transcriptional promoter, an optional operator to control
the onset of transcription, transcription ~nh~ncers to
elevate the level of mRNA e~?reSsion~ a sequence that
encodes a suitable ribosome b; n~; ng site, and sequences
that term;n~te transcription and translation. Expression
vectors also usually contain an origin of replication
that allows the vector to replicate independently of the
host cell.
The vectors of this invention contain DNA which
encodes a human BAS-l antigen, or a fragment thereof
encoding a biologically active polypeptide. The DNA can
CA 022~4843 1998-11-13
W097l44452 PCT~S97/07648--
2q
be under the control of a viral promoter and can encode a
selection marker. This invention further contemplates
use of such expression vectors which are capable of
expressing eukaryotic cDNA coding for a human BAS-l
antigen in a prokaryotic or eukaryotic host, where the
vector is compatible with the host and where the
eukaryotic cDNA coding for the antigen is inserted into
the vector such that growth of the host cont~; n;ng the
vector expresses the cDNA in auestion. Usually,
expression vectors are designed for stable replication in
their host cells or for amplification to greatly increase
the total number of copies of the desirable gene per
cell. It is not always necessary to require that an
expression vector replicate in a host cell, e.g., it is
possible to effect transient expression of the antigen or
its fragments in various hosts using vectors that do not
contain a replication origin that is recognized by the
host cell. It is also possible to use vectors that cause
integration of the human BAS-l gene or its fragments into
the host DNA by recombination.
Vectors, as used herein, comprise plasmids, viruses,
bacteriophage, integratable DNA fragments, and other
vehicles which enable the integration of DNA fragments
into the genome of the host. Expression vectors are
specialized vectors which contain genetic control
elements that effect expression of operably linked genes.
Plasmids are the most commonly used form of vector but
all other forms of vectors which serve an equivalent
function and which are, or become, known in the art are
suitable for use herein. See, e.g., Pouwels, et al.
(1985 and Supplements) Clon~ n~ Vectors: A Laboratorv
~1, Elsevier, N.Y., and Rodriquez, et al.
(1988)(eds.) Vectors: A SurveY of Molecular Clonina
Vectors ~nd Th~; r Uses, Buttersworth, Boston, MA.
Transformed cells are cells, preferably mammalian,
that have been transformed or transfected with human BAS-
1 vectors constructed using recombinant DNA techniques.
Transformed host cells usually express the antigen or its
fragments, but for purposes of cloning, amplifying, and
.. . " . _ .................. .. . .
CA 022~4843 1998-11-13
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3~
manipulating its DNA, do not need to express the protein.
This invention further contemplates culturing transformed
cells in a nutrient medium, thus permitting the protein
to accumulate in the culture. The protein can be
recovered, either from the culture or from the culture
medium.
For purposes of this invention, DNA sequences are
operably linked when they are functionally related to
each other. For example, DNA for a presequence or
secretory leader is operably linked to a polypeptide if
it is expressed as a preprotein or participates in
directing the polypeptide to the cell membrane or in
secretion of the polypeptide. A promoter is operably
linked to a coding sequence if it controls the
transcription of the polypeptide; a ribosome binding site
is operably linked to a coding sequence if it is
positioned to permit translation. Usually, operably
linked means contiguous and in reading frame, however,
certain genetic elements such as repressor genes are not
contiguously linked but still bind to operator sequences
that in turn control expression.
Suitable host cells include prokaryotes, lower
eukaryotes, and higher eukaryotes. Prokaryotes include
both gram negative and gram positive org~n;~m~, e.g., E.
coli and B. subtilis. Lower eukaryotes include yeasts,
e.g., S. cerevisiae and Pichia, and species of the genus
Dictyostelium. Higher eukaryotes include established
tissue culture cell lines from ~n;m~l cells, both of
non-mammalian origin, e.g., insect cells, and birds, and
of ~=~-lian origin, e.g., human, primates, and rodents.
Prokaryotic host-vector systems include a wide
variety of vectors for many different species. As used
herein, E. coli and its vectors will be used generically
to include equivalent vectors used in other prokaryotes.
A representative vector for amplifying DNA is pBR322 or
many of its derivatives. Vectors that can be used to
express the human BAS-l antigens or its fragments
include, but are not limited to, such vectors as those
cont~ining the lac promoter (pUC-series); trp promoter
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31
(pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or
pR promoters (pOTS); or hybrid promoters such as ptac
(pDR540). See Brosius, et al. (1988) "Expression Vectors
Employing Lambda-, trp-, lac-, and Ipp-derived
Promoters", in Rodriguez and Denhardt (eds.) Vectors: A
Surve~ of Molecular Cloninq Vectors and Their Uses,
Buttersworth, Boston, Chapter 10, pp. 205-236.
Lower eukaryotes, e.g., yeasts and Dictyostelium,
may be transformed with human BAS-l antigen sequence
cont~;n;ng vectors. For purposes of this invention, the
most common lower eukaryotic host is the baker's yeast,
Saccharomyces cerevisiae. It will be used to generically
represent lower eukaryotes although a number of other
strains and species are also available. Yeast vectors
typically consist of a replication origin (unless of the
integrating type), a selection gene, a promoter, DNA
encoding the desired protein or its fragments, and
sequences for translation term;n~tion, polyadenylation,
and transcription tPrm;n~tion. Suitable expression
vectors for yeast include such constitutive promoters as
3-phosphoglycerate kinase and various other glycolytic
enzyme gene promoters or such inducible promoters as the
alcohol dehydrogenase 2 promoter or metallothionine
promoter. Suitable vectors include derivatives of the
following types: self-replicating low copy number (such
as the YRp-series), self-replicating high copy number
(such as the YEp-series); integrating types (such as the
YIp-series), or mini-chromosomes (such as the
YCp-series).
Higher eukaryotic tissue culture cells are the
preferred host cells for expression of the functionally
active human BAS-l antigen protein. In principle, many
higher eukaryotic tissue culture cell lines are workable,
e.g., insect baculovirus expression systems, whether from
an invertebrate or vertebrate source. However, m~mm~lian
cells are preferred, in that the processing, both
cotranslationally and posttranslationally.
Transformation or transfection and propagation of such
cells has become a routine procedure. Examples of useful
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3~
cell lines include HeLa cells, C~;ne~e hamster ovary
(CHO) cell lines, baby rat kidney (BRK) cell lines,
insect cell lines, bird cell lines, and monkey (COS) cell
lines. Expression vectors for such cell lines usually
include an origin of replication, a promoter, a
translation initiation site, RNA splice sites (if genomic
DNA is used), a polyadenylation site, and a transcription
term;n~tion site. These vectors also usually contain a
selection gene or amplification gene. Suitable
expression vectors may be plasmids, viruses, or
retroviruses carrying promoters derived, e.g., from such
sources as from adenovirus, SV40, parvoviruses, vaccinia
virus, or cytomegalovirus. ~epresentative examples of
suitable expression vectors include pCDNAl; pCD, see
Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142;
pMClneo Poly-A, see Thomas, et al. (1987) Cell 51:503-
512; and a baculovirus vector such as pAC 373 or pAC 610.
It will often be desired to express a human BAS-l
antigen polypeptide in a system which provides a specific
or defined glycosylation pattern. In this case, the
usual pattern will be that provided naturally by the
expression system. However, the pattern will be
modifiable by exposing the polypeptide, e.g., an
unglycosylated form, to appropriate glycosylating
proteins introduced into a heterologous expression
system. For example, the BAS-l antigen gene may be co-
transformed with one or more genes encoding mammalian or
other glycosylating enzymes. Using this approach,
certain mAm~lian glycosylation patterns will be
achievable or approximated in prokaryote or other cells.
The BAS-l antigen might also be produced in a form
which is phosphatidyl inositol (PI) linked, but can be
~u~ved from membranes by treatment with a phosphatidyl
inositol cleaving enzyme, e.g., phosphatidyl inositol
phospholipase-C. This releases the antigen in a
biologically active form, and allows purification by
stAn~rd procedures of protein chemistry. See, e.g., Low
(1989) Biochim. Bio~hvs. Acta 988:427-454; Tse, et al.
(1985) Science 230:1003-1008; and Brunner, et al. (1991)
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33
J. Cell Biol. 114:1275-1283. Alternatively, purification
segments may be engineered into the sequence, e.g., at
the N-terminus or C-terminus, to assist in the
purification or detection of the protein product. Means
to remove such segments may also be engineered, e.g.,
protease cleavage sites.
Now that the entire sequence is known, the human
BAS-1 antigens, fragments, or derivatives thereof can be
prepared by conventional processes for synthesizing
peptides. These include processes such as are described
in Stewart and Young ~1984) Solid Phase Peptide
Svnthesis, Pierce Chemical Co., Rockford, IL; Bodanszky
and Bodanszky (1984) The Practice of Pe~tide Synthesis,
Springer-Verlag, New York; and Bodanszky (1984~ The
Princi~les of Pe~tide Synthesis, Springer-Verlag, New
York. For example, an azide process, an acid chloride
process, an acid anhydride process, a mixed anhydride
process, an active ester process (for example, p-
nitrophenyl ester, N-hydroxysuccinimide ester, or
cyanomethyl ester), a carbodiimidazole process, an
oxidative-reductive process, or a
dicyclohexylcarbodiimide (DCCD)/additive process can be
used. Solid phase and solution phase syntheses are both
applicable to the foregoing processes.
The human BAS-1 antigens, fragments, or derivatives
are suitably prepared in accordance with the above
processes as typically employed in peptide synthesis,
generally either by a so-called stepwise process which
comprises condensing an amino acid to the t~rm; n~ 1 amino
acid, one by one in sequence, or by coupling peptide
fragments to the t~nm; n~l amino acid. Amino groups that
are not being used in the coupling reaction must be
protected to prevent coupling at an incorrect location.
If a solid phase synthesis is adopted, the C-
ter~;n~1 amino acid is bound to an insoluble carrier or
support through its carboxyl group. The insoluble
carrier is not particularly limited as long as it has a
bi n~; ng capability to a reactive carboxyl group.
Examples of such insoluble carriers include halomethyl
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3 ~
resins, such as chloromethyl resin or bLol"ol.lethyl resin,
hydroxymethyl resins, phenol resins, tert-
alkyloxycarbonyl-hydrazidated resins, and the like.
An amino group-protected amino acid is bound in
sequence through condensation of its activated carboxyl
group and the reactive amino group of the previously
formed peptide or chain, to synthesize the peptide step
by step. After synthesizing the complete sequence, the
peptide is split off from the insoluble carrier to
produce the peptide. This solid-phase approach is
generally described by Merrifield, et al. (1963) in J.
Am. Chem. Soc. 85:2149-2156.
The prepared antigen and fragments thereof can be
isolated and purified from the reaction mixture by means
of peptide separation, for example, by extraction,
precipitation, electrophoresis and various forms of
chromatography, and the like. The human BAS-1 antigens
of this invention can be obtained in varying degrees of
purity dep~n~; n~ upon its desired use. Purification can
be accomplished by use of the protein purification
techniques disclosed herein or by the use of the
antibodies herein described in immunoabsorbant affinity
chromatography. This immunoabsorbant affinity
chromatography is carried out by first linking the
antibodies to a solid support and then contacting the
linked antibodies with solubilized lysates of small cell
lung cancer cells, lysates of other cells expressing the
BAS-1 antigens, or lysates or supernatants of cells
producing the BAS-1 antigens as a result of DNA
techniques, see below.
VIII. Uses
The present invention provides reagents which will
find use in ~ nostic applications as described
elsewhere herein, e.g., in the general description for
developmental or physiological abnormalities, or below in
the description of kits for ~;~gnosis.
This invention also provides reagents with
significant therapeutic value. The human BAS-1
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(naturally occurring or recombinant), fragments thereof
and antibodies thereto, along with compounds identified
as having binding affinity to human BAS-l, should be
useful in the treatment of conditions associated with
abnormal B cell response, including abnormal
proliferation, e.g., cancerous conditions, or
degenerative conditions. Abnormal proliferation,
regeneration, degeneration, and atrophy may be modulated
by appropriate therapeutic treatment using the
compositions provided herein. For example, a disease or
disorder associated with abnormal expression or abnormal
triggering of BAS-l should be a likely target for an
agonist or antagonist of the antigen. BAS-l likely plays
a role in activation or regulation of B cells, which
affect immunological responses, e.g., auto;mm-lne
disorders or allergic responses.
In addition, the BAS-l:BAS-l binding partner
interaction may be involved in T:B cell interactions that
permit the activtion, proliferation, and/or
differentiation of naive B cells as observed in the
hyper-IgM syndrome. If so, treatment may result form
interference with the BAS-l:BAS-l binding partner signal
transduction. Blocking of the signal may be effected,
e.g., by soluble BAS-l or antibodies to BAS-l.
The BAS-l:BAS-l hln~;ng partner interaction may also
be involved in initiating and/or regulation of the
massive proliferation of B cell blasts and centroblasts
in the dark zone of the germ;nAl centers. See, e.g.,
RAn~h~reaU and Rosset (1992) Adv. Immunol. 125:868-877.
The cell surface interactions involved in the signal to
initiate and/or regulate the Ig somatic mutation process
may also involve BAS-l, and may be regulated by agonistic
or antagonistic intervention.
Other abnormal developmental conditions are known in
each of the cell types shown to possess BAS-l mRNA by
Northern blot analysis. See Berkow (ed.) The Merck
nl1Al of Dia~n~sis and Thera~y, Merck & Co., Rahway,
N.J.; and Thorn, et al. Harrison's Principles of Tnternal
Medicine, McGraw-Hill, N.Y. For example, therapeutic
.... ~ , ....... . . . . .
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3~
;mml~nosuppression may be achieved by blocking T
lymphocyte and B lymphocyte interaction through this
molecule. It will represent an important therapy for
controlling autoimmune diseases and graft rejection
during transplantation. Alternatively, anti-B cell tumor
therapy may be achieved by blocking the growth and
survival effects of BAS-l on B cells. The blockage may
be effected by blocking binding compositions, e.g.,
neutralizing antibodies, or by molecules which interfere
with the interaction of BAS-l with its counterreceptor.
Soluble BAS-l may block the counterreceptor interaction.
Recombinant BAS-l or BAS-l antibodies can be
purified and then ~m; n;~tered to a patient. These
reagents can be combined for therapeutic use with
additional active ingredients, e.g., in conventional
ph~rm~ceutically acceptable carriers or diluents, e.g.,
;mml~nogenic adjuvants, along with physiologically
innocuous stabilizers and excipients. These combinations
can be sterile filtered and placed into dosage forms as
by lyophilization in dosage vials or storage in
stabilized aqueous preparations. This invention also
contemplates use of antibodies or b;n~ing fragments
thereof which are not complement b;n~;ng.
Drug screening using BAS-l or fragments thereof can
be performed to identify compounds having b;n~;ng
affinity to BAS-l, including isolation of associated
components. Subsequent biological assays can then be
utilized to det~rm;ne whether the compound has intrinsic
stimulating activity and is therefore a blocker or
antagonist in that it blocks the activity of a ligand.
Likewise, a compound having intrinsic stimulating
activity can activate the antigen and is thus an agonist
in that it simulates the activity of BAS-l. This
invention further contemplates the therapeutic use of
antibodies to BAS-l as antagonists. This approach should
be particularly useful with other BAS-l species variants.
The quantities of reagents necessary for effective
therapy will depend upon many different factors,
including means of ~m;n;~tration, target site,
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physiological state of the patient, and other medicants
~m; n; stered. Thus, treatment dosages should be titrated
to optimize safety and efficacy. Typically, dosages used
in vitro may provide useful guidance in the amounts
useful for in situ administration of these reagents.
~n;m~l testing of effective doses for treatment of
particular disorders will provide further predictive
indication of human dosage. Various considerations are
described, e.g., in Gilman, et al. (eds.) (1990) Goodman
and Gilman's: The Pharmacoloaical Bases of Therapeutics,
8th Ed., Pergamon Press; and Reminqton's Pharmaceutical
Sciences, 17th ed. (1990), Mack Publishing Co., Easton,
Penn. Methods for administration are discussed therein
and below, e.g., for oral, intravenous, intraperitoneal,
or intramuscular ~m; n; stration, transdermal diffusion,
and others. Pharmaceutically acceptable carriers will
include water, saline, buffers, and other compounds
described, e.g., in the Merck Index, Merck ~ Co., Rahway,
New Jersey. Dosage ranges would ordinarily be expected
to be in amounts lower than 1 mM concentrations,
typically less than about 10 ~M concentrations, usually
less than about 100 nM, preferably less than about 10 pM
(picomolar), and most preferably less than about 1 fM
(femtomolar), with an appropriate carrier. Slow release
formulations, or a slow release apparatus will often be
utilized for continuous ~m;n;stration.
Human BAS-l, fragments thereof, and antibodies to it
or its fragments, antagonists, and agonists, may be
a~m;n;stered directly to the host to be treated or,
dep~n~;ng on the size of the compounds, it may be
desirable to conjugate them to carrier proteins such as
ovalbumin or serum albumin prior to their ~m;n;~tration.
Therapeutic formulations may be ~m;n;stered in many
conventional dosage formulations. While it is possible
for the active ingredient to be ~m;n;stered alone, it is
preferable to present it as a ph~rm~ceutical formulation.
Formulations typically comprise at least one active
ingredient, as defined above, together with one or more
acceptable carriers thereof. Each carrier should be both
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3~
pharmaceutically and physiologically acceptable in the
sense of being compatible with the other ingredients and
not injurious to the patient. Formulations include those
suitable for topical, oral, rectal, nasal, or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration. The formulations may
conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of
p~rm~Cy. See, e.g., Gilman, et al. (eds.) (1990)
Goodman and Gilman's: The ph~rm~oloaical Bases of
Thera~eutics, 8th Ed., Pergamon Press; and Reminqton's
Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing
Co., Easton, Penn. The therapy of this invention may be
combined with or used in association with other
chemotherapeutic or chemopreventive agents.
Both the naturally occurring and the recomb;n~nt
form of the BAS-1 antigens of this invention are
particularly useful in kits and assay methods which are
capable of screening compounds for b; n~; ng activity to
the proteins. Several methods of automating assays have
been developed in recent years so as to permit screening
of tens of thousands of compounds in a short period.
See, e.g., Fodor, et al. (1991) Science 251:767-773,
which describes means for testing of hln~;n~ affinity by
a plurality of defined polymers synthesized on a solid
substrate. The development of suitable assays can be
greatly facilitated by the availability of large amounts
of purified, soluble BAS-1 as provided by this invention.
For example, antagonists can normally be found once
the BAS-1 has been structurally defined. Testing of
potential ligand antagonists is now possible upon the
development of highly automated assay methods using a
purified BAS-1. In particular, new agonists and
antagonists will be discovered by using screening
techniques made available herein. Of particular
importance are compounds found to have a combined b; n~; n~
affinity for multiple BAS-1 proteins, e.g., compounds
which can serve as antagonists for allelic variants of
BAS-1.
.
CA 022~4843 1998-11-13
W097/~452 PCT~S97/07648-
Moreover, since the signaling through the BAS-l:BAS-
1 b; n~; n~ partner may function in combination with other
signals, e.g., the CD2 8 /CTLA-4: B7 /B7 0 and/or the
CD40: CD40 ligand pathways, combination compositions or
combination therapy with such pathways will also be
considered. Thus, antagonism of multiple signal
pathways, or stimulation with multiple pathways may be
useful.
This invention is particularly useful for screening
compounds by using the recombinant antigens in any of a
variety of drug screening techniques. The advantages of
using a recombinant protein in screening for specific
ligands include: (a) improved renewable source of the
sAS-l from a specific source; (b~ potentially greater
number of antigen molecules per cell giving better signal
to noise ratio in assays; and (c) species variant
specificity (theoretically giving greater biological and
disease specificity).
One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably transformed with
recombinant DNA molecules expressing the BAS-l. Cells
may be isolated which express a BAS-l in isolation from
others. Such cells, either in viable or fixed form, can
be used for st~n~rd antigen/ligand bin~;ng assays. See
also, Parce, et al. (1989) Science 246:243-247; and
Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-
4011, which describe sensitive methods to detect cellular
responses. Competitive assays are particularly useful,
where the cells (source of BAS-l) are contacted and
incubated with a labeled ligand having known bin~;ng
affinity to the antigen, such as 125I-ligand, and a test
compound whose b;n~;ng affinity to the BAS-l is being
measured. The bound ligand and free ligand are then
separated to assess the degree of ligand b;n~;n~. The
amount of test compound bound is inversely proportional
to the amount of labeled ligand b;n~;ng measured. Any
one of numerous techniques can be used to separate bound
from free ligand to assess the degree of ligand b;n~ing.
This separation step could typically involve a procedure
. , ,~ . .. . ....... . ..
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yD
such as adhesion to filters followed by washing, adhesion
to plastic followed by washing, or centrifugation of the
cell membranes. Viable cells could also be used to
screen for the effects of drugs on BAS-l mediated
functions, e.g., second messenger levels, i.e., Ca++;
cell proliferation; inositol phosphate pool changes; and
others. Some detection methods allow for elimination of
a separation step, e.g., a proximity sensitive detection
system. Calcium sensitive dyes will be useful for
detecting Ca++ levels, with a fluorimeter or a
fluorescence cell sorting apparatus.
Another method utilizes membranes from transformed
eukaryotic or prokaryotic host cells as the source of the
human BAS-l. These cells are stably transformed with DNA
vectors directing the expression of human BAS-l antigen.
Essentially, the membranes would be prepared from the
cells and used in any receptor/ligand b;n~;ng assay such
as the competitive assay set forth above.
Still another approach is to use solubilized,
unpurified or solubilized, purified BAS-l from
transformed eukaryotic or prokaryotic host cells. This
allows for a "molecular" b;n~;n~ assay with the
advantages of increased specificity, the ability to
automate, and high drug test throughput.
Another techni~ue for drug screening involves an
approach which provides high throughput screening for
compounds having suitable b; n~; ng affinity to human BAS-l
and is described in detail in Geysen, European Patent
Application 84/03564, publ;~he~ on September 13, 1984.
First, large numbers of different small peptide test
compounds are synthesized on a solid substrate, e.g.,
plastic pins or some other a~L~riate surface, see
Fodor, et al. (1991). Then all the pins are reacted with
solubilized, unpurified or solubilized, purified BAS-l,
and washed. The next step involves detecting bound BAS-
1.
Rational drug design may also be based upon
structural studies of the molecular shapes of the BAS-l
and other effectors or ligands. Effectors may be other
CA 022~4843 1998-11-13
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~1
proteins which mediate other functions in response to
ligand binding, or other proteins which normally interact
with the antigen. One means for det~rmi n; n~ which sites
interact with specific other proteins is a physical
structure determ.ination, e.g., x-ray crystallography or 2
~;m~n~ional NMR techniques. These will provide guidance
as to which amino acid residues form molecular contact
regions. For a detailed description of protein
structural det~rm;n~tion, see, e.g., Blundell and Johnson
(1976) Protein Crvstallo~ra~hy, ACA~1Pm;C Press, New York.
Purified BAS-1 can be coated directly onto plates
for use in the aforementioned drug screening techniques.
However, non-neutralizing antibodies to these antigens
can be used as capture antibodies to immobilize the
respective BAS-1 on the solid phase.
IX. Kits
This invention also contemplates use of BAS-1
proteins, fragments thereof, peptides, and their fusion
products in a variety of diagnostic kits and methods for
detecting the presence of BAS-1 or a ligand. Typically
the kit will have a compartment cont~; n; ng either a
defined BAS-1 peptide or gene segment or a reagent which
recognizes one or the other.
A kit for determ;n;ng the b;n~;n~ affinity of a test
compound to a BAS-1 would typically comprise a test
compound; a labeled compound, for ~r' e a ligand or
antibody having known b;n~;n~ affinity for the BAS-1; a
source of BAS-1 (naturally occurring or rec~mh;nAnt); and
a means for separating bound from free labeled compound,
such as a solid phase for immobilizing the BAS-1. Once
compounds are screened, those having suitable b;n~;ng
affinity to the BAS-1 can be evaluated in suitable
biological assays, as are well known in the art, to
det~rm;ne whether they act as agonists or antagonists.
The availability of recombinant BAS-1 polypeptides also
provide well defined st~n~rds for calibrating such
assays.
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4~
A preferred kit for determ;n;ng the concentration
of, for example, a BAS-l in a sample would typically
comprise a labeled compound, e.g., ligand or antibody,
having known binding affinity for the BAS-l, a source of
BAS-l (naturally occurring or recombinant) and a means
for separating the bound from free labeled compound, for
example, a solid phase for immobilizing the BAS-l.
Compartments cont~;n;ng reagents, and instructions, will
normally be provided.
One method for detPrm;n; n~ the concentration of BAS-
1 in a sample would typically comprise the steps of: (1)
preparing membranes from a sample comprised of a BAS-l
source; (2) w~h;n~ the membranes and suspending them in
a bufferi (3) solubilizing the BAS-l by incubating the
membranes in a culture medium to which a suitable
detergent has been added; (4) adjusting the detergent
concentration of the solubilized BAS-l; (5) contacting
and incubating said dilution with radiolabeled ligand to
form complexes; (6) recovering the complexes such as by
filtration through polyethyleneimine treated filters; and
(7) measuring the radioactivity of the recovered
complexes.
Antibodies, including antigen b;n~;ng fragments,
specific for human BAS-1 or BAS-1 fragments are useful in
diagnostic applications, e.g., to detect the presence of
elevated levels of BAS-1 and/or its fragments. Such
diagnostic assays can employ lysates, live cells, fixed
cells, immunofluorescence, cell cultures, body fluids,
and further can involve the detection of antigens related
to the BAS-1 in serum, or the like. Diagnostic assays
may be homogeneous (without a separation step between
free reagent and antigen-ligand complex) or heterogeneous
(with a separation step). Various commercial assays
exist, such as radioimmunoassay (RIA), enzyme-linked
immunosorbent assay (ELISA), enzyme immunoassay (EIA),
enzyme-multiplied ;~lmoassay technique (EMI~),
substrate-labeled fluorescent ;mmllnQassay (SLFIA), and
the like. For example, unlabeled antibodies can be
employed by using a second antibody which is labeled and
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648.
which recognizes the antibody to a BAS-1 or to a
particular fragment thereof. These assays have also been
extensively discussed in the literature. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratorv Manual,
CSH.
Anti-idiotypic antibodies may have similar use to
diagnose presence of antibodies against a human BAS-1, as
such may be diagnostic of various abnormal states. For
example, overproduction of BAS-1 may result in production
of various immunological reactions which may be
diagnostic of abnormal physiological states, particularly
in proliferative cell conditions such as cancer or
abnormal differentiation.
Frequently, the reagents for diagnostic assays are
supplied in kits, so as to optimize the sensitivity of
the assay. For the subject invention, depending upon the
nature of the assay, the protocol, and the label, either
labeled or unlabeled antibody, or labeled BAS-1 is
provided. This is usually in conjunction with other
additives, such as buffers, stabilizers, materials
necessary for signal production such as substrates for
enzymes, and the like. Preferably, the kit will also
contain instructions for proper use and disposal of the
contents after use. Typically the kit has compartments
for each useful reagent. Desirably, the reagents are
provided as a dry lyophilized powder, where the reagents
may be reconstituted in an aqueous medium providing
appropriate concentrations of reagents for performing the
assay.
Any of the aforementioned constituents of the drug
screening and the diagnostic assays may be used without
modification or may be modified in a variety of ways.
For example, labeling may be achieved by covalently or
non-covalently joining a moiety which directly or
indirectly provides a detectable signal. In any of these
assays, the ligand, test compound, BAS-1, or antibodies
thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups:
radiolabels such as 125I, enzymes (U.S. Pat. No.
CA 022~4843 1998-11-13
W O 97/44452 PCT~US97/07648--
A~
3,645,090) such as peroxidase and alkaline phosphatase,
and fluorescent labels (U.S. Pat. No. 3,940,475) capable
of monitoring the change in fluorescence intensity,
wavelength shift, or fluorescence polarization. Both of
the patents are incorporated herein by reference.
Possibilities for indirect labeling include biotinylation
of one constituent followed by binding to avidin coupled
to one of the above label groups.
There are also numerous methods of separating the
bound from the free ligand, or alternatively the bound
from the free test compound. The BAS-l can be
immobilized on various matrixes followed by washing.
Suitable matrixes include plastic such as an ELISA plate,
filters, and beads. Methods of immobilizing the BAS-l to
a matrix include, without limitation, direct adhesion to
plastic, use of a capture antibody, chemical coupling,
and biotin-avidin. The last step in this approach
involves the precipitation of antigen/ligand complex by
any of several methods including those utilizing, e.g.,
an organic solvent such as polyethylene glycol or a salt
such as ammonium sulfate. Other suitable separation
techniques include, without limitation, the fluorescein
antibody magnetizable particle method described in
Rattle, et al. ~1984) Clin. Chem. 30:1457-1461, and the
double antibody magnetic particle separation as described
in U.S. Pat. No. 4,659,678.
The methods for linking proteins or their fragments
to the various labels have been extensively reported in
the literature. Many of the techniques involve the use
of activated carboxyl groups either through the use of
carbodiimide or active esters to form peptide bonds, the
formation of thioethers by reaction-of a mercapto group
with an activated halogen such as chloroacetyl, or an
activated olefin such as maleimide, for linkage, or the
like. Fusion proteins will also find use in these
applications.
Another ~;~gnostic aspect of this invention involves
use of polynucleotide or oligonucleotide sequences taken
from the sequence of a BAS-l. These sequences can be
CA 022~4843 1998-11-13
W097/~452 PCT~S97/07648.
used as probes for detecting levels of the antigen in
samples from patients suspected of having an abnormal
condition, e.g., cancer or developmental problem. The
preparation of both RNA and DNA nucleotide se~uences, the
labeling of the sequences, and the preferred size of the
sequences has received ample description and discussion
in the literature. Normally an oligonucleotide probe
should have at least a~out 14 nucleotides, usually at
least about 18 nucleotides, and the polynucleotide probes
may be up to several kilobases. Various labels may be
employed, most c~monly radionuclides, particularly 32p.
However, other techniques may also be employed, such as
using biotin modified nucleotides for introduction into a
polynucleotide. The biotin then serves as the site for
binding to avidin or antibodies, which may be labeled
with a wide variety of labels, such as radionuclides,
~1uorescers, enzymes, or the like. Alternatively,
antibodies may be employed which can recognize specific
duplexes, including DNA duplexes, RNA duplexes, DNA-RNA
hybrid duplexes, or DNA-protein duplexes. The antibodies
in turn may be labeled and the assay carried out where
the duplex is bound to a surface, so that upon the
formation of duplex on the surface, the presence of
antibody bound to the duplex can be detected. The use of
probes to the novel anti-sense RNA may be carried out in
any conventional techniques such as nucleic acid
hybridization, plus and minus screening, recombinational
probing, hybrid released translation (HRT), and hybrid
arrested translation (HART). This also includes
amplification techniques such as polymerase chain
reaction (PCR).
Diagnostic kits which also test for the qualitative
or guantitative presence of other markers are also
contemplated. Diagnosis or prognosis may depend on the
combination of multiple indications used as markers.
Thus, kits may test for combinations of markers. See,
e.g., Viallet, et al. (1989) Pro~ress in Growth Factor
Res. 1:89-97.
X. Ligand or Counterreceptor
~.
CA 022~4843 1998-11-13
W O 97144452 PCT~US97/07648 .-
The description of the BAS-1 protein herein provides
means to identify a counterreceptor or ligand. Such
ligand or counterreceptor should bind specifically to the
BAS-l with reasonably high affinity. Various constructs
are made available which allow either labeling of the
BAS-l to detect its partner. For example, directly
labeling BAS-l, fusing onto it markers for secondary
labeling, e.g., FLAG or other epitope tags, Ig ~omA;n
fusions, etc., will allow detection of b;n~;n~ partners.
This can be histological, as an affinity method for
biochemical purification, or labeling or selection in an
expression cloning approach. A two-hybrid selection
system may also be applied making appropriate constructs
with the available BAS-l sequences. See, e.g., Fields
and Song (1989) Nature 340:245-246.
The broad scope of this invention is best understood
with reference to the following examples, which are not
intended to limit the invention to specific embodiments.
EXAMPLES
I. General Methods
Some of the stAn~Ard methods are described or
referenced, e.g., in Maniatis, et al. (1982) Molecular
Clonina, A Laboratorv Manual, Cold Spring Harbor
La~oratory, Cold Spring Harbor Press; Sambrook, et al.
(1989) Molecular Clonina: A Laboratorv Manual, ~2d ed.),
vols 1-3, CSH Press, NY; Ausubel, et al., Bioloay, Greene
Publish;ng Associates, Brooklyn, NY; or Ausubel, et al.
(1987 and Supplements) Current Protocols in Molecular
Bioloav, GreenetWiley, New York. Methods ror protein
purification include such methods as Amm~n;um sulfate
precipitation, column chromatography, electrophoresis,
centrifugation, crystallization, and others. See, e.g.,
Ausubel, et al. (1987 and periodic supplements);
Deutscher (1990) "Guide to Protein Purification" in
Methods in Enzvmoloqv, vol. 182, and other volumes in
this series; and manufacturer's literature on use of
CA 022~4843 1998-11-13
W 097/44452 PCTAUS97/07648.
't'1
protein purification products, e.g., Pharmacia,
Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination
with recombinant techniques allow fusion to appropriate
segments, e.g., to a FLAG sequence or an equivalent which
can be fused via a protease-removable sequence. See,
e.g., Hochuli (1989) Chemische Industrie 12:69-70;
Hochuli (1990) "Purification of Recombinant Proteins with
Metal Chelate Absorbent" in Setlow (ed.) Genetic
Eng;neerin~, Princi~le and Methods 12:87-98, Plenum
Press, N.Y.; and Crowe, et al. (1992) OIAex~ress: The
Hi~h Level Expression & Protein Purification System
QUIAGEN, Inc., Chatsworth, CA.
Computer sequence analysis is performed, e.g., using
available software programs, including those from the GCG
(U. Wisconsin) and GenBank sources. Public sequence
databases were also used, e.g., from GPnRAnk and others.
II. Amplification of human BAS-l fragment by PCR
Two primers were designed according to the mouse
RP105 sequences. See Miyake, et al (1995) J. Immunol.
154:3333-3340. Nucleotide designations are counted from
a CTT sequence therein. 5' primer A (TTG...) corresponds
to 24 nucleotides from position 162 to 185 and 3' primer
F (...TTC) corresponds to 24 nucleotides from position
1462 to 1485. No PCR products were obt~;neA from human
tonsillar B cells.
Three 5' primers were made: primer B (ACA...)
covering nucleotide positions 321-344, primer C ~AAG...)
covering nucleotide positions 568-591, and primer D
(TCT... ) covering nucleotide positions 859-882. Three
other 3' primers were also made: primer G (... TTA)
covering nucleotide position 1765-1788, primer H (...GAA)
covering nucleotide position 2024-2047, and primer E
(.... ....TGG) covering nucleotide position 1164-1187.
To increase the chances of obt~;n;n~ PCR products
from human tonsillar B cells, the PCR reactions were
performed by using one 5' primer with the co~b;n~tion of
the four 3' primers. Out of these four groups of PCR
reactions, only one clear product of 300 bp was obt~;ne~,
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W O 97144452 PCTrUS97tO7648 --
which spanned from 5' primer D to 3' primer E. This
product was purified, subcloned into pCRTM vector
(Invitrogen, San Diego CA), and then sequenced. See SEQ
ID NO: 1.
According to the 300 bp human sequence, 3 primers
were designed: 5' primers K; 3' primers I and L. Using a
similar PCR method, another 300 bp human fragment was
obtained with the com~bination of primers C and I (see SEQ
ID NO: 1).
III. Tissue distribution of human BAS-l
The PCR product amplified by the primer pair D-E
(see SEQ ID NO: 1) was used as a probe (D-E probe) to
study the tissue distribution of BAS-l in human tissues.
A 2.6 kb mRNA was detected in human spleen and at a lower
level in human heart by Northern blot. This was not
readily detected in human brain, thymus, lung, liver,
skeletal muscle, kidney, prostate, testis, ovary, small
intestine, colon, and peripheral blood leukocyte. In
addition, the 2.6 kb message was detected by Northern
blot in a sIgD+ lym~homA cell line B104 and at a lower
level in a Burkitt's lymphoma cell line BL2, but not in
kidney epithelial carcinoma cell line CHA, lung
epithelial carcinoma cell line MRC-5, EBV cell line JY,
or monocyte cell line U937.
By PCR, human BAS-l message RNA was found in
sIgD+CD38~ naive B cells, sIgD+CD38+ germ;nAl center B
cells, sIgD~CD38+ g~r~;n~l center B cells, sIgD~CD38-
memory B cells, sCD38++CD20l~W plasma cells, dendritic
cells generated from CD14+ monocytes cultured with GM-CSF
plus IL-4, and dendritic cells generated from CD34+ cord
blood cells cultured with GM-CSF plus TNF-a. This
message was not detectable by PCR in the human T cell
line MT9.
IV. Screening of a plasmid cDNA library derived from a
human sIgD+ lymphom~ cell line B104
From the result of Northern blot analysis, BAS-l
expression was detected in 6 ~g of poly-A mRNA of the
B104 cell line. Accordingly, a B104 cDNA library was
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W097/44452 PCT~S97/07648 -
~ 7
constructed in pSPORTl plasmid (Gibco Life Technologies,
Cergy Pontoise, France). After screening of 150,000
bacteria colonies, no positive clone was obtained by
using the D-E probe. This may reflect that the
expression of BAS-l in human cells may be very low.
V. Screening a ~gtlO bacteriophage library of human
spleen
In order to screen more clones, a ~gtlO
bacteriophage library of human spleen (Clontech) was
used. After screening 2.4 x 106 clones, 8 positive
clones were identified. These clones were isolated,
purified, and subcloned into pME18S plasmid (DNAX, Palo
Alto, CA). After seguencing, two clones S2 and Ll were
found to represent full length human BAS-l (see SEQ ID
NO: 1). The full length human BAS-l cDNA isolated
contains about 2635 bp, and encodes a protein of about
661 aa. This protein product contains about 11 potential
sites for glycosylation and about 22 leucine-rich repeat
motifs. The coding region of this human BAS-l cDNA
shares about 72~ homology with the coding region of the
mouse RP105 sequence, and this complete BAS-l cDNA shares
about 67% homology with the complete mouse cDNA sequence.
The amino acid homology between human BAS-l and mouse
RP105 is about 73.5%.
VI. Expression of human BAS-l protein
All constructions were made by eliminating the 5'
and 3' non-coding region and by adding the Marylin Kozak
(Ref: J. Biol. Chem. 266: 19867, 1991) consensus sequence
(ACCATGG; SEQ ID NO: 3) to ~nh~nce the translational
level.
Soluble BAS-l-FLAG protein has been transiently
expressed of in Cos 7 cells, as follows. A recombinant
form of BAS-l displaying the FLAG peptide at the carboxy
ter~;n~ (Hoppe, et al. (1988) Biotechnoloov 6:1205-1210)
was introduced into the expression vector pME18S and
subsequently transfected into Cos-7 cells by
electroporation. Electroporated cells were grown in DMEM
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W O 97144452 PCTrUS97/07648--
medium supplemented either with 1% Nutridoma HU
tBoehringer MAnnhe;m, M~nnh~im, Germany) or DMEM medium
alone for 5 days.
VII. Purification of soluble BAS-l Flag protein
Typically, 280 ml of supernatant cont~;n;ng soluble
BAS-l FLAG was passed on a 20 ml column of Cu++ ions
attached to a Chelating Sepharose Fast Flow matrix
(phArm~cia, Upsalla, Sweden). After washing with 16
volumes of b;n~;ng buffer (His-Bind Buffer kit, Novagen,
Madison, WI), the proteins retained by the metal ions
were eluted with a gradient of 20-100 mM Imidazole. The
content of human BAS-l FLAG in the eluted fractions was
determined by dot blot using the anti-FLAG monoclonal
antibody M2 (Eastman Kodak, New Haven, CT) and by
Coomassie blue and silver st~;n;ng of reducing SDS-PAGE.
The BAS-l FLAG protein cont~;n-ng fractions were then
pooled and dialyzed against PBS. By Western blot, the
enriched human BAS-l correspond to about 95 and 97 kd
proteins, as predicted from the amino acid sequences.
VIII. Stable expression of membrane BAS-l in NIH-3T3
cells
A native membrane form was subcloned into the
expression vector pM~Mneo (Clontech, Palo Alto,
California), which contains the RSV-LTR ~nhAncer linked
to the ~e~m~thasone-inducible MMTV-LTR promoter. This
construct was then transfected into NIH-3T3 cells by
electroporation. Transfected NIH-3T3 cells were seeded
in selective 0.5 mg/ml Geneticin (G418) (Boehringer-
~nnhe;m, Mannheim, Germany) DMEM supplemented with 10%
Fetal Calf Serum.
Biochemical characterization of membrane BAS-l
protein in these stable transfected NIH-3T3 cells has
been with metabolic labeling. Cells were then cultivated
for 24 hours in DMEM medium supplemented with 10% Fetal
Calf Serum and 1 ~M final dexamethasone (Sigma, Saint
Quentin Fallavier, France). Cells were then incubated
with 35S-Met and 35S-Cys during the last 12 hours in
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~ 1
order to label cellular proteins. Analysis of the
proteins under reducing conditlons on SDS-PAGE showed the
predicted 110 kDa protein in the lysate of the
transfected NIH-3T3 cells, but not in the lysate of
untransfected NIH-3T3 cells.
IX. Preparation of antibodies specific for BAS-l
Inbred Balb/c mice have been ;mmllntzed
intraperitoneally with recombinant forms of the human
protein. The first received the purified soluble BAS-l
FLAG and the second received the stable transfected NIH-
3T3 cells. ~nimAls are boosted at appropriate time
points with protein, with or without additional adjuvant,
to further stimulate antibody production. Serum is
collected, or hybridomas produced with harvested spleens.
Alternatively, Balb/c mice are immlln;zed with cells
transformed with the gene or fragments thereof, either
endogenous or exogenous cells, or with isolated membranes
enriched for expression of the antigen. Serum is
collected at the appropriate time, typically after
numerous further ~m; n; strations. Various gene therapy
techniques may be useful, e.g., in producing protein in
situ, for generating an immune response.
Monoclonal antibodies may be made. ~or example,
splenocytes are fused with an appropriate fusion partner
and hybridomas are selected in growth medium by st~n~rd
procedures. Hybridoma supernatants are screened for the
presence of antibodies which bind to the human BAS-l,
e.g., by ELISA or other assay. Antibodies which
specifically recognize human BAS-l but not species
variants may also be selected or prepared.
In another method, synthetic peptides or purified
protein are presented to an ;~ ne system to generate
monoclonal or polyclonal antibodies. See, e.g., Coligan
(1991) Current Protocols in T~ oloqv Wiley/Greene; and
Harlow and Lane ~1989) Antibodies: A Laboratorv Manual
Cold Spring Harbor Press. In a~o~liate situations, the
binding reagent is either labeled as described above,
e.g., fluorescence or otherwise, or immobilized to a
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648~-
5~
substrate for p~nn;n~ methods. Nucleic acids may also be
introduced into cells in an ~n;m~l to produce the
antigen, which serves to elicit an immune response. See,
e.g., Wang, et al. (1993) Proc. Nat'l. Acad. Sci.
90:4156-4160; Barry, et al. ~1994) BioTechniaues 16:616-
619; and Xiang, et al. (1995) Immunitv 2: 129-135.
X. Large scale production of BAS-1 in NS0 cells
Large quantities of soluble BAS-1 and soluble BAS-1
FLAG can be produced from stable transformants of NS0
cells prepared, e.g., according to a methodology
developed by Celltech (Slough, Berkshire, UK;
International Patent Applications W086/05807, W087/04462,
WO89/01036 and WO89/10404). Both BAS-1 and BAS-1-FLAG
were subcloned into pEE12 and subsequently transfected
into NS0 cells by electroporation. Transfected NS0 cells
were seeded in selective glutAm;ne-free DMEM supplemented
with 10~ Fetal Calf Serum as described in Celltech's
protocol. Supernatants from the best producing lines are
used in biological assays and purification of soluble
BAS-1 and soluble BAS-1-FLAG.
Other expression systems can be used to produce
large quantities of recombinant proteins.
XI. Production of fusion proteins with BAS-1
Various fusion constructs are made with the BAS-1
extracellular ~m~; n. This portion of the gene is fused
to another protein, e.g., a FLAG epitope tag, an Ig
~om~ ; n ~ or to a two hybrid system construct. See, e.g.,
Fields and Song (1989) Nature 340:245-246.
The epitope tag may be used in an expression cloning
procedure with detection with anti-FLAG antibodies to
detect a binding partner, e.g., ligand for the BAS-1.
Alternatively, the Ig ~o~;n may be used to purify using
antigen-like affinity for ligand. The two hybrid system
may also be used to isolate proteins which specifically
bind to BAS-1.
XII. Mapping of human BAS-1 by in situ hybridization
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W 097/44452 PCT~US97/07648'-
~3
Chromosome spreads were prepared. In situ
hybridization was performed on chromosome preparations
obtained from phytohemagglutinin-stimulated human
lymphocytes cultured for 72 h. 5-bromodeoxyuridine was
added for the final seven hours of culture (60 ~g/ml of
medium), to ensure a posthybridization chromosomal
banding of good quality.
A 655 base pair PCR fragment, amplified with the
help of primers, e.g., corresponding to nucleotides 395-
411 and 1027-1050, on total B cells cDNA template, was
cloned into an appropriate vector. The vector was
labeled by nick-translation with 3H. The radiolabeled
probe was hybridized to met~ph~e spreads at final
concentration of 200 ng/ml of hybridization solution as
described in Mattei, et al. (1985) Hum. Genet. 69:327-
331.
After coating with nuclear track emulsion (KODAK
NTB2), slides were exposed for 18 days at 4~ C. To avoid
any slipping of silver grains during the b~n~;ng
procedure, chromosome spreads were first stained with
buffered Giemsa solution and met~rh~e photographed. R-
h~n~;ng was then performed by the fluorochrome-
photolysis-Giemsa (FPG) method and metaphases
rephotographed before analysis.
In 100 metA~h~e cells examined after in situ
hybridization, 20% of silver grains were located on
chromosome 5. The distribution of grains was not random,
67% of them mapped to the qll.2-ql3.1 region of
chromosome 5 long arm. This maps the human BAS-1 to the
5qll.2-ql3.1 region of the human genome.
XIII. Isolation of a Receptor for Human BAS-1
A human BAS-1 can be used as a specific b;n~;ng
reagent to identify its bi~;ng partner, by taking
advantage of its specificity of bin~; n~, much like an
antibody would be used. A b;n~;n~ reagent is either
labeled as described above, e.g., fluorescence or
otherwise, or immobilized to a substrate for pAnn;ng
methods.
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648--
5~
The binding composition is used to screen an
expression library made from a cell line which expresses
a b;n~;ng partner, i.e. receptor. St~n~rd st~;ning
techniques are used to detect or sort intracellular or
surface expressed receptor, or surface expressing
transformed cells are screened by p~nn;n~ Screening of
intracellular expression is performed by various st~;n;ng
or ;m~t2nofluorescence procedures. See also M~M~h~n, et
al. (1991) EMBO J. 10:2821-2832.
For example, on day 0, precoat 2-chamber permanox
slides with 1 ml per chamber of fibronectin, 10 ng/ml in
PBS, for 30 min at room temperature. Rinse once with
PBS. Then plate COS cells at 2-3 x 105 cells per chamber
in 1.5 ml of growth media. Incubate overnight at 37- C.
On day 1 for each sample, prepare 0.5 ml of a
solution of 66 ~g/ml DEAE-dextran, 66 ~M chloroquine, and
4 ~g DNA in serum free DME. For each set, a positive
control is prepared, e.g., of human BAS-1-FLAG cDNA at 1
and 1/200 dilution, and a negative mock. Rinse cells
with serum free DME. Add the DNA solution and incubate 5
hr at 37- C. Remove the medium and add 0.5 ml 10% DMSO
in DME for 2.5 min. Remove and wash once with DME. Add
1.5 ml growth medium and incubate overnight.
On day 2, change the medium. On days 3 or 4, the
cells are fixed and stained. Rinse the cells twice with
Hank's Buffered Saline Solution (HBSS) and fix in 4%
paraformaldehyde (PFA)/glucose for 5 min. Wash 3X with
B SS. The slides may be stored at -80- C after all
li~uid is removed. For each chamber, 0.5 ml incubations
are performed as follows. Add B SS/saponin (0.1%) with
32 ~l/ml of 1 M NaN3 for 20 min. Cells are then washed
with HBSS/saponin lX. Add human BAS-1 or BAS-1/antibody
complex to cells and incubate for 30 min. Wash cells
twice with HBSS/saponin. If appropriate, add first
antibody for 30 min. Add second antibody, e.g., Vector
anti-mouse antibody, at 1/200 dilution, and incubate for
30 min. Prepare ELISA solution, e.g., Vector Elite ABC
horseradish peroxidase solution, and preincubate for 30
min. Use, e.g., 1 drop of solution A (avidin) and 1 drop
CA 022~4843 1998-11-13
W097/44452 PCT~S97/07648 -
5 ~
solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells
twice with HBSS/saponin. Add ABC HRP solution and
incubate for 30 min. Wash cells twice with HBSS, second
wash for 2 min, which closes cells. Then add Vector
diaminobenzoic acid (DAB) for 5 to 10 min. Use 2 drops
of buffer plus 4 drops DAB plus 2 drops of H2~2 per 5 ml
of glass distilled water. Carefully remove chamber and
rinse slide in water. Air dry for a few minutes, then
add 1 drop of Crystal Mount and a cover slip. Bake for 5
min at 85-90 C.
Evaluate positive st~;n;ng of pools and
progressively subclone to isolation of single genes
responsible for the binding.
Alternatively, BAS-1 reagents are used to affinity
purify or sort out cells expressing a receptor. See,
e.g., Sambrook, et al. or Ausubel, et al.
Another strategy is to screen for a membrane bound
receptor by p~nn; ng . The receptor cDNA is constructed as
described above. The ligand can be immobilized and used
to immobilize expressing cells. Immobilization may be
achieved by use of appropriate antibodies which
recognize, e.g., a FLAG se~uence of a BAS-1 fusion
construct, or by use of antibodies raised against the
first antibodies. Recursive cycles of selection and
amplification lead to enrichment of appropriate clones
and eventual isolation of receptor expressing clones.
Phage expression libraries can be screened by human
BAS-1. Appropriate label techniques, e.g., anti-FLAG
antibodies, will allow specific labeling of appropriate
clones.
All citations herein are incorporated herein by
reference to the same extent as if each individual
publication or patent application was specifically and
individually indicated to be incorporated by reference.
Many modifications and variations of this invention can
be made without departing from its spirit and scope, as will
be apparent to those skilled in the art. The specific
embodiments described herein are offered by way of example
only, and the invention is to be limited by the terms of the
CA 02254843 1998-11-13
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SC~
appended claims, along with the full scope of equivalents to
which such claims are entitled.
CA 022~4843 l998-ll-l3
WO 97/44452 PCT/IJS97107648
51
SEQUENCE LISTING
5 (1) GENERAL INFORMATION:
(i) APPLICANT: Schering Corporation
(ii) TITLE OF INVENTION: HUMAN B CELL ANTIGENS; RELATED REAGENTS
(iii) NUMBER OF SEQUENCES: 3
~iv) CORRESP~N~N~ ADDRESS:
(A) ADDRESSEE: Schering-Plough Corporation
(B) STREET: 2000 Galloping Hill Road
(C) CITY: Kenilworth
(D) STATE: New Jersey
(E) COUNTRY: USA
(F) ZIP: 07033
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: Macintosh
(C) OPERATING SYSTEM: 7.5.3
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NVMBER: US 08/649,553
(B) FILING DATE: 17-MAY-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Cynthia L. Foulke, Esq.
(B) REGISTRATION NUMBER: 32,364
(C) REFERENCE/DOCKET NUMBER: DX0580PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 908-298-2987
(B) TELEFAX: 908-298-5388
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2635 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 97..2082
(xi) ~Qu~-~ DESCRIPTION: SEQ ID NO:1:
ATTCCGGCGG CTGCCAAATT GCCAGGGCCT TCACAGTTTG ATTCCATTTC TCAGCTCCAA
. .
CA 022~4843 l998-ll-l3
WO 97/44452 PCT/US97/07648
5&-
GCATTAGGTA AACCCACCAA GCAATCCTAG CCTGTG ATG GCG TTT GAC GTC AGC
114
Met Ala Phe Asp Val Ser
1 5
TGC TTC TTT TGG GTG GTG CTG TTT TCT GCC GGC TGT AAA GTC ATC ACC
162
Cys Phe Phe Trp Val Val Leu Phe Ser Ala Gly Cys Lys Val Ile Thr
TCC TGG GAT CAG ATG TGC ATT GAG AAA GAA GCC AAC AAA ACA TAT AAC
210
Ser Trp Asp Gln Met Cys Ile Glu Lys Glu Ala Asn Lys Thr Tyr Asn
TGT GAA AAT TTA GGT CTC AGT GAA ATC CCT GAC ACT CTA CCA AAC ACA
258
Cys Glu Asn Leu Gly Leu Ser Glu Ile Pro Asp Thr Leu Pro Asn Thr
ACA GAA TTT TTG GAA TTC AGC TTT AAT TTT TTG CCT ACA ATT CAC AAT
306
Thr Glu Phe Leu Glu Phe Ser Phe Asn Phe Leu Pro Thr Ile His Asn
AGA ACC TTC AGC AGA CTC ATG AAT CTT ACC TTT TTG GAT TTA ACT AGG
354
Arg Thr Phe Ser Arg Leu Met Asn Leu Thr Phe Leu Asp Leu Thr Arg
TGC CAG ATT AAC TGG ATA CAT GAA GAC ACT TTT CAA AGC CAT CAT CAA
402
Cys Gln Ile Asn Trp Ile His Glu Asp Thr Phe Gln Ser His His Gln
100
TTA AGC ACA CTT GTG TTA ACT GGA AAT CCC CTG ATA TTC ATG GCA GAA
450
Leu Ser Thr Leu Val Leu Thr Gly Asn Pro Leu Ile Phe Net Ala Glu
105 110 115
ACA TCG CTT AAT GGG CCC AAG TCA CTG AAG CAT CTT TTC TTA ATC CAA
498
Thr Ser Leu Asn Gly Pro Lys Ser Leu Lys His Leu Phe Leu Ile Gln
120 125 130
AC& GGA ATA TCC AAT CTC GAG TTT ATT CCA GTG CAC AAT CTG GAA AAC
546
Thr Gly Ile Ser Asn Leu Glu Phe Ile Pro Val His Asn Leu Glu Asn
135 140 145 150
TTG GAA AGC TTG TAT CTT GGA AGC AAC CAT ATT TCC TCC ATT AAG TTC
594
Leu Glu Ser Leu Tyr Leu Gly Ser Asn His Ile Ser Ser Ile Lys Phe
155 160 165
CCC AAA GAC TTC CCA GCA CGG AAT CTG AAA GTA CTG GAT TTT CAG AAT
642
Pro Lys Asp Phe Pro Ala Arg Asn Leu Lys Val Leu Asp Phe Gln Asn
170 175 180
AAT GCT ATA CAC TAC ATC TCT AGA GAA GAC ATG AGG TCT CTG GAG CAG
690
Asn Ala Ile His Tyr Ile Ser Arg Glu Asp Met Arg Ser Leu Glu Gln
185 190 195
_ .
CA 022~4843 l998-ll-l3
W O 97/44452 PCTrUS97/07648
3 q
GCC ATC AAC CTA AGC CTG AAC TTC AAT GGC AAT AAT GTT AAA GGT ATT
738
Ala Ile Asn Leu Ser Leu Asn Phe Asn Gly Asn Asn Val Lys Gly Ile
200 205 210
GAG CTT GGG GCT TTT GAT TCA ACG GTC TTC CAA AGT TTG AAC TTT GGA
786
Glu Leu Gly Ala Phe Asp Ser Thr Val Phe Gln Ser Leu Asn Phe Gly
0 215 220 225 230
GGA ACT CCA AAT TTG TCT GTT ATA TTC AAT GGT CTG CAG AAC TCT ACT
834
Gly Thr Pro Asn Leu Ser Val Ile Phe Asn Gly Leu Gln Asn Ser Thr
235 240 245
ACT CAG TCT CTC TGG CTG GGA ACA TTT GAG GAC ATT GAT GAC GAA GAT
882
Thr Gln Ser Leu Trp Leu Gly Thr Phe Glu Asp Ile Asp Asp Glu Asp
250 255 260
ATT AGT TCA GCC ATG CTC AAG GGA CTC TGT GAA ATG TCT GTT GAG AGC
930
Ile Ser Ser Ala Met Leu Lys Gly Leu Cys Glu Net Ser Val Glu Ser
265 270 275
CTC AAC CTG CAG GAA CAC CGC TTC TCT GAC ATC TCA TCC ACC ACA TTT
978
Leu Asn Leu Gln Glu His Arg Phe Ser Asp Ile Ser Ser Thr Thr Phe
280 285 290
CAG TGC TTC ACC CAA CTC CAA GAA TTG GAT CTG ACA GCA ACT CAC TTG
1026
Gln Cys Phe Thr Gln Leu Gln Glu Leu Asp Leu Thr Ala Thr His Leu
295 300 305 310
AAA GGG TTA CCC TCT GGG ATG AAG GGT CTG AAC TTG CTC AAG AAA TTA
1074
Lys Gly Leu Pro Ser Gly Met Lys Gly Leu Asn Leu Leu Lys Lys Leu
315 320 325
GTT CTC AGT GTA AAT CAT TTC GAT CAA TTG TGT CAA ATC AGT GCT GCC
1122
Val Leu Ser Val Asn His Phe Asp Gln Leu Cys Gln Ile Ser Ala Ala
330 335 340
AAT TTC CCC TCC CTT ACA CAC CTC TAC ATC AGA GGC AAC GTG AAG AAA
1170
Asn Phe Pro Ser Leu Thr His Leu Tyr Ile Arg Gly Asn Val Lys Lys
345 350 355
CTT CAC CTT GGT GTT GGC TGC TTG GAG AAA CTA GGA AAC CTT CAG ACA
1218
Leu His Leu Gly Val Gly Cys Leu Glu Lys Leu Gly Asn Leu Gln Thr
360 365 370
CTT GAT TTA AGC CAT AAT GAC ATA GAG GCT TCT GAC TGC TGC AGT CTG
1266
Leu Asp Leu Ser His Asn Asp Ile Glu Ala Ser Asp Cys Cys Ser Leu
375 380 385 390
CAA CTC AAA AAC CTG TCC CAC TTG CAA ACC TTA AAC CTG AGC CAC AAT
1314
Gln Leu Lys Asn Leu Ser His Leu Gln Thr Leu Asn Leu Ser His Asn
. , ,
CA 022~4843 1998-11-13
W O 97/44452 PCT~US97/07648--
6~
395 400 405
GAG CCT CTT GGT CTC CAG AGT CAG GCA TTC AAA GAA TGT CCT CAG CTA
1362
5 Glu Pro Leu Gly Leu Gln Ser Gln Ala Phe Lys Glu Cys Pro Gln Leu
410 415 420
GAA CTC CTC GAT TTG GCA TTT ACC CGC TTA CAC ATT AAT GCT CCA CAA
1410
0 Glu Leu Leu Asp Leu Ala Phe Thr Arg Leu His Ile Asn Ala Pro Gln
425 430 435
AGT CCC TTC CAA AAC CTC CAT TTC CTT CAG GTT CTG AAT CTC ACT TAC
1458
15 Ser Pro Phe Gln Asn Leu His Phe Leu Gln Val Leu Asn Leu Thr Tyr
440 445 450
TGC TTC CTT GAT ACC AGC AAT CAG CAT CTT CTA GCA GGC CTA CCA GTT
1506
20 Cys Phe Leu Asp Thr Ser Asn Gln His Leu Leu Ala Gly Leu Pro Val
455 460 465 470
CTC CGG CAT CTC AAC TTA AAA GGG AAT CAC TTT CAA GAT GGG ACT ATC
1554
25 Leu Arg His Leu Asn Leu Lys Gly Asn His Phe Gln Asp Gly Thr Ile
475 480 485
ACG AAG ACC AAC CTA CTT CAG ACC GTG GGC AGC TTG GAG GTT CTG ATT
1602
3 0 Thr Lys Thr Asn Leu Leu Gln Thr Val Gly Ser Leu Glu Val Leu Ile
490 495 500
TTG TCC TCT TGT GGT CTC CTC TCT ATA GAC CAG CAA GCA TTC CAC AGC
1650
3 5 Leu Ser Ser Cys Gly Leu Leu Ser Ile Asp Gln Gln Ala Phe His Ser
505 510 515
TTG GGA AAA ATG AGC CAT GTA GAC TTA AGC CAC AAC AGC CTG ACA TGC
1698
40 Leu Gly Lys Met Ser His Val Asp Leu Ser His Asn Ser Leu Thr Cys
520 525 530
GAC AGC ATT GAT TCT CTT AGC CAT CTT AAG GGA ATC TAC CTC AAT CTG
1746
45 Asp Ser Ile Asp Ser Leu Ser His Leu Lys Gly Ile Tyr Leu Asn Leu
535 540 545 550
GCT GCC AAC AGC ATT AAC ATC ATC TCA CCC CGT CTC CTC CCT ATC TTG
1794
50 Ala Ala Asn Ser Ile Asn Ile Ile Ser Pro Arg Leu Leu Pro Ile Leu
555 560 565
TCC CAG CAG AGC ACC ATT AAT TTA AGT CAT AAC CCC CTG GAC TGC ACT
1842
55 Ser Gln Gln Ser Thr Ile Asn Leu Ser His Asn Pro Leu Asp Cys Thr
570 575 580
TGC TCG AAT ATT CAT TTC TTA ACA TGG TAC AAA GAA AAC CTG CAC AAA
1890
60 Cys Ser Asn Ile His Phe Leu Thr Trp Tyr Lys Glu Asn Leu His Lys
585 590 595
CTT GAA GGC TCG GAG GAG ACA CGT GTG CAA AAC CCG CCA TCT CTA AGG
1938
CA 022~4843 l998-ll-l3
W O 97/44452 PCTrUS97/07648
6l
Leu Glu Gly Ser Glu Glu Thr Arg Val Gln Asn Pro Pro Ser Leu Arg
600 605 610
GGA GTT AAG CTA TCT GAT GTC AAG CTT TCC TGT GGG ATT ACA GCC ATA
1986
Gly Val Lys Leu Ser Asp Val Lys Leu Ser Cys Gly Ile Thr Ala Ile
615 620 625 630
GGC ATT TTC TTT CTC ATA GTA TTT CTA TTA TTG TTG GCT ATT CTG CTA
0 2034
Gly Ile Phe Phe Leu Ile Val Phe Leu Leu Leu Leu Ala Ile Leu Leu
635 640 645
TTT TTT GCA GTT AAA TAC CTT CTC AGG TGG AAA TAC CAA CAC TTT TAG
2082
Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp Ly6 Tyr Gln His Phe
650 655 660
TGCTGAAGGT TTCCAGAGAA AGCAAATAAG l~lG~llAGc AAAATTGCTC TAAGTGAAAG
2142
AACTGTCATC TGCTGGTGAC CAGACCAGAC TTTTCAGATT G~llC~lGGA A~lGGG~AGG
2202
GACTCACTGT G~ lGA GC,l~llACT C~ ~AGTC CCAGAGCTAA AGAACCTTCT
2262
AGGCAAGTAC ACCGAATGAC TCAGTCCAGA GGGTCAGATG CTGCTGTGAG AGGCACAGAG
2322
CC~lllCCGC ATGTGGAAGA GTGGGAGGAA GCAGAGGGAG GGACTGGGCA GGGACTGCCG
2382
GCCCCGGAGT CTCCCACAGG ~-AGGCCATTC CCCTTCTACT CACCGACATC CCTCCCAGCA
2442
CCACACACCC CGCCCCTGAA AGGAGATCAT CAGCCCCCAC AATTTGTCAG AGCTGAAGCC
2502
AGCCCACTAC CCACCCCCAC TACAGCATTG ~G~llGGGlC' TGGGTTCTCA GTAATGTAGC
2562
CATTTGAGAA ACTTACTTGG ~r.A~A~A~.TC TCAATCCTTA TTTTAAATGA AA~AAGAA~A
2622
GAAAAGCATA ATA
2635
~2) INFORMATION FOR SEQ ID NO:2:
(i) ~Q~N~ CHARACTERISTICS:
(A) LENGTH: 661 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) ~Qu~N~ DESCRIPTION: SEQ ID NO:2:
Met Ala Phe Asp Val Ser Cys Phe Phe Trp Val Val Leu Phe Ser Ala
1 5 10 15
Gly Cys Lys Val Ile Thr Ser Trp Asp Gln Met Cys Ile Glu Lys Glu
.
CA 022~4843 l998-ll-l3
W O 97/44452 PCTAUS97/07648 -
62
Ala Asn Lys Thr Tyr Asn Cys Glu Asn Leu Gly Leu Ser Glu Ile Pro
Asp Thr Leu Pro Asn Thr Thr Glu Phe Leu Glu Phe Ser Phe Asn Phe
Leu Pro Thr Ile His Asn Arg Thr Phe Ser Arg Leu Met Asn Leu Thr
65 70 75 80
Phe Leu Asp Leu Thr Arg Cys Gln Ile Asn Trp Ile His Glu Asp Thr
85 90 95
Phe Gln Ser His His Gln Leu Ser Thr Leu Val Leu Thr Gly Asn Pro
100 105 110
Leu Ile Phe Met Ala Glu Thr Ser Leu Asn Gly Pro Lys Ser Leu Lys
115 120 125
His Leu Phe Leu Ile Gln Thr Gly Ile Ser Asn Leu Glu Phe Ile Pro
130 135 140
Val His Asn Leu Glu Asn Leu Glu Ser Leu Tyr Leu Gly Ser Asn His
145 150 155 160
Ile Ser Ser Ile Lys Phe Pro Lys Asp Phe Pro Ala Arg Asn Leu Lys
- 165 170 175
3 0 Val Leu Asp Phe Gln Asn Asn Ala Ile His Tyr Ile Ser Arg Glu Asp
180 185 190
Met Arg Ser Leu Glu Gln Ala Ile Asn Leu Ser Leu Asn Phe Asn Gly
195 200 205
Asn Asn Val Lys Gly Ile Glu Leu Gly Ala Phe Asp Ser Thr Val Phe
210 215 220
Gln Ser Leu Asn Phe Gly Gly Thr Pro Asn Leu Ser Val Ile Phe Asn
225 230 235 240
Gly Leu Gln Asn Ser Thr Thr Gln Ser Leu Trp Leu Gly Thr Phe Glu
245 250 255
Asp Ile Asp Asp Glu Asp Ile Ser Ser Ala Met Leu Lys Gly Leu Cys
260 265 270
Glu Met Ser Val Glu Ser Leu Asn Leu Gln Glu His Arg Phe Ser Asp
275 280 285
Ile Ser Ser Thr Thr Phe Gln Cys Phe Thr Gln Leu Gln Glu Leu Asp
290 295 300
Leu Thr Ala Thr His Leu Lys Gly Leu Pro Ser Gly Met Lys Gly Leu
305 310 315 320
Asn Leu Leu Lys Lys Leu Val Leu Ser Val Asn His Phe Asp Gln Leu
325 330 335
60 Cys Gln Ile Ser Ala Ala Asn Phe Pro Ser Leu Thr His Leu Tyr Ile
340 345 350
Arg Gly Asn Val Lys Lys Leu His Leu Gly Val Gly Cys Leu Glu Lys
355 360 365
CA 022~4843 l998-ll-l3
W O 97/44452 PCT~US97/07648 -
~3
Leu Gly Asn Leu Gln Thr Leu Asp Leu Ser His Asn Asp Ile Glu Ala
370 375 380
Ser Asp Cys Cys Ser Leu Gln Leu Lys Asn Leu Ser His Leu Gln Thr
385 390 395 400
Leu Asn Leu Ser His Asn Glu Pro Leu Gly Leu Gln Ser Gln Ala Phe
405 410 415
Lys Glu Cys Pro Gln Leu Glu Leu Leu Asp Leu Ala Phe Thr Arg Leu
420 425 430
His Ile Asn Ala Pro Gln Ser Pro Phe Gln Asn Leu His Phe Leu Gln
435 440 445
Val Leu Asn Leu Thr Tyr Cys Phe Leu Asp Thr Ser Asn Gln His Leu
450 455 460
Leu Ala Gly Leu Pro Val Leu Arg His Leu Asn Leu Lys Gly Asn His
465 470 475 480
Phe Gln Asp Gly Thr Ile Thr Lys Thr Asn Leu Leu Gln Thr Val Gly
485 490 495
Ser Leu Glu Val Leu Ile Leu Ser Ser Cys Gly Leu Leu Ser Ile Asp
500 505 510
Gln Gln Ala Phe His Ser Leu Gly Lys Met Ser His Val Asp Leu Ser
515 520 525
His Asn Ser Leu Thr Cys Asp Ser Ile Asp Ser Leu Ser His Leu Lys
530 535 540
Gly Ile Tyr Leu Asn Leu Ala Ala Asn Ser Ile Asn Ile Ile Ser Pro
545 550 555 560
Arg Leu Leu Pro Ile Leu Ser Gln Gln Ser Thr Ile Asn Leu Ser His
565 570 575
Asn Pro Leu Asp Cys Thr Cys Ser Asn Ile His Phe Leu Thr Trp Tyr
580 585 590
Lys Glu Asn Leu His Lys Leu Glu Gly Ser Glu Glu Thr Arg Val Gln
595 600 605
Asn Pro Pro Ser Leu Arg Gly Val Lys Leu Ser Asp Val Lys Leu Ser
610 615 620
Cys Gly Ile Thr Ala Ile Gly Ile Phe Phe Leu Ile Val Phe Leu Leu
625 630 635 640
Leu Leu Ala Ile Leu Leu Phe Phe Ala Val Lys Tyr Leu Leu Arg Trp
645 650 655
Lys Tyr Gln His Phe
660
(2) INFORMATION FOR SEQ ID NO:3:
u~-~ CHARACTERISTICS:
(A) LENGTH: 7 base pairs
~B) TYPE: nucleic acid
~C) STR~Nn~nN~SS: single
CA 02254843 1998-11-13
W O 97/44452 PCT~US97tO7648 -
.' ~,~.
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) ~Q~ DESCRIPTION: SEQ ID NO:3:
ACCATGG
