Binding compounds specific for cysteine-rich soluble protein C23
United States Patent: 7,560,533
Issued: July 14, 2009
Karin (San Diego, CA), Gorman; Daniel M. (Newark, CA), McClanahan; Terrill
K. (Sunnyvale, CA)
Corporation (Kenilworth, NJ)
Appl. No.: 10/246,853
Filed: September 18, 2002
Nucleic acids encoding a new family of
small cysteine rich soluble proteins, from a mammal, reagents related
thereto, including specific antibodies, and purified proteins are
described. Methods of using said reagents and related diagnostic kits are
Description of the
The present invention provides DNA sequences encoding soluble mammalian
proteins which exhibit structural properties or motifs characteristic of a
small soluble protein, e.g., a defensin, growth factor, cytokine, or
chemokine. Because the proteins are cyteine rich, and the cysteine motifs
appear conserved, these proteins are referred to as Cysteine Rich Soluble
Proteins (CRSPs). The tissue expression distribution of these proteins
correlate with significant medical conditions.
For reviews on the defensins, see, e.g., Ganz, et al. (1998) Current Op. in
Immunol. 10:41-44; Hancock, et al. (1998) Trends Biotech. 16:82-88; Lehrer,
et al. (1996) Ann. NY Acad. Sci. 797:228-239; White, et al. (1995) Current
Op. Struct. Biol. 5:521-527; Ganz, et al. (1995) Pharmacol. Ther.
66:191-205; Harwig, et al. (1994) Methods in Enzymology 236:160-172; and
Lehrer, et al. (1993) Ann. Rev. Immunol. 11:105-128. The defensins exhibit
significant sequence similarity to the carboxy terminus of the CRSPs.
Processing of the defensins from inactive precursers follows a known
pathway. The amounts of protein expressed and the exon structure of the
defensins seems to match with that of the CRSPs.
For a review of the cytokines, see, e.g., Thompson (1994) The Cytokine
Handbook 2d ed., Academic Press, San Diego; and Aggarwal and Gutterman
(1992) Human Cytokines: Handbook for Basic and Clinical Research, Blackwell
Pub., Oxford. Many specific sequences and references are available, e.g.,
from the GenBank, and references providing gene and/or cytokine amino acid
sequence. Many receptor sequences are also available from GenBank. See also
Howard, et al. (1993) in Paul (ed.) (1993) Fundamental Immunology (3d ed.)
Raven Press, NY.
For reviews of the chemokine family, see, e.g., Lodi, et al. (1994) Science
263:1762-1767; Gronenborn and Clore (1991) Protein Engineering 4:263-269;
Miller and Kranger (1992) Proc. Nat'l Acad. Sci. USA 89:2950-2954;
Matsushima and Oppenheim (1989) Cytokine 1:2-13; Stoeckle and Baker (1990)
New Biol. 2:313-323; Oppenheim, et al. (1991) Ann. Rev. Immunol. 9:617-648;
Schall (1991) Cytokine 3:165-183; and The Cytokine Handbook Academic Press,
NY. The proteins described herein are designated Cysteine Rich Soluble
Proteins because they were initially recognized as soluble proteins. These
proteins share a highly conserved pattern of cysteine motifs, e.g.,
structural motifs, distinct from the other known groups of soluble protein
The best characterized embodiment of this family of proteins were discovered
from a human sequence source. See Table 1 (see Original Patent). The
descriptions below are directed, for exemplary purposes, to primate
embodiments, e.g., human, but are likewise applicable to related embodiments
from other, e.g., natural, sources. These sources should include various
vertebrates, typically warm blooded animals, e.g., birds and mammals,
particularly domestic animals, and primates.
The CRSPs of this invention are defined in part by their physicochemical and
structural properties. The biological properties of the primate, e.g., human
CRSP described herein, are defined by their amino acid sequence, and mature
size. One of skill will readily recognize that some sequence variations may
be tolerated, e.g., conservative substitutions or positions remote from the
helical structures, without altering significantly the biological activity
of the molecule. The cysteines, being conserved across family members, are
probably relatively important structurally. It is likely that most, or all,
of them are disulfide linked in important pairings.
The cysteine rich nature makes the protein a good substrate for sulfhydryl
reagents. It will be useful as a control for cysteine incorporation, and as
a sample to test ability to sequence through those residues. The protein
will also find use as a carbon source.
In addition, the label may refer, in specific embodiments, to the nucleic
acids which may be isolated using PCR amplification from appropriate cells
of sequences using primers which flank the sequences described. Thus, even
if minor errors in the given sequences may have resulted from ambiguities or
uncertainties in sequencing, the PCR technology will allow isolation of
natural isolates of the described genes. In addition, the nucleotide
sequences in the regions corresponding to C23 residues 56-63 and 79-86 are
highly conserved across the known class members. It is likely that
additional human embodiments will be found using appropriate PCR primers
encoding such regions.
CRSPs are present in specific tissue types, as described below. Each
correlates with important conditions, which are suggestive of important
roles in immunological conditions. The interaction of the protein with a
receptor is likely to be important for mediating various aspects of cellular
physiology or development. The cellular types which express message encoding
CRSPs suggest that signals important in cell differentiation and development
are mediated by them. See, e.g., Gilbert (1991) Developmental Biology (3d
ed.) Sinauer Associates, Sunderland, Mass.; Browder, et al. (1991)
Developmental Biology (3d ed.) Saunders, Philadelphia, Pa.; Russo, et al.
(1992) Development: The Molecular Genetic Approach Springer-Verlag, New
York, N.Y.; and Wilkins (1993) Genetic Analysis of Animal Development (2d
ed.) Wiley-Liss, New York, N.Y. Moreover, CRSP expression correlates with
certain specific conditions. See below.
The CRSP producing profile of different cell types is elucidated herein.
These observations suggest that the CRSPs represent novel additions to the
chemokine/growth factor superfamily.
CRSP protein biochemistry was assessed in some mammalian expression systems.
CRSP member C23 was produced as a protein of Mr.about.8 kDa as evaluated by
reducing SDS-PAGE; control transfected supernatants contained no such
species. The absence of glycosylation motifs suggests that the natural
protein is not glycosylated, and that recombinant protein forms lacking
natural glycosylation should share similar biological activity.
Since the structure of the proteins are soluble, it is likely that the
entire spectrum of inflammatory, infectious, and immunoregulatory states
thought to involve other related cytokines or growth factors may function
through such proteins.
III. Nucleic Acids
This C23 human CRSP is exemplary of a larger class of structurally and
functionally related proteins. These soluble proteins will likely serve to
transmit signals between different cell types. The preferred embodiments, as
disclosed, will be useful in standard procedures to isolate genes from
different individuals or other species, e.g., warm blooded animals, such as
birds and mammals. Cross hybridization or other techniques will allow
isolation of related genes encoding proteins from individuals, strains, or
species. In fact, Applicants possess specific data that this gene hybridizes
across species to mouse embodiments. A number of different approaches are
available to successfully isolate a suitable nucleic acid clone based upon
the information provided herein.
While the overall coding sequence identity among mouse family members is in
the 40% range overall, specific portions exhibit over 80%. Southern blot
hybridization studies using specific portions might qualitatively determine
the presence of homologous genes in human, monkey, rat, dog, cow, and rabbit
genomes under specific hybridization conditions. PCR techniques may be
useful using, e.g., the conserved sequence regions, preferably at the
nucleotide level, but perhaps also at the protein level.
Complementary sequences will also be used as probes or primers. Based upon
identification of the likely amino terminus, other peptides should be
particularly useful, e.g., coupled with anchored vector or poly-A
complementary PCR techniques or with complementary DNA of other peptides.
Techniques for nucleic acid manipulation of genes encoding CRSP proteins,
such as subcloning nucleic acid sequences encoding polypeptides into
expression vectors, labeling probes, DNA hybridization, and the like are
described generally in Sambrook, et al. (1989) Molecular Cloning: A
Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold
Spring Harbor Press, NY, which is incorporated herein by reference. This
manual is hereinafter referred to as "Sambrook, et al."
There are various methods of isolating DNA sequences encoding CRSPs. For
example, DNA is isolated from a genomic or cDNA library using labeled
oligonucleotide probes having sequences identical or complementary to the
sequences disclosed herein. Full-length probes may be used, or
oligonucleotide probes may be generated by comparison of the sequences
disclosed. Such probes can be used directly in hybridization assays to
isolate DNA encoding CRSPs, or probes can be designed for use in
amplification techniques such as PCR, for the isolation of DNA encoding
CRSPs. Certain genomic searches of available sequence databases, public or
private, will also be useful to identify additional members.
To prepare a cDNA library, mRNA is isolated from cells which express a CRSP
protein. cDNA is prepared from the mRNA and ligated into a recombinant
vector. The vector is transfected into a recombinant host for propagation,
screening, and cloning. Methods for making and screening cDNA libraries are
well known. See Gubler and Hoffman (1983) Gene 25:263-269 and Sambrook, et
For a genomic library, e.g., the DNA can be extracted from tissue and either
mechanically sheared or enzymatically digested to yield fragments of about
12-20 kb. The fragments are then separated by gradient centrifugation and
cloned in bacteriophage lambda vectors. These vectors and phage are packaged
in vitro, as described in Sambrook, et al. Recombinant phage are analyzed by
plaque hybridization as described in Benton and Davis (1977) Science
196:180-182. Colony hybridization is carried out as generally described in
e.g., Grunstein, et al. (1975) Proc. Natl. Acad. Sci. USA. 72:3961-3965.
Modifications may be incorporated.
DNA encoding a CRSP can be identified in either cDNA or genomic libraries by
its ability to hybridize with the nucleic acid probes described herein,
e.g., in colony or plaque hybridization assays. The corresponding DNA
regions are isolated by standard methods familiar to those of skill in the
art. See, e.g., Sambrook, et al.
Various methods of amplifying target sequences, such as the polymerase chain
reaction, can also be used to prepare DNA encoding CRSPs. Polymerase chain
reaction (PCR) technology is used to amplify such nucleic acid sequences
directly from mRNA, from cDNA, and from genomic libraries or cDNA libraries.
The isolated sequences encoding CRSP proteins may also be used as templates
for PCR amplification. The sequences provided teach many appropriate
primers, and pairs.
Typically, in PCR techniques, oligonucleotide primers complementary to two
5' regions in the DNA region to be amplified are synthesized. The polymerase
chain reaction is then carried out using the two primers. See Innis, et al.
(eds.) (1990) PCR Protocols: A Guide to Methods and Applications Academic
Press, San Diego, Calif. Primers can be selected to amplify the entire
regions encoding a full-length CRSP protein or to amplify smaller DNA
segments as desired. Once such regions are PCR-amplified, they can be
sequenced and oligonucleotide probes can be prepared from sequence obtained
using standard techniques. These probes can then be used to isolate DNA's
encoding CRSP proteins.
Oligonucleotides for use as probes are usually chemically synthesized
according to the solid phase phosphoramidite triester method first described
by Beaucage and Carruthers (1983) Tetrahedron Lett. 22(20):1859-1862, or
using an automated synthesizer, as described in Needham-VanDevanter, et al.
(1984) Nucleic Acids Res. 12:6159-6168. Purification of oligonucleotides is
performed e.g., by native acrylamide gel electrophoresis or by
anion-exchange HPLC as described in Pearson and Regnier (1983) J. Chrom.
255:137-149. The sequence of the synthetic oligonucleotide can be verified
using, e.g., the chemical degradation method of Maxam, A. M. and Gilbert, W.
in Grossman, L. and Moldave (eds.) (1980) Methods in Enzymology 65:499-560
Academic Press, New York.
An isolated nucleic acid encoding a human CRSP has been identified. The
nucleotide sequence and corresponding open reading frame of this embodiment
is provided in SEQ ID NO: 1.
Notably, the different genes of the family exhibit great conservation of
spacing of the cysteine residues. In particular, the spacing is
CX11CX8CXCX3CX10CXCXCX9CC. See Table 1.
Based upon the structural modeling and insights in the binding regions of
the collective group, it is predicted that residues in the mature protein,
lacking a signal of about 18 residues, exhibit structural features as
described in Table 1. The soluble protein possesses one helical structure,
and six beta strands. The structure suggests that the sheets form a plane,
and that the underside of the plane is probably the receptor binding
surface. Thus, residue substitutions at positions on the upper surface, or
in the helical region away from the sheet surface will not affect biological
Fragments of at least about 8-10 residues in the cysteine rich region would
be especially interesting peptides, e.g., starting at residue positions of
the mature protein 1, 2, 3, etc. However, the cysteine rich region begins at
33-86. Those fragments would typically end at the ends of the helical or
sheet strands should be important. Other interesting peptides of various
lengths would include ones which begin or end in other positions of the
protein, e.g., at residues 87, 86, etc., with lengths ranging, e.g., from
about 8 to 20, 25, 30, 35, 40, etc.
This invention provides isolated DNA or fragments to encode a CRSP protein.
In addition, this invention provides isolated or recombinant DNA which
encodes a protein or polypeptide which is capable of hybridizing under
appropriate conditions, e.g., high stringency, with the DNA sequences
described herein. Said biologically active protein or polypeptide can be an
intact ligand, or fragment, and have an amino acid sequence as disclosed in
SEQ ID NO: 2. Preferred embodiments will be full length natural sequences,
from isolates, e.g., about 7-8K daltons in size when unglycosylated, or
fragments of at least about 1,000 daltons, more preferably at least about
3,000 daltons. In glycosylated forms, which appear unnatural, the protein
may be larger. Further, this invention contemplates the use of isolated or
recombinant DNA, or fragments thereof, which encode proteins which are
homologous to a CRSP or which were isolated using cDNA encoding a CRSP as a
probe. The isolated DNA can have the respective regulatory sequences in the
5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and
IV. Making CRSPs
DNAs which encode a CRSP or fragments thereof can be obtained by chemical
synthesis, screening cDNA libraries, or by screening genomic libraries
prepared from a wide variety of cell lines or tissue samples.
These DNAs can be expressed in a wide variety of host cells for the
synthesis of a full-length protein 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 CRSP 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 contaminants, other than 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, e.g., CRSP, or portions thereof, may be expressed as fusions with
other proteins or possessing an epitope tag. Forms with a carboxy terminal
FLAG.RTM. expression system epitope tag have been produced.
Expression vectors are typically self-replicating DNA or RNA constructs
containing the desired antigen gene or its fragments, usually operably
linked to appropriate genetic control elements that are recognized in a
suitable host cell. 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 enhancers to elevate the level of mRNA
expression, a sequence that encodes a suitable ribosome binding site, and
sequences that terminate transcription and translation. Expression vectors
also usually contain an origin of replication that allows the vector to
replicate independently from the host cell.
The vectors of this invention contain DNAs which encode a CRSP, or a
fragment thereof, typically encoding, e.g., a biologically active
polypeptide, or protein. The DNA can 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 CRSP in a prokaryotic or eukaryotic host, where
the vector is compatible with the host and where the eukaryotic cDNA coding
for the protein is inserted into the vector such that growth of the host
containing the vector expresses the cDNA in question. 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 protein 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 a
CRSP gene or its fragments into the host DNA by recombination, or to
integrate a promoter which controls expression of an endogenous gene.
Vectors, as used herein, contemplate 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 many other forms of vectors which serve an equivalent
function are suitable for use herein. See, e.g., Pouwels, et al. (1985 and
Supplements) Cloning Vectors: A Laboratory Manual Elsevier, N.Y.; and
Rodriquez, et al. (eds.) (1988) Vectors: A Survey of Molecular Cloning
Vectors and Their Uses Buttersworth, Boston, Mass.
Suitable host cells include prokaryotes, lower eukaryotes, and higher
eukaryotes. Prokaryotes include both gram negative and gram positive
organisms, 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 animal
cells, both of non-mammalian origin, e.g., insect cells, and birds, and of
mammalian 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 its derivatives.
Vectors that can be used to express CRSPs or CRSP fragments include, but are
not limited to, such vectors as those containing the lac promoter (pUC-series);
trp promoter (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 Survey of Molecular
Cloning Vectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.
Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformed with
CRSP sequence containing vectors. For purposes of this invention, the most
common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae.
It will be used generically to 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 termination, polyadenylation, and
transcription termination. 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
Higher eukaryotic tissue culture cells are typically the preferred host
cells for expression of the functionally active CRSP protein. In principle,
many higher eukaryotic tissue culture cell lines may be used, e.g., insect
baculovirus expression systems, whether from an invertebrate or vertebrate
source. However, mammalian cells are preferred to achieve proper processing,
both cotranslationally and posttranslationally. Transformation or
transfection and propagation of such cells is routine. Useful cell lines
include HeLa cells, Chinese 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
(e.g., if genomic DNA is used), a polyadenylation site, and a transcription
termination site. These vectors also may 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.
Representative examples of suitable expression vectors include pcDNA1; pCD,
see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1neo Poly-A, see
Thomas, et al. (1987) Cell 51:503-512; and a baculovirus vector such as pAC
373 or pAC 610.
It is likely that CRSPs need not be glycosylated to elicit biological
responses. However, it will occasionally be desirable to express a CRSP
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., in unglycosylated form, to appropriate glycosylating
proteins introduced into a heterologous expression system. For example, the
CRSP gene may be co-transformed with one or more genes encoding mammalian or
other glycosylating enzymes. It is further understood that over
glycosylation may be detrimental to CRSP biological activity, and that one
of skill may perform routine testing to optimize the degree of glycosylation
which confers optimal biological activity.
A CRSP, or a fragment thereof, may be engineered to be phosphatidyl inositol
(PI) linked to a cell membrane, but can be removed 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 standard procedures of protein chemistry.
See, e.g., Low (1989) Biochem. Biophys. Acta 988:427-454; Tse, et al. (1985)
Science 230:1003-1008; and Brunner, et al. (1991) J. Cell Biol.
Now that CRSPs have been characterized, 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 Synthesis Pierce Chemical Co., Rockford, Ill.; Bodanszky and
Bodanszky (1984) The Practice of Peptide Synthesis Springer-Verlag, New
York, N.Y.; and Bodanszky (1984) The Principles of Peptide Synthesis
Springer-Verlag, New York, N.Y. 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 prepared protein 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 CRSPs of this invention can be obtained in
varying degrees of purity depending upon its desired use. Purification can
be accomplished by use of known protein purification techniques or by the
use of the antibodies or binding partners herein described, e.g., 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 appropriate source cells, lysates of other cells expressing the ligand,
or lysates or supernatants of cells producing the CRSPs as a result of
recombinant DNA techniques, see below.
Multiple cell lines may be screened for one which expresses a CRSP at a high
level compared with other cells. Various cell lines, e.g., a mouse thymic
stromal cell line TA4, is screened and selected for its favorable handling
properties. Natural CRSPs can be isolated from natural sources, or by
expression from a transformed cell using an appropriate expression vector.
Purification of the expressed protein is achieved by standard procedures, or
may be combined with engineered means for effective purification at high
efficiency from cell lysates or supernatants. Epitope or other tags, e.g.,
FLAG.RTM. expression system tap sequences or His6 segments, can be used for
such purification features.
Antibodies can be raised to various CRSPs, including individual,
polymorphic, allelic, strain, or species variants, and fragments thereof,
both in their naturally occurring (full-length) forms and in their
recombinant forms. Additionally, antibodies can be raised to CRSPs in either
their active forms or in their inactive forms. Anti-idiotypic antibodies may
also be used.
A. Antibody Production
A number of immunogens may be used to produce antibodies specifically
reactive with CRSP proteins. Recombinant protein is the preferred immunogen
for the production of monoclonal or polyclonal antibodies. Naturally
occurring protein may also be used either in pure or impure form. Synthetic
peptides, made using the primate CRSP protein sequence described herein, may
also used as an immunogen for the production of antibodies to CRSPs.
Recombinant protein can be expressed in eukaryotic or prokaryotic cells as
described herein, and purified as described. Naturally folded or denatured
material can be used, as appropriate, for producing antibodies. Either
monoclonal or polyclonal antibodies may be generated for subsequent use in
immunoassays to measure the protein.
Methods of producing polyclonal antibodies are known to those of skill in
the art. Typically, an immunogen, preferably a purified protein, is mixed
with an adjuvant and animals are immunized with the mixture. The animal's
immune response to the immunogen preparation is monitored by taking test
bleeds and determining the titer of reactivity to the CRSP protein of
interest. When appropriately high titers of antibody to the immunogen are
obtained, usually after repeated immunizations, blood is collected from the
animal and antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the protein can be done if desired. See,
e.g., Harlow and Lane; or Coligan.
Monoclonal antibodies may be obtained by various techniques familiar to
those skilled in the art. Typically, spleen cells from an animal immunized
with a desired antigen are immortalized, commonly by fusion with a myeloma
cell (see, Kohler and Milstein (1976) Eur. J. Immunol. 6:511-519,
incorporated herein by reference). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or retroviruses,
or other methods known in the art. Colonies arising from single immortalized
cells are screened for production of antibodies of the desired specificity
and affinity for the antigen, and yield of the monoclonal antibodies
produced by such cells may be enhanced by various techniques, including
injection into the peritoneal cavity of a vertebrate host. Alternatively,
one may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B cells
according, e.g., to the general protocol outlined by Huse, et al. (1989)
Antibodies, including binding fragments and single chain versions, against
predetermined fragments of CRSPs can be raised by immunization of animals
with conjugates of the fragments with carrier proteins as described above.
Monoclonal antibodies are prepared from cells secreting the desired
antibody. These antibodies can be screened for binding to normal or
defective CRSPs, or screened for agonistic or antagonistic activity, e.g.,
mediated through a receptor. These monoclonal antibodies will usually bind
with at least a K.sub.D of about 1 mM, more usually at least about 300 .mu.M,
typically at least about 10 .mu.M, more typically at least about 30 .mu.M,
preferably at least about 10 .mu.M, and more preferably at least about 3 .mu.M
In some instances, it is desirable to prepare monoclonal antibodies from
various mammalian hosts, such as mice, rodents, primates, humans, etc.
Description of techniques for preparing such monoclonal antibodies may be
found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology (4th
ed.) Lange Medical Publications, Los Altos, Calif., and references cited
therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)
Academic Press, New York, N.Y.; and particularly in Kohler and Milstein
(1975) Nature 256:495-497, which discusses one method of generating
monoclonal antibodies. Summarized briefly, this method involves injecting an
animal with an immunogen. The animal 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 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 animal generated in response to a
specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of libraries of antibodies in
phage or similar vectors. See, e.g., Huse, et al. (1989) "Generation of a
Large Combinatorial Library of the Immunoglobulin Repertoire in Phage
Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-546.
The polypeptides and antibodies of the present invention may be used with or
without modification, including chimeric or humanized 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, teaching the use of such labels
include U.S. Pat. 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. Pat. No. 4,816,567; and Queen, et al. (1989)
Proc. Nat'l Acad. Sci. USA 86:10029-10033.
The antibodies of this invention are useful for affinity chromatography in
isolating CRSP 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 or supernatant may be passed through the
column, the column washed, followed by increasing concentrations of a mild
denaturant, whereby purified CRSP protein will be released.
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 to CRSPs may be used for the identification of cell populations
expressing CRSPs. By assaying the expression products of cells expressing
CRSPs it is possible to diagnose disease, e.g., immune-compromised
Antibodies raised against each CRSP will also be useful to raise anti-idiotypic
antibodies. These will be useful in detecting or diagnosing various
immunological conditions related to expression of the respective antigens.
A particular protein can be measured by a variety of immunoassay methods.
For a review of immunological and immunoassay procedures in general, see
Stites and Terr (eds.) (1991) Basic and Clinical Immunology (7th ed.).
Moreover, the immunoassays of the present invention can be performed in many
configurations, which are reviewed extensively in Maggio (ed.) (1980) Enzyme
Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985) "Practice and Theory
of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular
Biology, Elsevier Science Publishers B.V., Amsterdam; and Harlow and Lane
Antibodies, A Laboratory Manual, supra, each of which is incorporated herein
by reference. See also Chan (ed.) (1987) Immunoassay: A Practical Guide
Academic Press, Orlando, Fla.; Price and Newman (eds.) (1991) Principles and
Practice of Immunoassays Stockton Press, NY; and Ngo (ed.) (1988)
Non-isotopic Immunoassays Plenum Press, NY.
Immunoassays for measurement of CRSP proteins can be performed by a variety
of methods known to those skilled in the art. In brief, immunoassays to
measure the protein can be either competitive or noncompetitive binding
assays. In competitive binding assays, the sample to be analyzed competes
with a labeled analyte for specific binding sites on a capture agent bound
to a solid surface. Preferably the capture agent is an antibody specifically
reactive with CRSP proteins produced as described above. The concentration
of labeled analyte bound to the capture agent is inversely proportional to
the amount of free analyte present in the sample.
In a competitive binding immunoassay, the CRSP protein present in the sample
competes with labeled protein for binding to a specific binding agent, for
example, an antibody specifically reactive with the CRSP protein. The
binding agent may be bound to a solid surface to effect separation of bound
labeled protein from the unbound labeled protein. Alternately, the
competitive binding assay may be conducted in liquid phase and a variety of
techniques known in the art may be used to separate the bound labeled
protein from the unbound labeled protein. Following separation, the amount
of bound labeled protein is determined. The amount of protein present in the
sample is inversely proportional to the amount of labeled protein binding.
Alternatively, a homogeneous immunoassay may be performed in which a
separation step is not needed. In these immunoassays, the label on the
protein is altered by the binding of the protein to its specific binding
agent. This alteration in the labeled protein results in a decrease or
increase in the signal emitted by label, so that measurement of the label at
the end of the immunoassay allows for detection or quantitation of the
Quantitation of CRSP proteins may also be performed using many of a variety
of noncompetitive immunoassay methods. For example, a two-site, solid phase
sandwich immunoassay may be used. In this type of assay, a binding agent for
the protein, for example an antibody, is attached to a solid support. A
second protein binding agent, which may also be an antibody, and which binds
the protein at a different site, is labeled. After binding at both sites on
the protein has occurred, the unbound labeled binding agent is removed and
the amount of labeled binding agent bound to the solid phase is measured.
The amount of labeled binding agent bound is directly proportional to the
amount of protein in the sample.
Western blot analysis can be used to determine the presence of CRSP proteins
in a sample. Electrophoresis is carried out, for example, on a tissue sample
suspected of containing the protein. Following electrophoresis to separate
the proteins, and transfer of the proteins to a suitable solid support,
e.g., a nitrocellulose filter, the solid support is incubated with an
antibody reactive with the protein. This antibody may be labeled, or
alternatively may be detected by subsequent incubation with a second labeled
antibody that binds the primary antibody.
The immunoassay formats described above employ labeled assay components. The
label may be coupled directly or indirectly to the desired component of the
assay according to methods well known in the art. A wide variety of labels
and methods may be used. Traditionally, a radioactive label incorporating
.sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P was used.
Non-radioactive labels include ligands which bind to labeled antibodies,
fluorophores, chemiluminescent agents, enzymes, and antibodies which can
serve as specific binding pair members for a labeled ligand. The choice of
label depends on sensitivity required, ease of conjugation with the
compound, stability requirements, and available instrumentation. For a
review of various labeling or signal producing systems which may be used,
see U.S. Pat. No. 4,391,904, which is incorporated herein by reference.
Antibodies reactive with a particular protein can also be measured by a
variety of immunoassay methods. For a review of immunological and
immunoassay procedures applicable to the measurement of antibodies by
immunoassay techniques, see Stites and Terr (eds.) Basic and Clinical
Immunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; and
Harlow and Lane Antibodies, A Laboratory Manual, supra.
In brief, immunoassays to measure antisera reactive with CRSP proteins can
be either competitive or noncompetitive binding assays. In competitive
binding assays, the sample analyte competes with a labeled analyte for
specific binding sites on a capture agent bound to a solid surface.
Preferably the capture agent is a purified recombinant CRSP protein produced
as described above. Other sources of CRSP proteins, including isolated or
partially purified naturally occurring protein, may also be used.
Noncompetitive assays include sandwich assays, in which the sample analyte
is bound between two analyte-specific binding reagents. One of the binding
agents is used as a capture agent and is bound to a solid surface. The
second binding agent is labeled and is used to measure or detect the
resultant complex by visual or instrument means. A number of combinations of
capture agent and labeled binding agent can be used. A variety of different
immunoassay formats, separation techniques, and labels can be also be used
similar to those described above for the measurement of CRSP proteins.
VI. Purified CRSPs
Specific embodiments of primate CRSP amino acid sequences are provided in
SEQ ID NO: 2. The human sequence hybridizes to genes of mouse origin.
Purified protein or defined peptides are useful for generating antibodies by
standard methods, as described above. Synthetic peptides or purified protein
can be presented to an immune system to generate polyclonal and monoclonal
antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology
Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual
Cold Spring Harbor Press, NY, which are incorporated herein by reference.
Alternatively, a CRSP receptor can be useful as a specific binding reagent,
and advantage can be taken of its specificity of binding, for, e.g.,
purification of a CRSP ligand.
The specific binding composition can be used for screening an expression
library made from a cell line which expresses a CRSP. Many methods for
screening are available, e.g., standard staining of surface expressed ligand,
or by panning. Screening of intracellular expression can also be performed
by various staining or immunofluorescence procedures. The binding
compositions could be used to affinity purify or sort out cells expressing
The peptide segments, along with comparison to homologous genes, can also be
used to produce appropriate oligonucleotides to screen a library. The
genetic code can be used to select appropriate oligonucleotides useful as
probes for screening. In combination with polymerase chain reaction (PCR)
techniques, synthetic oligonucleotides will be useful in selecting desired
clones from a library, including natural allelic an polymorphic variants.
The peptide sequences allow preparation of peptides to generate antibodies
to recognize such segments, and allow preparation of oligonucleotides which
encode such sequences. The sequence also allows for synthetic preparation,
e.g., see Dawson, et al. (1994) Science 266:776-779. Since CRSPs appear to
be secreted proteins, the gene will normally possess an N-terminal signal
sequence, which is removed upon processing and secretion, and the putative
cleavage site is between amino acids as indicated in Table 1, though it may
be slightly in either direction.
VII. Physical Variants
This invention also encompasses proteins or peptides having substantial
amino acid sequence similarity with an amino acid sequence of a CRSP.
Natural variants include individual, polymorphic, allelic, strain, or
Amino acid sequence similarity, or sequence identity, is determined by
optimizing residue matches, if necessary, by introducing gaps as required.
This changes when considering conservative substitutions as matches.
Physiocochemical conservative residue 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. Homologous
amino acid sequences include natural polymorphic, allelic, and interspecies
variations in each respective protein sequence. Typical homologous proteins
or peptides will have from 50-100% similarity (if gaps can be introduced),
to 75-100% similarity (if conservative substitutions are included) with the
amino acid sequence of the CRSP. Similarity measures will be at least about
50%, generally at least 60%, more generally at least 65%, usually at least
70%, more usually at least 75%, preferably at least 80%, and more preferably
at least 80%, and in particularly preferred embodiments, at least 85% or
more. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff,
et al. (1983) Time Warps, String Edits, and Macromolecules: The Theory and
Practice of Sequence Comparison Chapter One, Addison-Wesley, Reading, Mass.;
and software packages from IntelliGenetics, Mountain View, Calif.; and the
University of Wisconsin Genetics Computer Group, Madison, Wis.
Nucleic acids encoding primate CRSP proteins will often hybridize to the
nucleic acid sequence of SEQ ID NO: 1 under stringent conditions. For
example, nucleic acids encoding mouse CRSP proteins will normally hybridize
to the nucleic acid of SEQ ID NO: 1 under stringent hybridization
conditions. Generally, stringent conditions are selected to be about
10.degree. C. lower than the thermal melting point (Tm) for the probe
sequence at a defined ionic strength and pH. The Tm is the temperature
(under defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent conditions
will be those in which the salt concentration is about 0.2 molar at pH 7 and
the temperature is at least about 50.degree. C. Other factors may
significantly affect the stringency of hybridization, including, among
others, base composition and size of the complementary strands, the presence
of organic solvents such as formamide, and the extent of base mismatching. A
preferred embodiment will include nucleic acids which will bind to disclosed
sequences in 50% formamide and 200 mM NaCl at 42.degree. C.
An isolated CRSP nucleic acid sequence can be readily modified by nucleotide
substitutions, nucleotide deletions, nucleotide insertions, and short
inversions of nucleotide stretches. These modifications result in novel DNA
sequences which encode CRSP antigens, their derivatives, or proteins having
highly similar physiological, immunogenic, or antigenic activity.
Modified sequences can be used to produce mutant antigens or to enhance
expression. Enhanced expression may involve gene amplification, increased
transcription, increased translation, and other mechanisms. Such mutant CRSP
derivatives include predetermined or site-specific mutations of the
respective protein or its fragments. "Mutant CRSP" encompasses a polypeptide
otherwise falling within the homology definition of the CRSP as set forth
above, but having an amino acid sequence which differs from that of a CRSP
as found in nature, whether by way of deletion, substitution, or insertion.
In particular, "site specific mutant CRSP" generally includes proteins
having significant similarity with a protein having a sequence of SEQ ID NO:
2, and as sharing various biological activities, e.g., antigenic or
immunogenic, with those sequences, and in preferred embodiments contain most
or all of the disclosed sequence. This applies also to polymorphic variants
from different individuals. Similar concepts apply to different CRSP
proteins, particularly those found in various warm blooded animals, e.g.,
mammals and birds. As stated before, it is emphasized that descriptions are
generally meant to encompass other CRSP proteins, not limited to the mouse
embodiments specifically discussed.
Although site specific mutation sites are predetermined, mutants need not be
site specific. CRSP 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 may
include amino- or carboxyl-terminal fusions, e.g., epitope tags. Random
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 DNA having a known sequence are well
known in the art, e.g., by M13 primer mutagenesis or polymerase chain
reaction (PCR) techniques. 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 immunoglobulin with
a CRSP 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. One preferred
embodiment is fusion of an Ig domain to the carboxy terminus of the protein.
A similar concept applies to heterologous nucleic acid sequences.
In addition, new constructs may be made from combining similar functional
domains from other proteins. Protein-binding or other segments may be
"swapped" between different new fusion polypeptides or fragments, e.g.,
different CRSP embodiments. 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 chimeric polypeptides exhibiting new combinations of specificities
will result from the functional linkage of protein-binding specificities and
other functional domains.
VIII. Binding Agent:CRSP Protein Complexes
A CRSP protein that specifically binds to or that is specifically
immunoreactive with an antibody generated against a defined immunogen, such
as an immunogen consisting of the amino acid sequence of SEQ ID NO: 2, is
typically determined in an immunoassay. The immunoassay uses a polyclonal
antiserum which was raised to a protein of SEQ ID NO: 2. This antiserum is
selected to have low crossreactivity against other chemokines and any such
crossreactivity is removed by immunoabsorbtion prior to use in the
In order to produce antisera for use in an immunoassay, the protein of SEQ
ID NO: 2, is isolated as described herein. For example, recombinant protein
may be produced in a mammalian cell line. An inbred strain of mice or rats,
such as balb/c mice, is immunized with a protein of SEQ ID NO: 2, using a
standard adjuvant, such as Freund's adjuvant, and a standard immunization
protocol (see Harlow and Lane, supra). Alternatively, a synthetic peptide,
preferably near full length, derived from the sequences disclosed herein and
conjugated to a carrier protein can be used an immunogen. Polyclonal sera
are collected and titered against the immunogen protein in an immunoassay,
for example, a solid phase immunoassay with the immunogen immobilized on a
solid support. Polyclonal antisera with a titer of 10.sup.4 or greater are
selected and tested for their cross reactivity against, e.g., known proteins
exhibiting sequence similarity such as cytokines or growth factors, using a
competitive binding immunoassay such as the one described in Harlow and
Lane, supra, at pages 570-573. Preferably two family members are used in
this determination in conjunction with various embodiments, e.g., mouse
Various forms of CRSPs are used to identify antibodies which are
specifically bound. These proteins can be produced as recombinant proteins
and isolated using standard molecular biology and protein chemistry
techniques as described herein. Moreover, since the CRSPs seem to lack
glycosylation sites, problems of post-translational modifications is
Immunoassays in the competitive binding format can be used for the
crossreactivity determinations. For example, a protein of SEQ ID NO: 2 can
be immobilized to a solid support. Proteins added to the assay compete with
the binding of the antisera to the immobilized antigen. The ability of the
above proteins to compete with the binding of the antisera to the
immobilized protein is compared to the protein of SEQ ID NO: 2. The percent
crossreactivity for the above proteins is calculated, using standard
calculations. Those antisera with less than 10% crossreactivity with each of
the proteins listed above are selected and pooled. The cross-reacting
antibodies are then removed from the pooled antisera by immunoabsorbtion
with the above-listed proteins.
The immunoabsorbed and pooled antisera are then used in a competitive
binding immunoassay as described above to compare a second protein to the
immunogen protein (e.g., the CRSP cysteine rich motifs of SEQ ID NO: 2). In
order to make this comparison, the two proteins are each assayed at a wide
range of concentrations and the amount of each protein required to inhibit
50% of the binding of the antisera to the immobilized protein is determined.
If, e.g., the amount of the second protein required is less than twice the
amount of the protein of SEQ ID NO: 2 that is required, then the second
protein is said to specifically bind to an antibody generated to the
It is understood that CRSP proteins are a family of homologous proteins that
comprise multiple genes. For a particular gene product, such as the C23 CRSP
protein, the term refers not only to the amino acid sequences disclosed
herein, but also to other proteins that are polymorphic, allelic,
non-allelic, or species variants. It is also understood that the term
"primate CRSP" includes nonnatural mutations introduced by deliberate
mutation using conventional recombinant technology such as single site
mutation, or by excising very short sections of DNA encoding CRSP proteins,
or by substituting new amino acids, or adding new amino acids. Such minor
alterations must substantially maintain a particular feature, e.g., the
immunoidentity of the original molecule and/or a biological activity. Thus,
these alterations include proteins that are specifically immunoreactive with
a designated naturally occurring CRSP protein, for example, the C23 CRSP
protein shown in SEQ ID NO: 2. The biological properties of the altered
proteins can be determined by expressing the protein in an appropriate cell
line and measuring, e.g., a chemotactic effect. Particular protein
modifications considered minor would include conservative substitution of
amino acids with similar chemical properties, as described above for the
CRSP family as a whole. By aligning, e.g., a protein optimally with the
protein of SEQ ID NO: 2, and by using the conventional immunoassays
described herein to determine immunoidentity, or by using chemotaxis assays,
one can determine the protein compositions of the invention.
IX. Functional Variants
The blocking of physiological response to CRSPs may result from the
inhibition of binding of the protein to its receptor, e.g., through
competitive inhibition. Thus, in vitro assays of the present invention will
often use isolated protein, membranes from cells expressing a recombinant
membrane associated CRSP, soluble fragments comprising receptor binding
segments of these proteins, or fragments attached to solid phase substrates.
These assays will also allow for the diagnostic determination of the effects
of either binding segment mutations and modifications, or protein mutations
and modifications, e.g., protein analogs. This invention also contemplates
the use of competitive drug screening assays, e.g., where neutralizing
antibodies to antigen or receptor fragments compete with a test compound for
binding to the protein. In this manner, the antibodies can be used to detect
the presence of a polypeptide which shares one or more antigenic binding
sites of the protein and can also be used to occupy binding sites on the
protein that might otherwise interact with a receptor.
"Derivatives" of CRSP 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 CRSP amino acid side chains 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 terminus, or of residues containing carboxyl side chains, O-acyl
derivatives of hydroxyl group-containing residues, and N-acyl derivatives of
the amino terminal amino acid or amino-group containing residues, e.g.,
lysine or arginine. Acyl groups are selected from the group of
alkyl-moieties including C3 to C18 normal alkyl, thereby forming alkanoyl
aroyl species. Covalent attachment to carrier proteins may be important when
immunogenic moieties are haptens.
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. While the primary sequences
suggest the absence of glycosylation sites, the sequences may be modified to
incorporate such. Particularly preferred means for accomplishing this are by
exposing the polypeptide to glycosylating enzymes derived from cells which
normally provide such processing, e.g., mammalian 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, or other moieties, including ribosyl
groups or cross-linking reagents.
A major group of derivatives are covalent conjugates of the CRSP or
fragments thereof with other proteins or 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 protein derivatization
sites with cross-linking agents are at free amino groups, carbohydrate
moieties, and cysteine residues.
Fusion polypeptides between CRSPs and other homologous or heterologous
proteins are also provided. Many growth factors and cytokines are
homodimeric entities, and a repeat construct may have various advantages,
including lessened susceptibility to proteolytic degradation. Moreover, many
receptors require dimerization to transduce a signal, and various dimeric
proteins or domain repeats can be desirable. Heterologous polypeptides may
be fusions between different surface markers, resulting in, e.g., a hybrid
protein exhibiting receptor binding specificity. 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 domain of a
protein, e.g., a receptor-binding segment, so that the presence or location
of the fused protein may be easily determined. See, e.g., Dull, et al., U.S.
Pat. No. 4,859,609. Other gene fusion partners include bacterial .beta.-galactosidase,
trpE, Protein A, .beta.-lactamase, alpha amylase, alcohol dehydrogenase, and
yeast alpha mating factor. See, e.g., Godowski, et al. (1988) Science
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.
This invention also contemplates the use of derivatives of CRSPs 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 the three classes: (1) salts, (2) side chain
and terminal residue covalent modifications, and (3) adsorption complexes,
for example with cell membranes. Such covalent or aggregative derivatives
are useful as immunogens, as reagents in immunoassays, or in purification
methods such as for affinity purification of ligands or other binding
ligands. For example, a CRSP 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-CRSP antibodies or its receptor. The CRSPs can also be
labeled with a detectable group, e.g., radioiodinated by the chloramine T
procedure, covalently bound to rare earth chelates, or conjugated to another
fluorescent moiety for use in diagnostic assays. Purification of CRSPs may
be effected by immobilized antibodies or receptor.
Isolated CRSP genes will allow transformation of cells lacking expression of
corresponding CRSPs, e.g., either species types or cells which lack
corresponding proteins 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 CRSP
receptor proteins. Subcellular fragments, e.g., cytoplasts or membrane
fragments, can be isolated and used.
The present invention provides reagents which will find use in diagnostic
applications as described elsewhere herein, e.g., in the general description
for developmental abnormalities, or below in the description of kits for
diagnosis. Each of these embodiments of the family are associated rather
specifically with an inflammatory or immunologially active tissue.
The cysteine rich nature makes the protein a good substrate for sulfhydryl
reagents. It will be useful as a control for cysteine incorporation, and as
a sample to test ability to sequence through those residues. The protein
will also find use as a carbon source.
CRSP nucleotides, e.g., DNA or RNA, may be used as a component in a
diagnostic assay. For instance, the nucleotide sequences provided may be
labeled using, e.g., .sup.32P or biotin and used to probe standard
restriction fragment polymorphism blots, providing a measurable character to
aid in distinguishing between individuals. Such probes may be used in
well-known forensic techniques such as genetic fingerprinting. In addition,
nucleotide probes made from CRSP sequences may be used in in situ assays to
detect chromosomal abnormalities. For instance, rearrangements in the mouse
chromosome encoding a CRSP gene may be detected via well-known in situ
techniques, using CRSP probes in conjunction with other known chromosome
Antibodies and other binding agents directed towards CRSP proteins or
nucleic acids may be used to purify the corresponding CRSP molecule. As
described in the Examples below, antibody purification of CRSP components is
both possible and practicable. Antibodies and other binding agents may also
be used in a diagnostic fashion to determine whether CRSP components are
present in a tissue sample or cell population using well-known techniques
described herein. Specific medical conditions correlating with expression of
the respective embodiments is described. The ability to attach a binding
agent to a CRSP provides a means to diagnose disorders associated with CRSP
misregulation. Antibodies and other CRSP binding agents may also be useful
as histological markers. As described in the examples below, CRSP expression
is limited to specific tissue types. By directing a probe, such as an
antibody or nucleic acid to a CRSP it is possible to use the probe to
distinguish tissue and cell types in situ or in vitro.
This invention also provides reagents with significant therapeutic value.
The CRSPs (naturally occurring or recombinant), fragments thereof, and
antibodies thereto, along with compounds identified as having binding
affinity to a CRSP, are useful in the treatment of conditions associated
with abnormal physiology or development, including abnormal proliferation,
e.g., inflammatory conditions, 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 signaling by a CRSP is a target for an agonist or
antagonist of the protein. The proteins likely play a role in regulation or
development of various cells, e.g., lymphoid cells, which affect
Other abnormal developmental conditions are known in cell types shown to
possess CRSP mRNA by northern blot analysis. See Berkow (ed.) The Merck
Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; and Thorn, et
al. Harrison's Principles of Internal Medicine, McGraw-Hill, NY.
Developmental or functional abnormalities, e.g., of the immune system, cause
significant medical abnormalities and conditions which may be susceptible to
prevention or treatment using compositions provided herein. The role of
epithelial cells in such conditions may be important.
Recombinant CRSP or CRSP antibodies can be purified and then administered to
a patient. These reagents can be combined for therapeutic use with
additional active or inert ingredients, e.g., in conventional
pharmaceutically acceptable carriers or diluents, e.g., immunogenic
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 binding
fragments thereof, including forms which are not complement binding.
Drug screening using antibodies or receptor or fragments thereof can
identify compounds having binding affinity to CRSPs, including isolation of
associated components. Subsequent biological assays can then be utilized to
determine if the compound has intrinsic stimulating activity and is
therefore a blocker or antagonist in that it blocks the activity of the
protein. Likewise, a compound having intrinsic stimulating activity can
activate the receptor and is thus an agonist in that it simulates the
activity of a CRSP. This invention further contemplates the therapeutic use
of antibodies to CRSPs as antagonists. This approach should be particularly
useful with other CRSP species variants.
The quantities of reagents necessary for effective therapy will depend upon
many different factors, including means of administration, target site,
physiological state of the patient, and other medicants administered. 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. Animal 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
Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press; and (1990)
Remington's Pharmaceutical Sciences (17th ed.) Mack Publishing Co., Easton,
Pa. Methods for administration are discussed therein and below, e.g., for
oral, intravenous, intraperitoneal, or intramuscular administration,
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, N.J. Dosage ranges would ordinarily be
expected to be in amounts lower than 1 mM concentrations, typically less
than about 10 .mu.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
CRSPs, fragments thereof, and antibodies to it or its fragments,
antagonists, and agonists, may be administered directly to the host to be
treated or, depending 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 administration. Therapeutic formulations may be administered in any
conventional dosage formulation. While it is possible for the active
ingredient to be administered alone, it is preferable to present it as a
pharmaceutical 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 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 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 pharmacy. See, e.g., Gilman, et al. (eds.)
(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8th
ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences (17th
ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY; Lieberman,
et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, NY; and
Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse
Systems Dekker, NY. The therapy of this invention may be combined with or
used in association with other therapeutic agents.
Both the naturally occurring and the recombinant forms of the CRSPs of this
invention are particularly useful in kits and assay methods which are
capable of screening compounds for binding 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, and other descriptions of
chemical diversity libraries, which describe means for testing of binding
affinity by a plurality of compounds. The development of suitable assays can
be greatly facilitated by the availability of large amounts of purified,
soluble CRSP as provided by this invention.
For example, antagonists can normally be found once the protein has been
structurally defined. Testing of potential protein analogs is now possible
upon the development of highly automated assay methods using a purified
receptor. In particular, new agonists and antagonists will be discovered by
using screening techniques described herein. Of particular importance are
compounds found to have a combined binding affinity for multiple CRSP
receptors, e.g., compounds which can serve as antagonists for species
variants of a CRSP.
This invention is particularly useful for screening compounds by using
recombinant protein in 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 CRSP from a specific source;
(b) potentially greater number of ligands 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 a
CRSP receptor. Cells may be isolated which express a receptor in isolation
from any others. Such cells, either in viable or fixed form, can be used for
standard ligand/receptor binding 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 CRSP)
are contacted and incubated with a labeled receptor or antibody having known
binding affinity to the ligand, such as .sup.125I-antibody, and a test
sample whose binding affinity to the binding composition is being measured.
The bound and free labeled binding compositions are then separated to assess
the degree of ligand binding. The amount of test compound bound is inversely
proportional to the amount of labeled receptor binding to the known source.
Any one of numerous techniques can be used to separate bound from free
ligand to assess the degree of ligand binding. This separation step could
typically involve a procedure 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 CRSP mediated functions, e.g., second messenger levels, i.e.,
Ca.sup.++; 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.sup.++ levels, with a fluorimeter or a fluorescence cell
Another method utilizes membranes from transformed eukaryotic or prokaryotic
host cells as the source of a CRSP. These cells are stably transformed with
DNA vectors directing the expression of a CRSP, e.g., an engineered membrane
bound form. Essentially, the membranes would be prepared from the cells and
used in a receptor/ligand binding assay such as the competitive assay set
Still another approach is to use solubilized, unpurified or solubilized,
purified CRSP from transformed eukaryotic or prokaryotic host cells. This
allows for a "molecular" binding assay with the advantages of increased
specificity, the ability to automate, and high drug test throughput.
Another technique for drug screening involves an approach which provides
high throughput screening for compounds having suitable binding affinity to
a CRSP antibody and is described in detail in Geysen, European Patent
Application 84/03564, published on Sep. 13, 1984. First, large numbers of
different small peptide test compounds are synthesized on a solid substrate,
e.g., plastic pins or some other appropriate surface, see Fodor, et al.,
supra. Then all the pins are reacted with solubilized, unpurified or
solubilized, purified CRSP antibody, and washed. The next step involves
detecting bound CRSP antibody.
Rational drug design may also be based upon structural studies of the
molecular shapes of the CRSP and other effectors or analogs. See, e.g.,
Methods in Enzymology vols 202 and 203. Effectors may be other proteins
which mediate other functions in response to ligand binding, or other
proteins which normally interact with the receptor. One means for
determining which sites interact with specific other proteins is a physical
structure determination, e.g., x-ray crystallography or 2 dimensional NMR
techniques. These will provide guidance as to which amino acid residues form
molecular contact regions. For a detailed description of protein structural
determination, see, e.g., Blundell and Johnson (1976) Protein
Crystallography Academic Press, NY.
A purified CRSP can be coated directly onto plates for use in the
aforementioned drug screening techniques. However, non-neutralizing
antibodies to these ligands can be used as capture antibodies to immobilize
the respective ligand on the solid phase.
This invention also contemplates use of CRSPs, fragments thereof, peptides,
and their fusion products in a variety of diagnostic kits and methods for
detecting the presence of CRSP or a CRSP receptor. Typically the kit will
have a compartment containing either a defined CRSP peptide or gene segment
or a reagent which recognizes one or the other, e.g., receptor fragments or
A kit for determining the binding affinity of a test compound to a CRSP
would typically comprise a test compound; a labeled compound, e.g., a
receptor or antibody having known binding affinity for the CRSP; a source of
CRSP (naturally occurring or recombinant); and a means for separating bound
from free labeled compound, such as a solid phase for immobilizing the CRSP.
Once compounds are screened, those having suitable binding affinity to the
CRSP can be evaluated in suitable biological assays, as are well known in
the art, to determine whether they act as agonists or antagonists to the
receptor. The availability of recombinant CRSP polypeptides also provide
well defined standards for calibrating such assays.
A preferred kit for determining the concentration of, for example, a CRSP in
a sample would typically comprise a labeled compound, e.g., receptor or
antibody, having known binding affinity for the CRSP, a source of CRSP
(naturally occurring or recombinant), and a means for separating the bound
from free labeled compound, for example, a solid phase for immobilizing the
CRSP. Compartments containing reagents, and instructions, will normally be
Antibodies, including antigen binding fragments, specific for the CRSP or
ligand fragments are useful in diagnostic applications to detect the
presence of elevated levels of CRSP 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 ligand in a body fluid, e.g., serum, or the like. Diagnostic
assays may be homogeneous (without a separation step between free reagent
and antigen-CRSP complex) or heterogeneous (with a separation step). Various
commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked
immunosorbentassay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied
immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA),
and the like. For example, unlabeled antibodies can be employed by using a
second antibody which is labeled and which recognizes the antibody to a CRSP
or to a particular fragment thereof. Similar assays have also been
extensively discussed in the literature. See, e.g., Harlow and Lane (1988)
Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987)
Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price and
Newman (eds.) (1991) Principles and Practice of Immunoassay Stockton Press,
NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press, NY.
Anti-idiotypic antibodies may have similar use to diagnose presence of
antibodies against a CRSP, as such may be diagnostic of various abnormal
states. For example, overproduction of CRSP may result in production of
various immunological or other medical reactions which may be diagnostic of
abnormal physiological states, e.g., in cell growth, activation, or
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 receptor, or labeled CRSP 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.
Many 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 protein, test compound, CRSP,
or antibodies thereto can be labeled either directly or indirectly.
Possibilities for direct labeling include label groups: radiolabels such as
.sup.125I, enzymes (U.S. Pat. No. 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. 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 CRSP can be
immobilized on various matrices followed by washing. Suitable matrices
include plastic such as an ELISA plate, filters, and beads. Methods of
immobilizing the CRSP 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
ligand/receptor or ligand/antibody 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.
Methods for linking proteins or their fragments to the various labels have
been extensively reported in the literature and do not require detailed
discussion here. 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 diagnostic aspect of this invention involves use of oligonucleotide
or polynucleotide sequences taken from the sequence of a CRSP. These
sequences can be used as probes for detecting levels of the CRSP message in
samples from natural sources, or patients suspected of having an abnormal
condition, e.g., cancer or developmental problem. The preparation of both
RNA and DNA nucleotide sequences, 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 about 14 nucleotides, usually at least about 18 nucleotides, and
the polynucleotide probes may be up to several kilobases. Various labels may
be employed, most commonly radionuclides, particularly .sup.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, fluorophores, 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 using many 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 quantitative 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) Progress in
Growth Factor Res. 1:89-97.
XII. Receptor Isolation
Having isolated a binding partner of a specific interaction, methods exist
for isolating the counter-partner. See, Gearing, et al. (1989) EMBO J.
8:3667-3676. For example, means to label a CRSP without interfering with the
binding to its receptor can be determined. For example, an affinity label or
epitope tag can be fused to either the amino- or carboxyl-terminus of the
ligand. An expression library can be screened for specific binding of the
CRSP, e.g., by cell sorting, or other screening to detect subpopulations
which express such a binding component. See, e.g., Ho, et al. (1993) Proc.
Nat'l Acad. Sci. USA 90:11267-11271. Alternatively, a panning method may be
used. See, e.g., Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA
84:3365-3369. A two-hybrid selection system may also be applied making
appropriate constructs with the available CRSP sequences. See, e.g., Fields
and Song (1989) Nature 340:245-246.
Protein cross-linking techniques with label can be applied to isolate
binding partners of a CRSP. This would allow identification of proteins
which specifically interact with a CRSP, e.g., in a ligand-receptor like
manner. It is likely that the receptor will be found by expression in a
system which is capable of expressing a membrane protein in a form capable
of exhibiting ligand binding capability.
Claim 1 of 11 Claims
1. An isolated binding compound
comprising an isolated antibody or an antigen binding portion thereof that
specifically binds to a polypeptide consisting of the amino acid sequence
of the C23 polypeptide of SEQ ID NO: 2.
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