Title: Treatment of
United States Patent: 7,419,663
Issued: September 2, 2008
Inventors: Ashkenazi; Avi
(San Mateo, CA), Helmy; Karim Yussef (San Francisco, CA), Fong; Sherman
(Alameda, CA), Goddard; Audrey (San Francisco, CA), Gurney; Austin L.
(Belmont, CA), Katschke, Jr.; Kenneth James (Millbrae, CA), Napier; Mary
A. (Hillsborough, CA), Tumas; Daniel (Orinda, CA), Van Lookeren; Menno
(San Francisco, CA), Wood; William I. (Hillsborough, CA)
Assignee: Genentech, Inc.
(South San Francisco, CA)
Appl. No.: 10/964,263
Filed: October 12, 2004
The present invention concerns a recently
discovered macrophage specific receptor, STIgMA, and its use in the
treatment of complement-associated disorders.
Description of the
The present invention
concerns the use of a novel macrophage-associated receptor with homology
to the A33 antigen and JAM1, which was cloned from a fetal lung library
and identified as a single transmembrane Ig superfamily member macrophage
associated (STIgMA) polypeptide. Native human STIgMA is expressed as two
spliced variants, one containing an N-terminal IgV like domain and a
C-terminal IgC2 like domain and a spliced form lacking the C-terminal
domain (SEQ ID NOs: 4 and 6, respectively). Both receptors have a single
transmembrane domain and a cytoplasmic domain, containing tyrosine
residues which are constitutively phosphorylated in macrophages in vitro.
A mouse homologue was found with 67% sequence homology to human STIgMA (SEQ
ID NO: 2). The full-length human STIgMA polypeptide also has a shorter
version, with an N-terminal segment missing (SEQ ID NO: 2).
As shown in the Examples below (see Original Patent), STIgMA binds
complement C3b and inhibits C3 convertase. STIgMA is selectively expressed
on tissue resident macrophages, and its expression is upregulated by
dexamethasone and IL-10, and down-regulated by LPS and IFN-.gamma., and
inhibits collagen- and antibody-induced arthritis independent of B or T
Complement plays a crucial role in the body's defense, and, together with
other components of the immune system, protect the individual from
pathogens invading the body. However, if not properly activated or
controlled, complement can also cause injury to host tissues.
Inappropriate activation of complement is involved in the pathogenesis of
a variety of diseases, referred to as complement associated diseases or
disorders, such as immune complex and autoimmune diseases, and various
inflammatory conditions, including complement-mediated inflammatory tissue
damage. The pathology of complement-associated diseases varies, and might
involve complement activation for a long or short period of time,
activation of the whole cascade, only one of the cascades (e.g. classical
or alternative pathway), only some components of the cascade, etc. In some
diseases complement biological activities of complement fragments result
in tissue injury and disease. Accordingly, inhibitors of complement have
high therapeutic potential. Selective inhibitors of the alternative
pathway would be particularly useful, because clearance of pathogens and
other organisms from the blood through the classical pathway will remain
C3b is known to covalently opsonize surfaces of microorganisms invading
the body, and act as a ligand for complement receptors present on
phagocytic cells, which ultimately leads to phagocytosis of the pathogens.
In many pathological situations, such as those listed above, complement
will be activated on cell surfaces, including the vascular wall, cartilage
in the joints, glomeruli in the liver or cells which lack intrinsic
complement inhibitors. Complement activation leads to inflammation caused
by the chemoattractant properties of the anaphylatoxins C3a and C5a and
can cause damage to self-cells by generating a membrane attack complex.
Without being bound by any particular theory, by binding C3b, STIgMA is
believed to inhibit C3 convertase, thereby preventing or reducing
complement-mediated diseases, examples of which have been listed
Compounds of the Invention
1. Native Sequence and Variant STIgMA Polypeptides
The preparation of native STIgMA molecules, along with their nucleic acid
and polypeptide sequences, have been discussed above. Example 1 shows the
cloning of full-length huSTIgMA of SEQ ID NO: 4. STIgMA polypeptides can
be produced by culturing cells transformed or transfected with a vector
containing STIgMA nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be employed to
prepare STIgMA. For instance, the STIgMA sequence, or portions thereof,
may be produced by direct peptide synthesis using solid-phase techniques
[see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman
Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc.,
85:2149-2154 (1963)]. In vitro protein synthesis may be performed using
manual techniques or by automation. Automated synthesis may be
accomplished, for instance, using an Applied Biosystems Peptide
Synthesizer (Foster City, Calif.) using manufacturer's instructions.
Various portions of STIgMA may be chemically synthesized separately and
combined using chemical or enzymatic methods to produce the fill-length
STIgMA variants can be prepared by introducing appropriate nucleotide
changes into the DNA encoding a native sequence STIgMA polypeptide, or by
synthesis of the desired STIgMA polypeptide. Those skilled in the art will
appreciate that amino acid changes may alter post-translational processes
of STIgMA, such as changing the number or position of glycosylation sites
or altering the membrane anchoring characteristics.
Variations in the native sequence STIgMA polypeptides described herein can
be made, for example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for instance, in
U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or
insertion of one or more codons encoding a native sequence or variant
STIgMA that results in a change in its amino acid sequence as compared
with a corresponding native sequence or variant STIgMA. Optionally the
variation is by substitution of at least one amino acid with any other
amino acid in one or more of the domains of a native sequence STIgMA
polypeptide. Guidance in determining which amino acid residue may be
inserted, substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the STIgMA with that of
homologous known protein molecules and minimizing the number of amino acid
sequence changes made in regions of high homology.
Amino acid substitutions can be the result of replacing one amino acid
with another amino acid having similar structural and/or chemical
properties, such as the replacement of a leucine with a serine, i.e.,
conservative amino acid replacements. Insertions or deletions may
optionally be in the range of 1 to 5 amino acids. The variation allowed
may be determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence and testing the resulting
variants for activity in the in vitro assay described in the Examples
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,
and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl. Acids
Res, 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
cassette mutagenesis [Wells et al., Gene, 34:315 (1985)], restriction
selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA,
317:415 (1986)] or other known techniques can be performed on the cloned
DNA to produce the STIgMA variant DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids that may be varied along a contiguous sequence. Among the
preferred scanning amino acids are relatively small, neutral amino acids.
Such amino acids include alanine, glycine, serine, and cysteine. Alanine
is typically a preferred scanning amino acid among this group because it
eliminates the side-chain beyond the beta-carbon and is less likely to
alter the main-chain conformation of the variant. Alanine is also
typically preferred because it is the most common amino acid. Further, it
is frequently found in both buried and exposed positions [Creighton, The
Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1
(1976)]. If alanine substitution does not yield adequate amounts of
variant, an isoteric amino acid can be used.
It has been found that removal or inactivation of all or part of the
transmembrane region and/or cytoplasmic region does not compromise STIgMA
biological activity. Therefore, transmembrane region and/or cytoplasmic
region deleted/inactivated STIgMA variants are specifically within the
scope herein. Similarly, the IgC2 region can be removed without
compromising biological activity, as demonstrated by the existence of a
biologically active native short form of huSTIgMA and a murine homologue.
Covalent modifications of native sequence and variant STIgMA polypeptides
are included within the scope of this invention. One type of covalent
modification includes reacting targeted amino acid residues of STIgMA with
an organic derivatizing agent that is capable of reacting with selected
side chains or the N- or C-terminal residues of the STIgMA polypeptide.
Derivatization with bifunctional agents is useful, for instance, for
crosslinking STIgMA to a water-insoluble support matrix or surface, for
example, for use in the method for purifying anti-STIgMA antibodies.
Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide
esters, for example, esters with 4-azidosalicylic acid, homobifunctional
imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate),
bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such
Other modifications include deamidation of glutaminyl and asparaginyl
residues to the corresponding glutamyl and aspartyl residues,
respectively, hydroxylation of proline and lysine, phosphorylation of
hydroxyl groups of seryl or threonyl residues, methylation of the
.alpha.-amino groups of lysine, arginine, and histidine side chains [T. E.
Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &
Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal
amine, and amidation of any C-terminal carboxyl group.
Another type of covalent modification of the STIgMA polypeptides included
within the scope of this invention comprises altering the native
glycosylation pattern of the polypeptides. "Altering the native
glycosylation pattern" is intended for purposes herein to mean deleting
one or more carbohydrate moieties found in native sequence STIgMA, and/or
adding one or more glycosylation sites that are not present in the native
sequence STIgMA, and/or alteration of the ratio and/or composition of the
sugar residues attached to the glycosylation site(s). A predicted native
glycosylation site on murine STIgMA is found at position 170 in the
Addition of glycosylation sites to the STIgMA polypeptide may be
accomplished by altering the amino acid sequence. The alteration may be
made, for example, by the addition of, or substitution by, one or more
serine or threonine residues to the native sequence STIgMA (for O-linked
glycosylation sites). The STIgMA amino acid sequence may optionally be
altered through changes at the DNA level, particularly by mutating the DNA
encoding the STIgMA polypeptide at preselected bases such that codons are
generated that will translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the
STIgMA polypeptide is by chemical or enzymatic coupling of glycosides to
the polypeptide. Such methods are described in the art, e.g., in WO
87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the a STIgMA polypeptide may
be accomplished chemically or enzymatically or by mutational substitution
of codons encoding for amino acid residues that serve as targets for
glycosylation. Chemical deglycosylation techniques are known in the art
and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys.,
259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981).
Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety of endo- and exo-glycosidases as
described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
Another type of covalent modification of STIgMA comprises linking the
STIgMA polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, for
example in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The native sequence and variant STIgMA of the present invention may also
be modified in a way to form a chimeric molecule comprising STIgMA,
including fragments of STIgMA, fused to another, heterologous polypeptide
or amino acid sequence. In one embodiment, such a chimeric molecule
comprises a fusion of STIgMA with a tag polypeptide which provides an
epitope to which an anti-tag antibody can selectively bind. The epitope
tag is generally placed at the amino- or carboxyl-terminus of the STIgMA
polypeptide. The presence of such epitope-tagged forms of the STIgMA
polypeptide can be detected using an antibody against the tag polypeptide.
Also, provision of the epitope tag enables the STIgMA polypeptide to be
readily purified by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds to the epitope tag. Various tag
polypeptides and their respective antibodies are well known in the art.
Examples include poly-histidine (poly-his) or poly-histidine-glycine
(poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5
[Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and
the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,
Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3
epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an .quadrature.-tubulin
epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)];
and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc.
Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
In another embodiment, the chimeric molecule may comprise a fusion of the
STIgMA polypeptide or a fragment thereof with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule, such a fusion could be to the Fc region of an IgG
molecule. These fusion polypeptides are antibody-like molecules which
combine the binding specificity of a heterologous protein (an "adhesin")
with the effector functions of immunoglobulin constant domains, and are
often referred to as immunoadhesins. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired binding
specificity which is other than the antigen recognition and binding site
of an antibody (i.e., is "heterologous"), and an immunoglobulin constant
domain sequence. The adhesin part of an immunoadhesin molecule typically
is a contiguous amino acid sequence comprising at least the binding site
of a receptor or a ligand. The immunoglobulin constant domain sequence in
the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1,
IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD
Chimeras constructed from a receptor sequence linked to an appropriate
immunoglobulin constant domain sequence (immunoadhesins) are known in the
art. lmmunoadhesins reported in the literature include fusions of the T
cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA, 84: 2936-2940
(1987)); CD4 (Capon et al., Nature 337: 525-531 (1989); Traunecker et al.,
Nature, 339: 68-70 (1989); Zettmeissl et al., DNA Cell Biol. USA, 9:
347-353 (1990); Byrn et al., Nature, 344: 667-670 (1990)); L-selectin
(homing receptor) ((Watson et al., J. Cell. Biol., 110:2221-2229(1990);
Watson et al., Nature, 349: 164-167 (1991)); CD44 (Aruffo et al., Cell,
61: 1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med., 173:
721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med. 174: 561-569 (1991));
CD22 (Stamenkovic et al., Cell, 66:1133-11144 (1991)); TNF receptor
(Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539(1991);
Lesslauer et al., Eur. J. Immunol., 27:2883-2886(1991); Peppel et al., J.
Exp. Med., 174:1483-1489(1991)); NP receptors (Bennett et al., J. Biol.
Chem. 266:23060-23067(1991)); and IgE receptor alpha. (Ridgway et al., J.
Cell. Biol., 115:abstr. 1448 (1991)).
The simplest and most straightforward immunoadhesin design combines the
binding region(s) of the "adhesin" protein with the hinge and Fc regions
of an immunoglobulin heavy chain. Ordinarily, when preparing the
STIgMA-immunoglobulin chimeras of the present invention, nucleic acid
encoding the extracellular domain of STIgMA will be fused C-terminally to
nucleic acid encoding the N-terminus of an immunoglobulin constant domain
sequence, however N-terminal fusions are also possible.
Typically, in such fusions the encoded chimeric polypeptide will retain at
least functionally active hinge and CH2 and CH3 domains of the constant
region of an immunoglobulin heavy chain. Fusions are also made to the
C-terminus of the Fc portion of a constant domain, or immediately
N-terminal to the CH1 of the heavy chain or the corresponding region of
the light chain.
The precise site at which the fusion is made is not critical; particular
sites are well known and may be selected in order to optimize the
biological activity, secretion or binding characteristics of the
In some embodiments, the STIgMA-immunoglobulin chimeras are assembled as
monomers, or hetero- or homo-multimer, and particularly as dimers or
tetramers, essentially as illustrated in WO 91/08298.
In a preferred embodiment, the STIgMA extracellular domain sequence is
fused to the N-terminus of the C-terminal portion of an antibody (in
particular the Fc domain), containing the effector functions of an
immunoglobulin, e.g. immunoglobulin G.sub.1 (IgG 1). It is possible to
fuse the entire heavy chain constant region to the STIgMA extracellular
domain sequence. However, more preferably, a sequence beginning in the
hinge region just upstream of the papain cleavage site (which defines IgG
Fc chemically; residue 216, taking the first residue of heavy chain
constant region to be 114, or analogous sites of other immunoglobulins) is
used in the fusion. In a particularly preferred embodiment, the STIgMA
amino acid sequence is fused to the hinge region and CH2 and CH3, or to
the CH1, hinge, CH2 and CH3 domains of an IgG1, gG2, or IgG3 heavy chain.
The precise site at which the fusion is made is not critical, and the
optimal site can be determined by routine experimentation.
In some embodiments, the STIgMA-immunoglobulin chimeras are assembled as
multimer, and particularly as homo-dimers or -tetramers. Generally, these
assembled immunoglobulins will have known unit structures. A basic four
chain structural unit is the form in which IgG, IgD, and IgE exist. A four
unit is repeated in the higher molecular weight immunoglobulins; IgM
generally exists as a pentamer of basic four units held together by
disulfide bonds. IgA globulin, and occasionally IgG globulin, may also
exist in multimeric form in serum. In the case of multimer, each four unit
may be the same or different.
Alternatively, the STIgMA extracellular domain sequence can be inserted
between immunoglobulin heavy chain and light chain sequences such that an
immunoglobulin comprising a chimeric heavy chain is obtained. In this
embodiment, the STIgMA sequence is fused to the 3' end of an
immunoglobulin heavy chain in each arm of an immunoglobulin, either
between the hinge and the CH2 domain, or between the CH2 and CH3 domains.
Similar constructs have been reported by Hoogenboom et al., Mol. Immunol.,
Although the presence of an immunoglobulin light chain is not required in
the immunoadhesins of the present invention, an immunoglobulin light chain
might be present either covalently associated to a STIgMA-immunoglobulin
heavy chain fusion polypeptide, or directly fused to the STIgMA
extracellular domain. In the former case, DNA encoding an immunoglobulin
light chain is typically coexpressed with the DNA encoding the
STIgMA-immunoglobulin heavy chain fusion protein. Upon secretion, the
hybrid heavy chain and the light chain will be covalently associated to
provide an immunoglobulin-like structure comprising two disulfide-linked
immunoglobulin heavy chain-light chain pairs. Methods suitable for the
preparation of such structures are, for example, disclosed in U.S. Pat.
No. 4,816,567 issued Mar. 28, 1989.
2. Preparation of Native Sequence and Variant STIgMA Polypeptides
DNA encoding native sequence STIgMA polypeptides may be obtained from a
cDNA library prepared from tissue believed to possess the STIgMA mRNA and
to express it at a detectable level. Accordingly, human STIgMA DNA can be
conveniently obtained from a cDNA library prepared from human tissue, such
as described in Example 1. The STIgMA-encoding gene may also be obtained
from a genomic library or by oligonucleotide synthesis.
Libraries can be screened with probes (such as antibodies to STIgMA or
oligonucleotides of at least about 20-80 bases) designed to identify the
gene of interest or the protein encoded by it. Screening the cDNA or
genomic library with the selected probe may be conducted using standard
procedures, such as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).
An alternative means to isolate the gene encoding STIgMA is to use PCR
methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995).
Example 1 describes techniques for screening a cDNA library. The
oligonucleotide sequences selected as probes should be of sufficient
length and sufficiently unambiguous that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected
upon hybridization to DNA in the library being screened. Methods of
labeling are well known in the art, and include the use of radiolabels
like .sup.32P-labeled ATP, biotinylation or enzyme labeling. Hybridization
conditions, including moderate stringency and high stringency, are
provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known sequences deposited and available in public
databases such as GenBank or other private sequence databases. Sequence
identity (at either the amino acid or nucleotide level) within defined
regions of the molecule or across the full-length sequence can be
determined through sequence alignment using computer software programs
such as BLAST, BLAST-2, ALIGN, DNAstar, and INHERIT which employ various
algorithms to measure homology.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic libraries using the deduced amino acid sequence
disclosed herein for the first time, and, if necessary, using conventional
primer extension procedures as described in Sambrook et al., supra, to
detect precursors and processing intermediates of mRNA that may not have
been reverse-transcribed into cDNA.
Host cells are transfected or transformed with expression or cloning
vectors described herein for STIgMA production and cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes encoding the
desired sequences. The culture conditions, such as media, temperature, pH
and the like, can be selected by the skilled artisan without undue
experimentation. In general, principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be found
in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL
Press, 1991) and Sambrook et al., supra.
Methods of transfection are known to the ordinarily skilled artisan, for
example, CaPO4 and electroporation. Depending on the host cell used,
transformation is performed using standard techniques appropriate to such
cells. The calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes or other cells that contain substantial cell-wall barriers.
Infection with Agrobacterium tumefaciens is used for transformation of
certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and
WO 89/05859 published 29 Jun. 1989. For mammalian cells without such cell
walls, the calcium phosphate precipitation method of Graham and van der Eb,
Virology, 52:456-457 (1978) can be employed. General aspects of mammalian
cell host system transformations have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out according
to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao
et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other
methods for introducing DNA into cells, such as by nuclear microinjection,
electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may also be used. For various
techniques for transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352
Suitable host cells for cloning or expressing the DNA in the vectors
herein include prokaryote, yeast, or higher eukaryote cells. Suitable
prokaryotes include but are not limited to eubacteria, such as
Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae
such as E. coli. Various E. coli strains are publicly available, such as
E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E.
coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi
or yeast are suitable cloning or expression hosts for STIgMA-encoding
vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host
Suitable host cells for the expression of glycosylated STIgMA are derived
from multicellular organisms. Examples of invertebrate cells include
insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant
cells. Examples of useful mammalian host cell lines include Chinese
hamster ovary (CHO) and COS cells. More specific examples include monkey
kidney CV1 cells transformed by SV40 (COS-7, ATCC CRL 1651); human
embryonic kidney cells (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese
hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci.
USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver
cells (Hep G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC
CCL51). The selection of the appropriate host cell is deemed to be within
the skill in the art.
The nucleic acid (e.g., cDNA or genomic DNA) encoding STIgMA may be
inserted into a replicable vector for cloning (amplification of the DNA)
or for expression. Various vectors are publicly available. The vector may,
for example, be in the form of a plasmid, cosmid, viral particle, or
phage. The appropriate nucleic acid sequence may be inserted into the
vector by a variety of procedures. In general, DNA is inserted into an
appropriate restriction endonuclease site(s) using techniques known in the
art. Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable vectors containing one or more of these
components employs standard ligation techniques which are known to the
The STIgMA polypeptides may be produced recombinantly not only directly,
but also as a fusion polypeptide with a heterologous polypeptide, which
may be a signal sequence or other polypeptide having a specific cleavage
site at the N-terminus of the mature protein or polypeptide. In general,
the signal sequence may be a component of the vector, or it may be a part
of the STIgMA DNA that is inserted into the vector. The signal sequence
may be a prokaryotic signal sequence selected, for example, from the group
of the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may be,
e.g., the yeast invertase leader, alpha factor leader (including
Saccharomyces and Kluyveromyces"--factor leaders, the latter described in
U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal
described in WO 90/13646 published 15 Nov. 1990. In mammalian cell
expression, mammalian signal sequences may be used to direct secretion of
the protein, such as signal sequences from secreted polypeptides of the
same or related species, as well as viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate in one or more selected host cells. Such
sequences are well known for a variety of bacteria, yeast, and viruses.
The origin of replication from the plasmid pBR322 is suitable for most
Gram-negative bacteria, the 2: plasmid origin is suitable for yeast, and
various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful
for cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene,
also termed a selectable marker. Typical selection genes encode proteins
that (a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not available
from complex media, e.g., the gene encoding D-alanine racemase for
An example of suitable selectable markers for mammalian cells are those
that enable the identification of cells competent to take up the STIgMA
nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell
when wild-type DHFR is employed is the CHO cell line deficient in DHFR
activity, prepared and propagated as described by Urlaub et al., Proc.
Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in
yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et
al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper
et al., Gene, 10:157 (1980)]. The trp1 gene provides a selection marker
for a mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked
to the STIgMA nucleic acid sequence to direct mRNA synthesis. Promoters
recognized by a variety of potential host cells are well known. Promoters
suitable for use with prokaryotic hosts include the .quadrature.-lactamase
and lactose promoter systems [Chang et al., Nature, 275:615 (1978);
Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057
(1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer
et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence
operably linked to the DNA encoding STIgMA.
Examples of suitable promoting sequences for use with yeast hosts include
the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol.
Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv.
Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as
enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription controlled by growth conditions, are the
promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism,
metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes
responsible for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP 73,657.
STIgMA transcription from vectors in mammalian host cells is controlled,
for example, by promoters obtained from the genomes of viruses such as
polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter
or an immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
Transcription of a DNA encoding the STIgMA polypeptides by higher
eukaryotes may be increased by inserting an enhancer sequence into the
vector. Enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp, that act on a promoter to increase its transcription. Many
enhancer sequences are now known from mammalian genes (globin, elastase,
albumin, .alpha.-fetoprotein, and insulin). Typically, however, one will
use an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270), the
cytomegalovirus early promoter enhancer, the polyoma enhancer on the late
side of the replication origin, and adenovirus enhancers. The enhancer may
be spliced into the vector at a position 5' or 3' to the STIgMA coding
sequence, but is preferably located at a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect,
plant, animal, human, or nucleated cells from other multicellular
organisms) will also contain sequences necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available from the 5' and, occasionally 3', untranslated regions of
eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated
portion of the mRNA encoding STIgMA.
Still other methods, vectors, and host cells suitable for adaptation to
the synthesis of STIgMA in recombinant vertebrate cell culture are
described in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,
Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
Gene amplification and/or expression may be measured in a sample directly,
for example, by conventional Southern blotting, Northern blotting to
quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA,
77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ
hybridization, using an appropriately labeled probe, based on the
sequences provided herein. Alternatively, antibodies may be employed that
can recognize specific duplexes, including DNA duplexes, RNA duplexes, and
DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn
may be labeled and the assay may be 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.
Gene expression, alternatively, may be measured by immunological methods,
such as immunohistochemical staining of cells or tissue sections and assay
of cell culture or body fluids, to quantitate directly the expression of
gene product. Antibodies useful for immunohistochemical staining and/or
assay of sample fluids may be either monoclonal or polyclonal, and may be
prepared in any mammal. Conveniently, the antibodies may be prepared
against a native sequence STIgMA polypeptide or against a synthetic
peptide based on the DNA sequences provided herein or against exogenous
sequence fused to STIgMA DNA and encoding a specific antibody epitope.
Forms of STIgMA may be recovered from culture medium or from host cell
lysates. If membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage.
Cells employed in expression of STIgMA can be disrupted by various
physical or chemical means, such as freeze-thaw cycling, sonication,
mechanical disruption, or cell lysing agents.
It may be desired to purify STIgMA from recombinant cell proteins or
polypeptides. The following procedures are exemplary of suitable
purification procedures: by fractionation on an ion-exchange column;
ethanol precipitation; reverse phase HPLC; chromatography on silica or on
a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex G-75;
protein A Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms of the STIgMA polypeptide.
Various methods of protein purification may be employed and such methods
are known in the art and described for example in Deutscher, Methods in
Enzymology, 182 (1990); Scopes, Protein Purification: Principles and
Practice, Springer-Verlag, New York (1982). The purification step(s)
selected will depend, for example, on the nature of the production process
used and the particular STIgMA produced.
3. Agonists of STIgMA Polypeptides
Agonists of the STIgMA polypeptides will mimic a qualitative biological
activity of a native sequence STIgMA polypeptide. Preferably, the
biological activity is the ability to bind C3b, and/or to affect
complement or complement activation, in particular to inhibit the
alternative complement pathway and/or C3 convertase. Agonists include, for
example, the immunoadhesins, peptide mimetics, and non-peptide small
organic molecules mimicking a qualitative biological activity of a native
STIgMA-Ig immunoadhesins have been discussed above.
Another group of STIgMA agonists are peptide mimetics of native sequence
STIgMA polypeptides. Peptide mimetics include, for example, peptides
containing non-naturally occurring amino acids provided the compound
retains STIgMA biological activity as described herein. Similarly, peptide
mimetics and analogs may include non-amino acid chemical structures that
mimic the structure of important structural elements of the STIgMA
polypeptides of the present invention and retain STIgMA biological
activity. The term "peptide" is used herein to refer to constrained (that
is, having some element of structure as, for example, the presence of
amino acids which initiate a .beta. turn or .beta. pleated sheet, or for
example, cyclized by the presence of disulfide bonded Cys residues) or
unconstrained (e.g., linear) amino acid sequences of less than about 50
amino acid residues, and preferably less than about 40 amino acids
residues, including multimers, such as dimers thereof or there between. Of
the peptides of less than about 40 amino acid residues, preferred are the
peptides of between about 10 and about 30 amino acid residues and
especially the peptides of about 20 amino acid residues. However, upon
reading the instant disclosure, the skilled artisan will recognize that it
is not the length of a particular peptide but its ability to bind C3b and
inhibit C3 convertase, in particular C3 convertase of the alternative
complement pathway, that distinguishes the peptide.
Peptides can be conveniently prepared using solid phase peptide synthesis
(Merrifield, J. Am. Chem. Soc. 85:2149 (1964); Houghten, Proc. Natl. Acad.
Sci. USA 82:5132 (1985)). Solid phase synthesis begins at the carboxyl
terminus of the putative peptide by coupling a protected amino acid to an
inert solid support. The inert solid support can be any macromolecule
capable of serving as an anchor for the C-terminus of the initial amino
acid. Typically, the macromolecular support is a cross-linked polymeric
resin (e.g., a polyamide or polystyrene resin), as shown in FIGS. 1-1 and
1-2 (see Original Patent), on pages 2 and 4 of Stewart and Young, supra.
In one embodiment, the C-terminal amino acid is coupled to a polystyrene
resin to form a benzyl ester. A macromolecular support is selected such
that the peptide anchor link is stable under the conditions used to
deprotect the .alpha.-amino group of the blocked amino acids in peptide
synthesis. If a base-labile alpha.-protecting group is used, then it is
desirable to use an acid-labile link between the peptide and the solid
support. For example, an acid-labile ether resin is effective for
base-labile Fmoc-amino acid peptide synthesis, as described on page 16 of
Stewart and Young, supra. Alternatively, a peptide anchor link and
.alpha.-protecting group that are differentially labile to acidolysis can
be used. For example, an aminomethyl resin such as the
phenylacetamidomethyl (Pam) resin works well in conjunction with Boc-amino
acid peptide synthesis, as described on pages 11-12 of Stewart and Young,
After the initial amino acid is coupled to an inert solid support, the
.alpha.-amino protecting group of the initial amino acid is removed with,
for example, trifluoroacetic acid (TFA) in methylene chloride and
neutralizing in, for example, triethylamine (TEA). Following deprotection
of the initial amino acid's .alpha.-amino group, the next .alpha.-amino
and sidechain protected amino acid in the synthesis is added. The
remaining .alpha.-amino and, if necessary, side chain protected amino
acids are then coupled sequentially in the desired order by condensation
to obtain an intermediate compound connected to the solid support.
Alternatively, some amino acids may be coupled to one another to form a
fragment of the desired peptide followed by addition of the peptide
fragment to the growing solid phase peptide chain.
The condensation reaction between two amino acids, or an amino acid and a
peptide, or a peptide and a peptide can be carried out according to the
usual condensation methods such as the axide method, mixed acid anhydride
method, DCC (N,N'-dicyclohexylcarbodiimide) or DIC (N,N'-diisopropylcarbodiimide)
methods, active ester method, p-nitrophenyl ester method, BOP
(benzotriazole-1-yl-oxy-tris [dimethylamino] phosphonium
hexafluorophosphate) method, N-hydroxysuccinic acid imido ester method,
etc, and Woodward reagent K method.
It is common in the chemical syntheses of peptides to protect any reactive
side-chain groups of the amino acids with suitable protecting groups.
Ultimately, these protecting groups are removed after the desired
polypeptide chain has been sequentially assembled. Also common is the
protection of the .alpha.-amino group on an amino acid or peptide fragment
while the C-terminal carboxyl group of the amino acid or peptide fragment
reacts with the free N-terminal amino group of the growing solid phase
polypeptide chain, followed by the selective removal of the .alpha.-amino
group to permit the addition of the next amino acid or peptide fragment to
the solid phase polypeptide chain. Accordingly, it is common in
polypeptide synthesis that an intermediate compound is produced which
contains each of the amino acid residues located in the desired sequence
in the peptide chain wherein individual residues still carry side-chain
protecting groups. These protecting groups can be removed substantially at
the same time to produce the desired polypeptide product following removal
from the solid phase.
.alpha.- and .epsilon.-amino side chains can be protected with
benzyloxycarbonyl (abbreviated Z), isonicotinyloxycarbonyl (iNOC), o-chlorobenzyloxycarbonyl
[Z(2Cl)], p-nitrobenzyloxycarbonyl [Z(NO.sub.2)], p-methoxybenzyloxycarbonyl
[Z(OMe))], t-butoxycarbonyl (Boc), t-amyloxycarbonyl (Aoc),
2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl (Fmoc),
methylsulfonyethoxycarbonyl (Msc), trifluoroacetyl, phthalyl, formyl,
2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt), and
dimethylphosphinothioyl (Mpt) groups, and the like.
Protective groups for the carboxyl functional group are exemplified by
benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzyl ester (ONb),
t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and the like. It is
often desirable that specific amino acids such as arginine, cysteine, and
serine possessing a functional group other than amino and carboxyl groups
are protected by a suitable protective group. For example, the guanidino
group of arginine may be protected with nitro, p-toluenesulfonyl,
benzyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzesulfonyl,
4-methoxy-2,6-dimethylbenzenesulfonyl (Nds), 1,3,5-trimethylphenysulfonyl
(Mts), and the like. The thiol group of cysteine can be protected with p-methoxybenzyl,
trityl, and the like.
Many of the blocked amino acids described above can be obtained from
commercial sources such as Novabiochem (San Diego, Calif.), Bachem CA (Torrence,
Calif.) or Peninsula Labs (Belmont, Calif.).
Stewart and Young, supra, provides detailed information regarding
procedures for preparing peptides. Protection of a-amino groups is
described on pages 14-18, and side chain blockage is described on pages
18-28. A table of protecting groups for amine, hydroxyl and sulfhydryl
functions is provided on pages 149-151.
After the desired amino acid sequence has been completed, the peptide can
be cleaved away from the solid support, recovered and purified. The
peptide is removed from the solid support by a reagent capable of
disrupting the peptide-solid phase link, and optionally deprotects blocked
side chain functional groups on the peptide. In one embodiment, the
peptide is cleaved away from the solid phase by acidolysis with liquid
hydrofluoric acid (HF), which also removes any remaining side chain
protective groups. Preferably, in order to avoid alkylation of residues in
the peptide (for example, alkylation of methionine, cysteine, and tyrosine
residues), the acidolysis reaction mixture contains thio-cresol and cresol
scavengers. Following HF cleavage, the resin is washed with ether, and the
free peptide is extracted from the solid phase with sequential washes of
acetic acid solutions. The combined washes are lyophilized, and the
peptide is purified.
4. Antagonists of STIgMA Polypeptides
Antagonists of native sequence STIgMA polypeptides find utility in the
treatment of condition benefiting from excessive complement activation,
including the treatment of tumors.
A preferred group of antagonists includes antibodies specifically binding
a native STIgMA. Exemplary antibodies include polyclonal, monoclonal,
humanized, bispecific and heteroconjugate antibodies.
Methods of preparing polyclonal antibodies are known to skilled artisan.
Polyclonal antibodies can be raised in a mammal, for example, by one or
more injections of an immunizing agent, and, if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in the
mammal by multiple subcutaneous or intraperitoneal injections. The
immunizing agent may include the STIgMA polypeptide of the invention or a
fragment or fusion protein thereof. It may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal being
immunized. Examples of such immunogenic proteins include but are not
limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin,
and soybean trypsin inhibitor. Examples of adjuvants which may be employed
include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl
Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol
may be selected by one skilled in the art without undue experimentation.
Antibodies which recognize and bind to the polypeptides of the invention
or which act as antagonists thereto may, alternatively be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma methods,
such as those described by Kohler and Milstein, Nature, 256:495 (1975). In
a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an immunizing agent to elicit lymphocytes that
produce or are capable of producing antibodies that will specifically bind
to the immunizing agent. Alternatively, the lymphocytes may be immunized
The immunizing agent will typically include the STIgMA polypeptide of the
invention, an antigenic fragment or a fusion protein thereof. Generally,
either peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then fused
with an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
Immortalized cell lines are usually transformed mammalian cells,
particularly myeloma cells of rodent, bovine and human origin. Usually,
rat or mouse myeloma cell lines are employed. The hybridoma cells may be
cultured in a suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the
culture medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent the
growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. More preferred immortalized cell lines are murine myeloma lines,
which can be obtained, for instance, from the Salk Institute Cell
Distribution Center, San Diego, Calif. and the American Type Culture
Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma
cell lines also have been described for the production of human monoclonal
antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of monoclonal antibodies directed against the
polypeptide of the invention or having similar activity as the polypeptide
of the invention. Preferably, the binding specificity of monoclonal
antibodies produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
Such techniques and assays are known in the art. The binding affinity of
the monoclonal antibody can, for example, be determined by the Scatchard
analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be
subcloned by limiting dilution procedures and grown by standard methods [Goding,
supra]. Suitable culture media for this purpose include, for example,
Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively,
the hybridoma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium or ascites fluid by conventional
immunoglobulin purification procedures such as, for example, protein A-Sepharose,
hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity
The monoclonal antibodies may also be made by recombinant DNA methods,
such as those described in U.S. Pat. No. 4,816,567. DNA encoding the
monoclonal antibodies of the invention can be readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding specifically to genes encoding the
heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA
may be placed into expression vectors, which are then transfected into
host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal antibodies in the recombinant host
cells. The DNA also may be modified, for example, by substituting the
coding sequence for human heavy and light chain constant domains in place
of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et
al., supra] or by covalently joining to the immunoglobulin coding sequence
all or part of the coding sequence for a non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for the
variable domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent antibody.
The antibodies are preferably monovalent antibodies. Methods for preparing
monovalent antibodies are well known in the art. For example, one method
involves recombinant expression of immunoglobulin light chain and modified
heavy chain. The heavy chain is truncated generally at any point in the Fc
region so as to prevent heavy chain crosslinking. Alternatively, the
relevant cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab
fragments, can be accomplished using routine techniques known in the art.
The antibodies of the invention may further comprise humanized antibodies
or human antibodies. Humanized forms of non-human (e.g., murine)
antibodies are chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal sequence
derived from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and capacity. In
some instances, Fv framework residues of the human immunoglobulin are
replaced by corresponding non-human residues. Humanized antibodies may
also comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and typically
two, variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will comprise at
least a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These non-human amino
acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can be
essentially performed following the method of Winter and coworkers [Jones
et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence from a
non-human species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in
the art, including phage display libraries [Hoogenboom and Winter, J. Mol.
Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The
techniques of Cole et al. and Boemer et al. are also available for the
preparation of human monoclonal antibodies (Cole et al., Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al.,
J. Immunol., 147(1):86-95 (1991); U.S. Pat. No. 5,750,373]. Similarly,
human antibodies can be made by introducing of human immunoglobulin loci
into transgenic animals, e.g., mice in which the endogenous immunoglobulin
genes have been partially or completely inactivated. Upon challenge, human
antibody production is observed, which closely resembles that seen in
humans in all respects, including gene rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and
in the following scientific publications: Marks et al., Bio/Technology 10,
779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison,
Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14,
845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding specificities for at least two different
antigens. In the present case, one of the binding specificities may be for
the polypeptide of the invention, the other one is for any other antigen,
and preferably for a cell-surface protein or receptor or receptor subunit.
Methods for making bispecific antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies is
based on the coexpression of two immunoglobulin heavy-chain/light-chain
pairs, where the two heavy chains have different specificities (Milstein
and Cuello, Nature, 305:537-539 ). Because of the random assortment
of immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of ten different antibody molecules, of which
only one has the correct bispecific structure. The purification of the
correct molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May 1993,
and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities
(antibody-antigen combining sites) can be fused to immunoglobulin constant
domain sequences. The fusion preferably is with an immunoglobulin
heavy-chain constant domain, comprising at least part of the hinge, CH2,
and CH3 regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light-chain binding present
in at least one of the fusions. DNAs encoding the immunoglobulin
heavy-chain fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are cotransfected into a
suitable host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210
Heteroconjugate antibodies are composed of two covalently joined
antibodies. Such antibodies have, for example, been proposed to target
immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089). It is
contemplated that the antibodies may be prepared in vitro using known
methods in synthetic protein chemistry, including those involving
crosslinking agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond. Examples of
suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.
Pat. No. 4,676,980.
It may be desirable to modify the antibody of the invention with respect
to effector function, so as to enhance the effectiveness of the antibody
in treating an immune related disease, for example. For example cysteine
residue(s) may be introduced in the Fc region, thereby allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated may have improved internalization capability and/or increased
complement-mediated cell killing and antibody-dependent cellular
cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992)
and Shopes, B., J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies
with enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al. Cancer
Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement lysis
and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design,
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin
(e.g. an enzymatically active toxin of bacterial, fungal, plant or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
Chemotherapeutic agents useful in the generation of such immunoconjugates
have been described above. Enzymatically active toxins and fragments
thereof which can be used include diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of radionuclides are
available for the production of radioconjugated antibodies. Examples
include .sup.212Bi, .sup.131I, .sup.131In, .sup.90Y and .sup.186Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT),
bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene
2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as described in Vitetta et al., Science 238: 1098 (1987).
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of radionucleotide to the antibody. See WO94/11026.
In another embodiment, the antibody may be conjugated to a "receptor"
(such streptavidin) for utilization in tissue pretargeting wherein the
antibody-receptor conjugate is administered to the patient, followed by
removal of unbound conjugate from the circulation using a clearing agent
and then administration of a "ligand" (e.g. avidin) which is conjugated to
a cytotoxic agent (e.g. a radionucleotide).
5. Target Diseases and Treatment Methods
The STIgMA polypeptides of the present invention and their agonists,
especially STIgMA-Ig immunoadhesins, find utility in the prevention and/or
treatment of complement-associated diseases and pathological conditions.
Such diseases and conditions include, without limitation, inflammatory and
Specific examples of complement-associated diseases include, without
limitation, rheumatoid arthritis (RA), acute respiratory distress syndrome
(ARDS), remote tissue injury after ischemia and reperfusion, complement
activation during cardiopulmonary bypass surgery, dermatomyositis,
pemphigus, lupus nephritis and resultant glomerulonephritis and vasculitis,
cardiopulmonary bypass, cardioplegia-induced coronary endothelial
dysfunction, type II membranoproliferative glomerulonephritis, IgA
nephropathy, acute renal failure, cryoglobulemia, antiphospholipid
syndrome, age-related macular degeneration, uveitis, diabetic retinopathy,
allo-transplantation, xeno-transplantation, hyperacute rejection,
hemodialysis, chronic occlusive pulmonary distress syndrome (COPD),
asthma, hereditary angioedema, paroxysma nocturnal hemoglobulinurea,
Alzheimers disease, atherosclerosis, and aspiration pneumonia.
A more extensive list of inflammatory conditions as examples of
complement-associated diseases includes, for example, inflammatory bowel
disease (IBD), systemic lupus erythematosus, rheumatoid arthritis,
juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma),
idiopathic inflammatory myopathies (dermatomyositis, polymyositis),
Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria),
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia), thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis,
tubulointerstitial nephritis), demyelinating diseases of the central and
peripheral nervous systems such as multiple sclerosis, idiopathic
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other nonhepatotropic viruses), autoimmune
chronic active hepatitis, primary biliary cirrhosis, granulomatous
hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung
diseases (e.g., cystic fibrosis), gluten-sensitive enteropathy, Whipple's
disease, autoimmune or immune-mediated skin diseases including bullous
skin diseases, erythema multiforme and contact dermatitis, psoriasis,
allergic diseases of the lung such as eosinophilic pneumonia, idiopathic
pulmonary fibrosis and hypersensitivity pneumonitis, transplantation
associated diseases including graft rejection and graft-versus host
In systemic lupus erythematosus, the central mediator of disease is the
production of auto-reactive antibodies to self proteins/tissues and the
subsequent generation of immune-mediated inflammation. Antibodies either
directly or indirectly mediate tissue injury. Though T lymphocytes have
not been shown to be directly involved in tissue damage, T lymphocytes are
required for the development of auto-reactive antibodies. The genesis of
the disease is thus T lymphocyte dependent. Multiple organs and systems
are affected clinically including kidney, lung, musculoskeletal system,
mucocutaneous, eye, central nervous system, cardiovascular system,
gastrointestinal tract, bone marrow and blood.
Rheumatoid arthritis (RA) is a chronic systemic autoimmune inflammatory
disease that mainly involves the synovial membrane of multiple joints with
resultant injury to the articular cartilage. The pathogenesis is T
lymphocyte dependent and is associated with the production of rheumatoid
factors, auto-antibodies directed against self IgG, with the resultant
formation of immune complexes that attain high levels in joint fluid and
blood. These complexes in the joint may induce the marked infiltrate of
lymphocytes and monocytes into the synovium and subsequent marked synovial
changes; the joint space/fluid is infiltrated by similar cells with the
addition of numerous neutrophils. Tissues affected are primarily the
joints, often in symmetrical pattern. However, extra-articular disease
also occurs in two major forms. One form is the development of extra-articular
lesions with ongoing progressive joint disease and typical lesions of
pulmonary fibrosis, vasculitis, and cutaneous ulcers. The second form of
extra-articular disease is the so called Felty's syndrome which occurs
late in the RA disease course, sometimes after joint disease has become
quiescent, and involves the presence of neutropenia, thrombocytopenia and
splenomegaly. This can be accompanied by vasculitis in multiple organs
with formations of infarcts, skin ulcers and gangrene. Patients often also
develop rheumatoid nodules in the subcutis tissue overlying affected
joints; the nodules late stages have necrotic centers surrounded by a
mixed inflammatory cell infiltrate. Other manifestations which can occur
in RA include: pericarditis, pleuritis, coronary arteritis, interstitial
pneumonitis with pulmonary fibrosis, keratoconjunctivitis sicca, and
Juvenile chronic arthritis is a chronic idiopathic inflammatory disease
which begins often at less than 16 years of age. Its phenotype has some
similarities to RA; some patients which are rheumatoid factor positive are
classified as juvenile rheumatoid arthritis. The disease is sub-classified
into three major categories: pauciarticular, polyarticular, and systemic.
The arthritis can be severe and is typically destructive and leads to
joint ankylosis and retarded growth. Other manifestations can include
chronic anterior uveitis and systemic amyloidosis.
Spondyloarthropathies are a group of disorders with some common clinical
features and the common association with the expression of HLA-B27 gene
product. The disorders include: ankylosing spondylitis, Reiter's syndrome
(reactive arthritis), arthritis associated with inflammatory bowel
disease, spondylitis associated with psoriasis, juvenile onset
spondyloarthropathy and undifferentiated spondyloarthropathy.
Distinguishing features include sacroileitis with or without spondylitis;
inflammatory asymmetric arthritis; association with HLA-B27 (a
serologically defined allele of the HLA-B locus of class I MHC); ocular
inflammation, and absence of autoantibodies associated with other
rheumatoid disease. The cell most implicated as key to induction of the
disease is the CD8+T lymphocyte, a cell which targets antigen presented by
class I MHC molecules. CD8+T cells may react against the class I MHC
allele HLA-B27 as if it were a foreign peptide expressed by MHC class I
molecules. It has been hypothesized that an epitope of HLA-B27 may mimic a
bacterial or other microbial antigenic epitope and thus induce a CD8+T
Systemic sclerosis (scleroderma) has an unknown etiology. A hallmark of
the disease is induration of the skin; likely this is induced by an active
inflammatory process. Scleroderma can be localized or systemic; vascular
lesions are common and endothelial cell injury in the microvasculature is
an early and important event in the development of systemic sclerosis; the
vascular injury may be immune mediated. An immunologic basis is implied by
he presence of mononuclear cell infiltrates in the cutaneous lesions and
the presence of anti-nuclear antibodies in many patients. ICAM-1 is often
upregulated on the cell surface of fibroblasts in skin lesions suggesting
that T cell interaction with these cells may have a role in the
pathogenesis of the disease. Other organs involved include: the
gastrointestinal tract: smooth muscle atrophy and fibrosis resulting in
abnormal peristalsis/motility; kidney: concentric subendothelial intimal
proliferation affecting small arcuate and interlobular arteries with
resultant reduced renal cortical blood flow, results in proteinuria,
azotemia and hypertension; skeletal muscle: atrophy, interstitial
fibrosis; inflammation; lung: interstitial pneumonitis and interstitial
fibrosis; and heart: contraction band necrosis, scarring/fibrosis.
Idiopathic inflammatory myopathies including dermatomyositis, polymyositis
and others are disorders of chronic muscle inflammation of unknown
etiology resulting in muscle weakness. Muscle injury/inflammation is often
symmetric and progressive. Autoantibodies are associated with most forms.
These myositis-specific autoantibodies are directed against and inhibit
the function of components, proteins and RNA's, involved in protein
Sjogren's syndrome is due to immune-mediated inflammation and subsequent
functional destruction of the tear glands and salivary glands. The disease
can be associated with or accompanied by inflammatory connective tissue
diseases. The disease is associated with autoantibody production against
Ro and La antigens, both of which are small RNA-protein complexes. Lesions
result in keratoconjunctivitis sicca, xerostomia, with other
manifestations or associations including bilary cirrhosis, peripheral or
sensory neuropathy, and palpable purpura.
Systemic vasculitis includes diseases in which the primary lesion is
inflammation and subsequent damage to blood vessels which results in
ischemia/necrosis/degeneration to tissues supplied by the affected vessels
and eventual end-organ dysfunction in some cases. Vasculitides can also
occur as a secondary lesion or sequelae to other immune-inflammatory
mediated diseases such as rheumatoid arthritis, systemic sclerosis, etc.,
particularly in diseases also associated with the formation of immune
complexes. Diseases in the primary systemic vasculitis group include:
systemic necrotizing vasculitis: polyarteritis nodosa, allergic angiitis
and granulomatosis, polyangiitis; Wegener's granulomatosis; lymphomatoid
granulomatosis; and giant cell arteritis. Miscellaneous vasculitides
include: mucocutaneous lymph node syndrome (MLNS or Kawasaki's disease),
isolated CNS vasculitis, Behet's disease, thromboangiitis obliterans (Buerger's
disease) and cutaneous necrotizing venulitis. The pathogenic mechanism of
most of the types of vasculitis listed is believed to be primarily due to
the deposition of immunoglobulin complexes in the vessel wall and
subsequent induction of an inflammatory response either via ADCC,
complement activation, or both.
Sarcoidosis is a condition of unknown etiology which is characterized by
the presence of epithelioid granulomas in nearly any tissue in the body;
involvement of the lung is most common. The pathogenesis involves the
persistence of activated macrophages and lymphoid cells at sites of the
disease with subsequent chronic sequelae resultant from the release of
locally and systemically active products released by these cell types.
Autoimmune hemolytic anemia including autoimmune hemolytic anemia, immune
pancytopenia, and paroxysmal noctural hemoglobinuria is a result of
production of antibodies that react with antigens expressed on the surface
of red blood cells (and in some cases other blood cells including
platelets as well) and is a reflection of the removal of those antibody
coated cells via complement mediated lysis and/or ADCC/Fc-receptor-mediated
In autoimmune thrombocytopenia including thrombocytopenic purpura, and
immune-mediated thrombocytopenia in other clinical settings, platelet
destruction/removal occurs as a result of either antibody or complement
attaching to platelets and subsequent removal by complement lysis, ADCC or
FC-receptor mediated mechanisms.
Thyroiditis including Grave's disease, Hashimoto's thyroiditis, juvenile
lymphocytic thyroiditis, and atrophic thyroiditis, are the result of an
autoimmune response against thyroid antigens with production of antibodies
that react with proteins present in and often specific for the thyroid
gland. Experimental models exist including spontaneous models: rats (BUF
and BB rats) and chickens (obese chicken strain); inducible models:
immunization of animals with either thyroglobulin, thyroid microsomal
antigen (thyroid peroxidase).
Type I diabetes mellitus or insulin-dependent diabetes is the autoimmune
destruction of pancreatic islet .beta. cells; this destruction is mediated
by auto-antibodies and auto-reactive T cells. Antibodies to insulin or the
insulin receptor can also produce the phenotype of
Immune mediated renal diseases, including glomerulonephritis and
tubulointerstitial nephritis, are the result of antibody or T lymphocyte
mediated injury to renal tissue either directly as a result of the
production of autoreactive antibodies or T cells against renal antigens or
indirectly as a result of the deposition of antibodies and/or immune
complexes in the kidney that are reactive against other, non-renal
antigens. Thus other immune-mediated diseases that result in the formation
of immune-complexes can also induce immune mediated renal disease as an
indirect sequelae. Both direct and indirect immune mechanisms result in
inflammatory response that produces/induces lesion development in renal
tissues with resultant organ function impairment and in some cases
progression to renal failure. Both humoral and cellular immune mechanisms
can be involved in the pathogenesis of lesions.
Demyelinating diseases of the central and peripheral nervous systems,
including Multiple Sclerosis; idiopathic demyelinating polyneuropathy or
Guillain-Barr syndrome; and Chronic Inflammatory Demyelinating
Polyneuropathy, are believed to have an autoimmune basis and result in
nerve demyelination as a result of damage caused to oligodendrocytes or to
myelin directly. In MS there is evidence to suggest that disease induction
and progression is dependent on T lymphocytes. Multiple Sclerosis is a
demyelinating disease that is T lymphocyte-dependent and has either a
relapsing-remitting course or a chronic progressive course. The etiology
is unknown; however, viral infections, genetic predisposition,
environment, and autoimmunity all contribute. Lesions contain infiltrates
of predominantly T lymphocyte mediated, microglial cells and infiltrating
macrophages; CD4+T lymphocytes are the predominant cell type at lesions.
The mechanism of oligodendrocyte cell death and subsequent demyelination
is not known but is likely T lymphocyte driven.
Inflammatory and Fibrotic Lung Disease, including eosinophilic pneumonia,
idiopathic pulmonary fibrosis and hypersensitivity pneumonitis may involve
a disregulated immune-inflammatory response. Inhibition of that response
would be of therapeutic benefit.
Autoimmune or Immune-mediated Skin Disease including Bullous Skin
Diseases, Erythema Multiforme, and Contact Dermatitis are mediated by
auto-antibodies, the genesis of which is T lymphocyte-dependent.
Psoriasis is a T lymphocyte-mediated inflammatory disease. Lesions contain
infiltrates of T lymphocytes, macrophages and antigen processing cells,
and some neutrophils. Allergic diseases, including asthma; allergic
rhinitis; atopic dermatitis; food hypersensitivity; and urticaria are T
lymphocyte dependent. These diseases are predominantly mediated by T
lymphocyte induced inflammation, IgE mediated-inflammation or a
combination of both.
Transplantation associated diseases, including Graft rejection and
Graft-Versus-Host-Disease (GVHD) are T lymphocyte-dependent; inhibition of
T lymphocyte function is ameliorative.
For the prevention, treatment or reduction in the severity of
complement-associated (immune related) disease, the appropriate dosage of
a compound of the invention will depend on the type of disease to be
treated, as defined above, the severity and course of the disease, whether
the agent is administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and response to the compound, and
the discretion of the attending physician. The compound is suitably
administered to the patient at one time or over a series of treatments.
Preferably, it is desirable to determine the dose-response curve and the
pharmaceutical composition of the invention first in vitro, and then in
useful animal models prior to testing in humans.
For example, depending on the type and severity of the disease, about 1 .mu.g/kg
to 15 mg/kg (e.g. 0.1-20 mg/kg) of polypeptide is an initial candidate
dosage for administration to the patient, whether, for example, by one or
more separate administrations, or by continuous infusion. A typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more, depending
on the factors mentioned above. For repeated administrations over several
days or longer, depending on the condition, the treatment is sustained
until a desired suppression of disease symptoms occurs. However, other
dosage regimens may be useful. The progress of this therapy is easily
monitored by conventional techniques and assays.
STIgMA antagonists, such as antibodies to STIgMA, can be used in
immunoadjuvant therapy for the treatment of tumors (cancer). It is now
well established that T cells recognize human tumor specific antigens. One
group of tumor antigens, encoded by the MAGE, BAGE and GAGE families of
genes, are silent in all adult normal tissues, but are expressed in
significant amounts in tumors, such as melanomas, lung tumors, head and
neck tumors, and bladder carcinomas. DeSmet, C. et al, (1996) Proc. Natl.
Acad. Sci. USA, 93:7149. It has been shown that costimulation of T cells
induces tumor regression and an antitumor response both in vitro and in
vivo. Melero, I. et al, Nature Medicine (1997) 3:682; Kwon, E. D. et al,
Proc. Natl. Acad. Sci. USA (1997) 94:8099; Lynch, D. H. et al, Nature
Medicine (1997) 3:625; Finn, O. J. and Lotze, M. T., J. Immunol. (1998)
21:114. The STIgMA antagonists of the invention can be administered as
adjuvants, alone or together with a growth regulating agent, cytotoxic
agent or chemotherapeutic agent, to stimulate T cell
proliferation/activation and an antitumor response to tumor antigens. The
growth regulating, cytotoxic, or chemotherapeutic agent may be
administered in conventional amounts using known administration regimes.
Immunostimulating activity by the STIgMA antagonists of the invention
allows reduced amounts of the growth regulating, cytotoxic, or
chemotherapeutic agents thereby potentially lowering the toxicity to the
Although some macrophages are involved in tumor eradication, many solid
tumors are known to contain macrophages that support tumor growth (Bingle
et al., J Pathol 196:254-265 (2002); Mantovani et al., Trends Immunol
23:549-555 (2002)). These macrophages may contain STIgMA on their surface
Antibodies that block the capacity of STIgMA to inhibit complement
activation could be used to activate complement on tumor cells and help
irradicate the tumor through complement-mediated lysis. This approach
would be particularly useful in tumors that contain STIgMA positive
Claim 1 of 24 Claims
1. A method for the treatment of an
alternative complement pathway-mediated inflammatory or autoimmune disease
or condition, comprising treating a subject in need of such treatment with
a therapeutically effective amount of a polypeptide comprising the STIgMA
polypeptide of SEQ ID NO: 4, or the extracellular region thereof.
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