Pharmaceutical compositions are designed based on the criticality of a portion of C9 for assembly of the C5b9 complex, which specifically modulate binding of CD59 to C9, either molecules structurally mimicking C9 amino acid residues 359 to 384 which bind to CD59 or molecules binding to C9 amino acid residues 359 to 384. Molecules which inhibit CD59 binding include peptides containing residues 359-384 which compete for binding with the other components of the C5b9 complex and anti-idiotypic antibodies immunoreactive with C9 amino acid residues 359 to 384. Molecules which prevent assembly of the C5b-9 complex include antibodies and antibody fragments immunoreactive with amino acid residues 359 to 384 of C9, peptides that bind to amino acid residues 359 to 384 of C9, and nucleotide molecules that bind to amino acid residues 359 to 384 of C9.

The complement system is a complex interaction of plasma proteins and 
membrane cofactors which act in a multi-step, multi-protein cascade 
sequence in conjunction with other immunological systems of the body to 
provide immunity from intrusion of foreign cells. Complement proteins 
represent up to about 10% of globulins in normal serum of man and other 
vertebrates. 
The classic complement pathway involves an initial antibody recognition of, 
and binding to, an antigenic site (SA) on a target cell. This surface 
bound antibody subsequently reacts with the first component of complement, 
C1q, forming a C1-antibody complex with Ca ++, C1r, and C1s which is 
proteolytically active. C1s cleaves C2 and C4 into active components, C2a 
and C4a. The C4b, 2a complex is an active protease called C3 convertase, 
and acts to cleave C3 into C3a and C3b. C3b forms a complex with C4b, 2a 
to produce C4b, 2a, 3b, which cleaves C5 into C5a and C5b. C5b combines 
with C6. The C5b, 6 complex combines with C7 to form the ternary complex 
C5b, 6, 7. The C5b, 6, 7 complex binds C8 at the surface of the cell, 
which may develop functional membrane lesions and undergo slow lysis. Upon 
binding of C9 to the C8 molecules in the C5b, 6, 7, 8 complex, lysis of 
bacteria and other foreign cells is rapidly accelerated. 
The C5b-9 proteins of the human plasma complement system have been 
implicated in non-lytic stimulatory responses from certain human vascular 
and blood cells. The capacity of C5b-9 to modify membrane permeability and 
to selectively alter ion conductance is thought to elicit these non-lytic 
responses from human cells. In the case of human blood platelets and 
vascular endothelium, assembly of the C5b-9 complex initiates a transient 
and reversible depolarization of the plasma membrane potential, a rise in 
cytosolic Ca2+, metabolic conversion of arachidonate to thromboxane or 
prostacyclin, and the activation of intracellular protein kinases. In 
addition, human platelets exposed to C5b-9 undergo shape changes, 
secretory fusion of intracellular storage granules with plasma membrane, 
and the vesiculation of membrane components from the cell surface. Human 
endothelial cells exposed to the human C5b-9 proteins secrete high 
molecular weight multimers of the platelet adhesion protein, von 
Willibrand Factor (vWF), and the intracellular granule membrane protein, 
GMP140, is translocated from the Weibel-Palade body to the endothelial 
surface. High molecular weight multimers of vWF have been implicated in 
the pathogenesis of vaso-occlusive platelet adherence to endothelium and 
cell surface GMP140 has been implicated in the adherence of inflammatory 
leukocytes to endothelium. 
These effects of complement proteins C5b-9 on platelet and endothelial 
cells alter the normal regulation of the enzymes of the plasma coagulation 
system at these cell surfaces. For example, the generation of platelet 
membrane microparticles by vesiculation is accompanied by the exposure of 
membrane binding sites for coagulation factor Va. Binding of factor Va to 
the platelet plasma membrane and to these membrane microparticle sites 
initiates assembly of the prothrombinase enzyme complex. This complex in 
turn accelerates coagulation factor Xa activation of prothrombin to 
thrombin which promotes plasma clotting. Similarly, C5b-9 binding to the 
endothelial cell results in the exposure of plasma membrane receptors for 
the prothrombinase complex, thereby accelerating the generation of 
thrombin from prothrombin at the endothelial surface. 
This interaction between components of the complement and coagulation 
systems at the surface of blood platelets and endothelium can generate 
inflammatory and chemotactic peptides at sites of vascular thrombus 
formation and may contribute to the altered hemostasis associated with 
immune disease states. In addition, immune reactions affecting blood 
platelets and endothelium can lead to platelet aggregation, the secretion 
of proteolytic enzymes and vasoactive amines from platelet storage 
granules, and increase adherence of platelets and leukocytes to the 
endothelial lining of blood vessels. 
Assembly of the C5b-9 complex is normally limited in plasma by the amount 
of C5b generated by proteolysis of C5 to its biologically-active fragments 
C5b and C5a. In addition to plasmin and other plasma or cell-derived 
proteases, two enzymes of the complement system can cleave C5 to C5a and 
C5b, the membrane-stabilized enzyme complexes C4b2a and C3bBb 
(C5-convertases). The activity of these two enzymes is normally inhibited 
on the surface of human blood and vascular membranes by the plasma 
membrane proteins, "membrane cofactor protein" (CD46), described by Lublin 
and Atkinson, Current Topics Microbiol. Immunol. 153:123 (1989) and 
"decay-accelerating factor" (CD55), Medof, et al., J. Exp. Med. 160:1558 
(1984). 
Platelet and endothelial cell activation by C5b-9 also has ramifications in 
autoimmune disorders and other disease states. The importance of 
spontaneous complement activation and the resulting exposure of platelets 
and endothelium to activated C5b-9 to the evolution of vaso-occlusive 
disease is underscored by consideration that a) leukocyte infiltration of 
the subendothelium, which is known to occur in regions of atheromatous 
degeneration and suggests localized generation of C5a at the vessel wall, 
is potentially catalyzed by adherent platelets and b) local intravascular 
complement activation resulting in membrane deposition of C5b-9 complexes 
accompanies coronary vessel occlusion and may affect the ultimate extent 
of myocardial damage associated with infarction. 
There is now considerable evidence that the human erythrocyte membrane as 
well as the plasma membranes of other human blood cells and vascular 
endothelium are normally protected from these effects of complement by 
cell-surface proteins that specifically inhibit activation of the C5b-9 
pore upon C9 binding to membrane C5b-8, as reported by Holguin, M. H., et 
al., J. Clin. Invest. 84, 7-17 (1989); Sims, P. J., et al., J. Biol. Chem. 
264, 19228-19235 (1989); Davies, A., et al., J. Exp. Med. 170, 637-654 
(1989); Rollins, S. A., and Sims, P. J. J. Immunol. 144, 3478-3483 (1990); 
and Hamilton, K. K., et al., Blood 76, 2572-2577 (1990). Plasma membrane 
constituents reported to exhibit this activity include homologous 
restriction factor (HRF) (C8 -binding protein), as described by Zalman, L. 
S., et al., Proc. Natl. Acad. Sci., U.S.A. 83, 6975-6979 (1986) and 
Schonermark, S., et al., J. Immunol. 136, 1772-1776 (1986), and the 
leukocyte antigen CD59, described by Sugita, Y., et al., J. Biochem. 
(Tokyo) 104, 633-637 (1988); Holguin, M. H., et al., (1989); Sims, P. J., 
et al., (1989); Davies, A., (1989); Rollins, S. A., and Sims, P. J. 
(1990); and Hamilton, K. K., et al., (1990). Accumulated evidence suggest 
that these two proteins exhibit quite similar properties, including the 
following: both HRF and CD59 are tethered to the cell surface by a 
glycolipid anchor, and are deleted from the membranes of the most 
hemolytically sensitive erythrocytes that arise in the stem cell disorder 
paroxysmal nocturnal hemoglobinuria; the activity of both inhibitors is 
species-restricted, showing selectivity for C8 and C9 that are derived 
from homologous (i.e. human) serum; and both HRF and CD59 appear to 
function by inhibiting the activation of C9 , decreasing the incorporation 
of C9 into the membrane C5b-9 complex, and limiting propagation of the C9 
homopolymer. 
In U.S. Pat. No. 5,136,916 to Sims and Wiedmer, Sims and Wiedmer disclose 
compositions and methods for use thereof relating to polypeptides having 
the ability to act as an inhibitor of complement C5b-9 complex activity. 
The compositions contain CD59, active derivatives or fragments thereof 
which act to inhibit the activity of C5b-9, anti-idiotypic antibodies 
mimicking the action of the inhibitor proteins or antibodies against C7 or 
C9 which block the formation of the C5b-9 complex. The compositions can be 
used in vitro to inhibit C5b-9 related stimulatory responses of platelets 
and vascular endothelium of perfused organs and tissues, thereby 
preventing the C5b-9 initiated cell necrosis or stimulated secretion of 
proteolytic enzymes and the exposure of the procoagulant membrane 
receptors during collection and in vitro storage. In one variation of this 
embodiment, the vascular endothelium of organs and tissues to be 
transplanted are treated with these compositions to protect these cells 
from complement activation after transplantation. In another embodiment, 
immune disease states are treated by administering an effective amount of 
a C5b-9 inhibitor to suppress C5b-9 mediated platelet activation in vivo. 
Also disclosed are methods for the production of isolated polypeptides 
that are able to suppress complement C5b-9 mediated platelet and 
endothelial cell activation. 
Human (hu).sup.1 CD59 antigen is a 18-21 kDa plasma membrane protein that 
functions as an inhibitor of the C5b-9 membrane attack complex (MAC) of hu 
complement. CD59 interacts with both the C8 and C9 components of MAC 
during its assembly at the cell surface, thereby inhibiting formation of 
the membrane-inserted C9 homopolymer responsible for MAC cytolytic 
activity. This serves to protect hu blood and vascular cells from injury 
arising through activation of complement in plasma. CD59's inhibitory 
activity is dependent upon the species of origin of C8 and C9, with 
greatest inhibitory activity observed when C9 is from hu or other 
primates. By contrast, CD59 exerts little or no inhibitory activity 
towards C8 or C9 of most other species, including rabbit (rb). Because the 
activity of CD59 is largely restricted to regulating hu C9, and the 
activity of analogous complement inhibitors expressed by cells of other 
species is likewise generally selective for homologous C9, xenotypic cells 
and tissue are particularly susceptible to complement-mediated destruction 
due to unregulated activity of MAC. This phenomenon underlies hyperacute 
immune rejection after xenotransplantation. 
Analysis of the physical association of CD59 with components of MAC 
suggested that separate binding sites for cD59 are contained within the 
.alpha.-chain of hu C8 and within hu C9. Within C9, this site(s) has been 
mapped to between residues 334-415. The complement-inhibitory activity of 
CD59 is species-selective, and is most effective towards C9 derived from 
human or other primate plasma. The species-selective activity of CD59 was 
recently used to map the segment of human C9 that is recognized by this 
MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain 
lytic function within the MAC Husler T, Lockert D. H., Kaufman K. M., 
Sodetz J. M., Sims P. J. (1995). J. Biol. Chem. 270:3483-3486!. These 
experiments suggested that the CD59 recognition domain was contained 
between residues 334-415 in human C9. 
It is apparent that additional or alternative inhibitors of the assembly of 
the C5b9 complex would be advantageous in inhibition of complement 
mediated inflammation. It is also clear that inhibitors which are 
extremely specific and which are directed to the most critical regions 
involved in assembly or function of the complex would be most effective as 
inhibitors of complement mediated inflammation, with the least likelihood 
of non-specific side effects. 
It is therefore an object of the present invention to provide a method and 
materials for specifically inhibiting complement mediated inflammation. 
It is another object of the present invention to provide a method and 
materials for determining the species specificity of C9 complement 
mediated activation and cytolysis. 
SUMMARY OF THE INVENTION 
CD59 interacts with a segment of human C9 (hu C9) between residues 334-415, 
immediately C-terminal to the predicted membrane-inserting domain of C9. 
This segment of C9 contains a region of markedly divergent sequence when 
hu C9 is compared to C9 of other species, with greatest divergence noted 
for the peptide segment contained within an internal Cys359-Cys384 
disulfide in hu C9. In order to determine whether sequence contained in 
this peptide loop represents a hu C9-specific motif that is selectively 
recognized by CD59, CD59's inhibitory activity toward various full-length 
C9 chimeras containing hu-unique or rabbit (rb)-unique sequence spanning 
this segment of the C9 polypeptide were analyzed. These experiments 
revealed that substitution of hu residues 359-391 into otherwise rb C9 
yielded a chimera indistinguishable from hu C9 in its regulation by CD59. 
C9 chimeras generated by substitution of hu C9 sequence flanking either 
side of residues 359-391 into rb C9 showed no consistent increase in 
inhibition by CD59. This indicates that only residues contained between 
359-391 of hu C9 are directly recognized by CD59. Moreover, truncation of 
the segment of hu C9 sequence in chimeric rb C9 from 359-391 to the 
putative recognition loop of hu 359-384 was accompanied by approximately 
35% reduction of CD59 inhibitory function. Further, CD59 specifically 
bound to a synthetic peptide corresponding to residues 359-384 of hu C9. 
IgG (Fab) specific for the hu C9 359-384 peptide inhibited the hemolytic 
activity of hu C9 (but not rb C9) in a manner analogous to CD59. 
Pharmaceutical compositions are designed based on the criticality of this 
portion of C9 for assembly of the C5b9 complex which specifically modulate 
binding of CD59 to C9, either molecules structurally mimicking C9 amino 
acid residues 359 to 384 which bind to CD59 or molecules binding to C9 
amino acid residues 359 to 384. Molecules which inhibit CD59 binding 
include peptides containing residues 359-384 which compete for binding 
with the other components of the C5b9 complex and anti-idiotypic 
antibodies immunoreactive with C9 amino acid residues 359 to 384. 
Molecules which prevent assembly of the C5b-9 complex include antibodies 
and antibody fragments immunoreactive with amino acid residues 359 to 384 
of C9, peptides that bind to amino acid residues 359 to 384 of C9, and 
nucleotide molecules that bind to amino acid residues 359 to 384 of C9.

DETAILED DESCRIPTION OF THE INVENTION 
I. C9 Peptide/CD59 C9 binding site Immunomodulators 
Peptide sequence in human complement protein C9 has been identified that 
contributes to the recognition of this protein by its naturally occurring 
inhibitor, CD59 . CD59 is known to bind to neo-epitopes that become 
exposed in complement C8 and C9 during assembly of the cytolytic membrane 
attack complex of proteins C5b through C9. Through this interaction, CD59 
interrupts assembly of the C5b-9 complex, protecting the target cell from 
destruction by these complement proteins. Data demonstrates that antibody 
raised against this human C9-derived peptide sequence is functionally 
inhibitory towards the lytic activity of the human C5b-9 complex. This 
permits design of reagents directed specifically at human C9 that mimic or 
inhibit the complement-inhibitory function of cell-surface CD59. 
Compounds which bind CD59 
As demonstrated by the following example, amino acid residues 359-384 of C9 
are critical for binding of CD59 to C9, resulting in inhibition of C5b-9 
complex assembly. Peptides can be as short as 26 amino acids, less than 
forty amino acids, or less than 56 amino acids (359 to 415 amino acid 
peptide fragment of C9). Substitutions based on conserved sequence (rabbit 
for human, amino acids with similar structure and charge), presence or 
absence of a disulfide bond between the cysteine residues, and elongation 
of the peptide through addition of supplemental amino acid sequence, were 
all shown not to significantly inhibit binding of CD59 to C9. Other 
derivatives that should also be active include covalently-cyclized 
derivatives, for example, disulfide-bonded and amide bonded peptides. 
The data indicates that CD59 inhibits C9 through binding to hu-specific 
residues contained within the Cys359-Cys384 disulfide loop of the 
polypeptide. Optimal interaction of CD59 with this binding site in hu C9 
appears to depend upon a few residues located immediately C-terminal to 
this segment of the protein. Although the specific role of this segment of 
C9 in membrane attack complex (MAC) assembly is unknown, the data 
indicates that ligand binding to this site abrogates the lytic activity of 
the C5b-9 complex, implicating these residues in the conversion of C9 from 
solution monomer to membrane-embedded polymer. CD59 specifically binds a 
human C9-derived peptide corresponding to residues 359-384, and antibody 
(Fab) raised against this C9-derived peptide inhibits the lytic activity 
of human MAC. Mutant human C9 in which Ala was substituted for Cys 359-384 
was found to express normal lytic activity and to be fully inhibited by 
CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond 
within C9 is not required to maintain the conformation of this segment of 
C9 for interaction with CD59. Other substitutions can also be made without 
decreasing activity. 
These compounds are effective as competitive inhibitors of CD59. Other 
compounds besides the peptides that can be used include anti-idiotypic 
antibodies and antibody fragments which bind to CD59, nucleotide 
molecules, and organic molecules that bind to the site on CD59 which binds 
amino acids 359-384 or 359 to 391. These can be identified using screening 
and computer assisted design, as described below. 
Compounds which Inhibit C5b-9 Assembly 
Data demonstrates that antibody raised against this human C9-derived 
peptide sequence is functionally inhibitory towards the lytic activity of 
the human C5b-9 complex. Other compounds besides antibodies and antibody 
fragments which also bind to this peptide portion of C9, thereby 
preventing assembly of the C5b-9 complex, include peptides, nucleotide 
molecules, and organic molecules that bind to amino acids 359-384 or 359 
to 391. These can be identified using screening and computer assisted 
design, as described below. 
Random generation of binding molecules 
Molecules with a given function, catalytic or ligand-binding, can be 
selected for from a complex mixture of random molecules in what has been 
referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992). One 
synthesizes a large pool of molecules bearing random and defined sequences 
and subjects that complex mixture, for example, approximately 10.sup.15 
individual sequences in 100 .mu.g of a 100 nucleotide RNA, to some 
selection and enrichment process. For example, by repeated cycles of 
affinity chromatography and PCR amplification of the molecules bound to 
the ligand on the column, Ellington and Szostak (1990) estimated that 1 in 
10.sup.10 RNA molecules folded in such a way as to bind a given ligand. 
DNA molecules with such ligand-binding behavior have been isolated 
(Ellington and Szostak, 1992; Bock et al, 1992). 
Computer assisted drug design 
Computer modeling technology allows visualization of the three-dimensional 
atomic structure of a selected molecule and the rational design of new 
compounds that will interact with the molecule. The three-dimensional 
construct typically depends on data from x-ray crystallographic analyses 
or NMR imaging of the selected molecule. The molecular dynamics require 
force field data. The computer graphics systems enable prediction of how a 
new compound will link to the target molecule and allow experimental 
manipulation of the structures of the compound and target molecule to 
perfect binding specificity. Prediction of what the molecule-compound 
interaction will be when small changes are made in one or both requires 
molecular mechanics software and computationally intensive computers, 
usually coupled with user-friendly, menu-driven interfaces between the 
molecular design program and the user. 
Examples of molecular modelling systems are the CHARMm and QUANTA programs, 
Polygen Corporation, Waltham, Mass. CHARMm performs the energy 
minimization and molecular dynamics functions. QUANTA performs the 
construction, graphic modelling and analysis of molecular structure. 
QUANTA allows interactive construction, modification, visualization, and 
analysis of the behavior of molecules with each other. 
A number of articles review computer modeling of drugs interactive with 
specific proteins, such as Rotivinen, et al., 1988 Acta Pharmaceutica 
Fennica 97, 159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinaly 
and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol. 29, 111-122; Perry and 
Davies, QSAR: Quantitative Structure-Activity Relationships in Drug Design 
pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc. 
Lond. 236, 125-140 and 141-162; and, with respect to a model receptor for 
nucleic acid components, Askew, et al., 1989 J. Am. Chem. Soc. 111, 
1082-1090. Other computer programs that screen and graphically depict 
chemicals are available from companies such as BioDesign, Inc., Pasadena, 
Calif., Allelix, Inc, Mississauga, Ontario, Canada, and Hypercube, Inc., 
Cambridge, Ontario. Although these are primarily designed for application 
to drugs specific to particular proteins, they can be adapted to design of 
drugs specific to regions of DNA or RNA, once that region is identified. 
Although described above with reference to design and generation of 
compounds which could alter binding, one could also screen libraries of 
known compounds, including natural products or synthetic chemicals, and 
biologically active materials, including proteins, for compounds which are 
inhibitors or activators. 
Nucleotide molecules which bind either CD59 or the C9 peptide can be 
generated in vitro, and then inserted into cells. Oligonucleotides can be 
synthesized on an automated synthesizer (e.g., Model 8700 automated 
synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B). 
(see, e.g., Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (in vivo 
inhibition of duck hepatitis B viral replication and gene expression by 
antisense phosphorothioate oligodeoxynucleotides); Rosenberg et al., PCT 
WO 93/01286 (synthesis of sulfurthioate oligonucleotides); Agrawal et al., 
1988 Proc. Natl. Acad. Sci. USA 85, 7079-7083 (synthesis of antisense 
oligonucleoside phosphoramidates and phosphorothioates to inhibit 
replication of human immunodeficiency virus-1); Sarin et al., 1989 Proc. 
Natl. Acad. Sci. USA 85, 7448-7794 (synthesis of antisense 
methylphosphonate oligonucleotides); Shaw et al., 1991 Nucleic Acids Res 
19, 747-750 (synthesis of 3' exonuclease-resistant oligonucleotides 
containing 3' terminal phosphoroamidate modifications); incorporated 
herein by reference). To reduce susceptibility to intracellular 
degradation, for example by 3' exonucleases, a free amine can be 
introduced to a 3' terminal hydroxyl group of oligonucleotides without 
loss of sequence binding specificity (Orson et al., 1991). Furthermore, 
more stable triplexes are formed if any cytosines that may be present in 
the oligonucleotide are methylated, and also if an intercalating agent, 
such as an acridine derivative, is covalently attached to a 5' terminal 
phosphate (e.g., via a pentamethylene bridge); again without loss of 
sequence specificity (Maher et al., (1989); Grigoriev et al., (1992). 
Methods to produce or synthesize oligonucleotides are well known in the 
art. Such methods can range from standard enzymatic digestion followed by 
nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5, 6) 
to purely synthetic methods, for example, by the cyanoethyl 
phosphoramidite method using a Milligen or Beckman System 1Plus DNA 
synthesizer (see also, Ikuta et al., in Ann. Rev. Biochem. 1984 53, 
323-356 (phosphotriester and phosphite-triester methods); Narang et al., 
in Methods Enzymol., 65, 610-620 (1980) (phosphotriester method). 
Preparation of Peptides 
Proteins can be expressed recombinantly and cleaved by enzymatic digest, 
expressed from a sequence encoding a peptide, or synthesized using 
standard techniques. It is a routine matter to make appropriate peptides, 
test for binding, and then utilize. The peptides can be as short as twenty 
six amino acids in length and up to 57 amino acids, and are easily 
prepared by standard techniques. They can also be modified to increase in 
vivo half-life, by chemical modification of the amino acids or by 
attachment to a carrier molecule or inert substrate. 
The peptides can also be conjugated to a carrier protein such as keyhole 
limpet hemocyanin by its N-terminal cysteine by standard procedures such 
as the commercial Imject kit from Pierce Chemicals or expressed as a 
fusion protein, which may have increased efficacy. As noted above, the 
peptides can be prepared by proteolytic cleavage of C9, or, preferably, by 
synthetic means. These methods are known to those skilled in the art. An 
example is the solid phase synthesis described by J. Merrifield, 1964 J. 
Am. Chem. Soc. 85, 2149, used in U.S. Pat. No. 4,792,525, and described in 
U.S. Pat. No. 4,244,946, wherein a protected alpha-amino acid is coupled 
to a suitable resin, to initiate synthesis of a peptide starting from the 
C-terminus of the peptide. Other methods of synthesis are described in 
U.S. Pat. No. 4,305,872 and 4,316,891. These methods can be used to 
synthesize peptides having identical sequence to the receptor proteins 
described herein, or substitutions or additions of amino acids, which can 
be screened for activity as described above. 
The peptide can also be administered as a pharmaceutically acceptable acid- 
or base- addition salt, formed by reaction with inorganic acids such as 
hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, 
thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids 
such as formic acid, acetic acid, propionic acid, glycolic acid, lactic 
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, 
and fumaric acid, or by reaction with an inorganic base such as sodium 
hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such 
as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. 
Peptides containing cyclopropyl amino acids, or amino acids derivatized in 
a similar fashion, can also be used. These peptides retain their original 
activity but have increased half-lives in vivo. Methods known for 
modifying amino acids, and their use, are known to those skilled in the 
art, for example, as described in U.S. Pat. No. 4,629,784 to Stammer. 
The peptides are generally active when administered parenterally in amounts 
above about 1 .mu.g/kg of body weight. Based on extrapolation from other 
proteins, for treatment of most inflammatory disorders, the dosage range 
will be between 0.1 to 70 mg/kg of body weight. This dosage will be 
dependent, in part, on whether one or more peptides are administered. 
Based on studies with other peptide fragments blocking binding, the 
IC.sub.50, the dose of peptide required to inhibit binding by 50%, ranges 
from about 50 .mu.M to about 300 .mu.M, depending on the peptides. These 
ranges are well within the effective concentrations for the in vivo 
administration of peptides, based on comparison with the RGD-containing 
peptides, described, for example, in U.S. Pat. No. 4,792,525 to 
Ruoslaghti, et al., used in vivo to alter cell attachment and 
phagocytosis. 
Antibodies 
Antibodies immunoreactive with the C9 peptide or an anti-idiotypic antibody 
to the antibodies immunoreactive with the C9 peptide can be prepared for 
use as described above. 
In vivo Immunization of Animals 
Animals such as mice may be immunized by administration of an amount of 
immunogen (either the C9 peptide or the antibody to the C9 peptide) 
effective to produce an immune response. Preferably a mouse is 
subcutaneously injected in the back with 100 micrograms of antigen, 
followed three weeks later with an intraperitoneal injection of 100 
micrograms of cocaine immunogen with adjuvant, most preferably Freund's 
complete adjuvant. Additional intraperitoneal injections every two weeks 
with adjuvant, preferably Freund's incomplete adjuvant, may be necessary 
until the proper titer in the mouse's blood is achieved. In order to use 
the mice for fusion and hybridoma production, a titer of at least 1:5000 
is preferred, and a titer of 1:100,000 or more is most preferred. 
In vitro Immunization 
The technique of in vitro immunization of human lymphocytes is frequently 
employed to generate a large variety of human monoclonal antibodies, since 
deliberate in vivo priming of humans with many antigens of interest is not 
feasible until approval by the Food and Drug Administration has been 
obtained. Techniques for in vitro immunization of human lymphocytes are 
well known to those skilled in the art. See, e.g., T. Inai, et al., 
Histochemistry (Germany), 99(5):335-362 (May 1993); A. Mulder, et al., 
Hum. Immunol., 36(3):186-192 (Mar. 1993); H. Harada, et al., J. Oral 
Pathol. Med. (Denmark), 22(4):145-152 (April 1993); N. Stauber, et al., J. 
Immunol. Methods (Netherlands), 161(2): 157-168 (May 26, 1993); and S. 
Venkateswaran, et al., Hybridoma, 11(6):729-739 (Dec. 1992), which are 
incorporated herein by reference. These techniques can be used to produce 
antigen-reactive human monoclonal antibodies, including antigen-specific 
IgG, and IgM human monoclonal antibodies. 
Humanization of Antibodies 
Because the methods for immunizing animals yield antibody which is not of 
human origin, the antibodies could elicit an adverse effect if 
administered to humans. Methods for "humanizing" antibodies, or generating 
less immunogenic fragments of non-human antibodies, are well known. A 
humanized antibody is one in which only the antigen-recognized sites, or 
complementarity-determining hypervariable regions (CDRs) are of non-human 
origin, whereas all framework regions (FR) of variable domains are 
products of human genes. These "humanized" antibodies present a lesser 
xenografic rejection stimulus when introduced to a human recipient. 
To accomplish humanization of a selected mouse monoclonal antibody, the CDR 
grafting method described by Daugherty, et al., Nucl. Acids Res., 
19:2471-2476 (1991), incorporated herein by reference, may be used. 
Briefly, the variable region DNA of a selected animal recombinant 
anti-idiotypic ScFv is sequenced by the method of C1ackson, T., et al., 
Nature, 352:624-688, 1991, incorporated herein by reference. Using this 
sequence, animal CDRs are distinguished from animal framework regions (FR) 
based on locations of the CDRs in known sequences of animal variable 
genes. Kabat, H.A., et al., Sequences of Proteins of Immunological 
Interest, 4th Ed. (U.S. Dept. Health and Human Services, Bethesda, MD, 
1987). Once the animal CDRs and FR are identified, the CDRs are grafted 
onto human heavy chain variable region framework by the use of synthetic 
oligonucleotides and polymerase chain reaction (PCR) recombination. Codons 
for the animal heavy chain CDRs, as well as the available human heavy 
chain variable region framework, are built in four (each 100 bases long) 
oligonucleotides. Using PCR, a grafted DNA sequence of 400 bases is formed 
that encodes for the recombinant animal CDR/human heavy chain FR 
protection. 
The immunogenic stimulus presented by the monoclonal antibodies so produced 
may be further decreased by the use of Pharmacia's (Pharmacia LKB 
Biotechnology, Sweden) "Recombinant Phage Antibody System" (RPAS), which 
generates a single-chain Fv fragment (ScFv) which incorporates the 
complete antigen-binding domain of the antibody. In the RPAS, antibody 
variable heavy and light chain genes are separately amplified from the 
hybridoma mRNA and cloned into an expression vector. The heavy and light 
chain domains are co-expressed on the same polypeptide chain after joining 
with a short linker DNA which codes for a flexible peptide. This assembly 
generates a single-chain Fv fragment (ScFv) which incorporates the 
complete antigen-binding domain of the antibody. Compared to the intact 
monoclonal antibody, the recombinant ScFv includes a considerably lower 
number of epitopes, and thereby presents a much weaker immunogenic 
stimulus when injected into humans. 
Pharmaceutical Compositions 
The compounds described above are preferably administered in a 
pharmaceutically acceptable vehicle. Suitable pharmaceutical vehicles are 
known to those skilled in the art. For parenteral administration, the 
compound will usually be dissolved or suspended in sterile water or 
saline. For enteral administration, the compound will be incorporated into 
an inert carrier in tablet, liquid, or capsular form. Suitable carriers 
may be starches or sugars and include lubricants, flavorings, binders, and 
other materials of the same nature. The compounds can also be administered 
locally by topical application of a solution, cream, gel, or polymeric 
material (for example, a Pluronic.TM., BASF). 
Alternatively, the compound may be administered in liposomes or 
microspheres (or microparticles). Methods for preparing liposomes and 
microspheres for administration to a patient are known to those skilled in 
the art. U.S. Pat. No. 4,789,734 describe methods for encapsulating 
biological materials in liposomes. Essentially, the material is dissolved 
in an aqueous solution, the appropriate phospholipids and lipids added, 
along with surfactants if required, and the material dialyzed or 
sonicated, as necessary. A review of known methods is by G. Gregoriadis, 
Chapter 14. "Liposomes", Drug Carriers in Biology and Medicine pp. 287-341 
(Academic Press, 1979). Microspheres formed of polymers or proteins are 
well known to those skilled in the art, and can be tailored for passage 
through the gastrointestinal tract directly into the bloodstream. 
Alternatively, the compound can be incorporated and the microspheres, or 
composite of microspheres, implanted for slow release over a period of 
time, ranging from days to months. See, for example, U.S. Pat. No. 
4,906,474 4,925,673, and 3,625,214. 
II. Methods of treament 
The effective amount of composition described above is that which achieves 
the desired effect: either to inhibit assembly of the C5b-9 complex by 
binding to C9 or to bind to the endogenous CD59 to prevent the CD59 from 
inhibiting assembly of the C5b-9 complex, thereby increasing complement 
mediated activation of cells. 
Inhibition of CD59 is useful as an adjuvant for tumor therapy and as a 
contraceptive since its been demonstrated that CD59 protects sperm from 
rejection by antibody and complement in the female genital tract and that 
CD59 expressed on human tumor cells protect those cells from complement 
mediated lysis. 
Inhibition of C5b-9 complex assembly is useful for all disorders 
characterized by excessive complement activation or complement mediated 
cytolysis, including, for example, immune disorders and diseases such as 
immunovasculitis, rheumatoid arthritis, scleroderma, disseminated 
intravascular coagulation, lupus, paroxysmal nocturnal hemoglobinuria, 
thrombotic thrombolytic purpura, vascular occlusion, reocclusion after 
surgery, coronary thrombosis, and myocardial infarction. 
The present invention will be further understood by reference to the 
following studies. 
EXAMPLE 1 
Demonstration of role of a disulfide bonded peptide loop within hu C9 in 
the species-selectivity of CD59 
EXPERIMENTAL PROCEDURES 
Materials 
Hu complement proteins C5b6, C7, C8, and C9, and hu erythrocyte membrane 
glycoprotein CD59 were purified and assayed as described by Davies, et al. 
Immunol. Res. 12, 258-275 (1993), Wiedmer and Sims, J. Membr. Biol. 84, 
249-258 (1985), and Wiedmer and Sims, J. Biol. Chem. 260, 8014-8019 
(1985). Hu C9 peptide 359-384 
(allyl-K!-CLGYHLDVSLAFSEISVGAEFNKDD-allyl-C), BSA-conjugated hu C9 
peptide 359-384, and affinity-purified rb IgG against hu C9 peptide 
359-384 were custom ordered from Quality Controlled Biochemicals 
(Hopkinton, Mass.). Full-length cDNA for hu C9 was a generous gift from 
Dr. J. Tschopp (University of Lausanne, Epalinges, Switzerland) and is 
described by Dupuis, et al., Mol. Immunol. 30, 95-100 (1993), the 
teachings of which are incorporated herein. Full length cDNA for rb C9 was 
isolated and cloned into pSVL as reported by Husler, et al., J. Biol. 
Chem. 270, 3483-3486 (1995), the teachings of which are incorporated 
herein. Chicken erythrocytes (chE) were from Cocalico Biologics, Inc. 
(Reamstown, Pa.); COS-7 cells were from American Tissue Culture Collection 
(Rockville, Md.); E. coli strain DH5.alpha. and Opti-MEM I were from Life 
Technologies Inc. (Gaithersburg, Md.), Dulbecco's Modified Eagle Medium 
was from Mediatech Inc. (Herndon, Va.), and heat-inactivated fetal bovine 
serum was from Biocell (Rancho Dominquez, Calif.). Oligonucleotides were 
synthesized by the Molecular Biology Core Laboratories, Blood Research 
Institute. 
Solutions 
MBS: 150 mM NaCl, 10 mM MOPS, pH7.4; GVBS: 150 mM NaCl, 3.3 mM sodium 
barbital, 0.15 mM CaCl.sub.2, 0.5 mM MgCl.sub.2, 0.1%(w/v) gelatin, pH 
7.4; GVBE:150 mM NaCl, 3.3 mM sodium barbital, 10 mM EDTA, 0.1%(w/v) 
gelatin, pH 7.4. 
Construction of chimeric C9 cDNA's 
cDNA's coding for hu/rb C9 chimeras were constructed essentially as 
described by Husler, et al. (1995). In brief, regions of sequence identity 
were determined from the aligned sequences of rb and hu C9, and used as 
junctions for chimeric cDNA construction. Based on these alignments, 
primers for PCR were designed to generate defined segments of rb and hu C9 
cDNA's. Primers annealing to 5'-or 3'-untranslated sequence with added 
Xbal (5'-end) or Sacl (3'-end) recognition sites were paired with chimeric 
primers (28-37 bp in length) and used to generate cDNA fragments that 
contained the desired overlapping sequence at either the 5'-or 3'-ends. 
These fragments were gel purified, mixed at a 1:1 molar ration, and used 
in a second amplification with primers located in the 5'-and 
3'-untranslated region to produce full length chimeric C9 cDNA's. 
Fragments were cloned into the Xbal/Sacl sites of pSVL for mammalian 
expression. PCR fidelity was confirmed by sequencing 3'-coding sequence in 
each construct, starting from the stop codon and continuing through all 
junctions of rb and hu sequence. In certain cases, chimeric constructs 
were further modified by site directed mutagenesis. 
Site Directed Mutagenesis 
C9 cDNA in pSVL served as a template for site-directed mutagenesis using 
the Chameleon mutagenesis kit (Stratagene, La Jolla, Calif.). Mutagenesis 
was performed using 0.25 pmol of template plasmid, 25 pmol of mutagenic 
primer and 25 pmol of selection primer, the latter chosen to modify Sall, 
Scal, or Xhol restriction sites unique to pSVL. The resulting mutagenized 
plasmids were subject to a minimum of two rounds of selection by 
restriction digest, and then transformed into E. coli XL1-Blue 
(Stratagene) for single colony isolation and plasmid purification. In all 
cases, mutations were confirmed by double stranded sequencing of each 
purified plasmid. 
Transfection of COS-7 cells 
Plasmid DNA used in transfections was obtained from purification over 
Qiagen-tips (Qiagen Inc., Chatsworth, Calif.). COS-7 cells were 
transfected using DEAE-dextran, then cultured for 24h in Dulbecco's 
Modified Eagle Medium (Mediatech Inc., Herndon, Va.) supplemented with 10% 
fetal bovine serum, after which this medium was replaced by Opti-MEM I 
(Life Technologies, Inc., Gaithersburg, Md.). Cell supernatants were 
harvested after 48-65h, PMSF (1 mM), benzamidine (1 mM) and EDTA (10 mM) 
were added and the supernatants concentrated at 4.degree. C. (Centricon 
30, Amicon). 
Immunoblotting 
C9 in the COS-7 supernatants was analyzed by quantitative dot blotting 
using murine monoclonal antibody P9-2T as described by Husler, et al. 
(1995). 
Biotin-CD59 
CD59 was biotinylated by incubation (1 h, room temperature) with a 20-fold 
molar excess of NHS-LC-biotin in 10 mM MOPS, 0.1% Nonidet P-40, pH 9.0 
followed by exhaustive dialysis against charcoal, as described by Chang, 
et al. J. Biol. Chem. 269, 26424-26430 (1994). 
Analysis of the inhibitory function of CD 59 towards recombinant C9 
constructs 
Hemolytic activity of each C9 construct was assayed using as target cells 
chE that were reconstituted with purified hu CD59, as described by Husler, 
et al., (1995). chE were washed extensively and suspended in GVBS, and the 
membrane C5b67 complex assembled by mixing cells (1.4.times.10.sup.9 /ml) 
with C5b6 (13 .mu.g/ml) followed by addition of C7 (1 .mu.g/ml). After 10 
min., the C5b67 chE were diluted to 1.4.times.10.sup.8 /ml in GVBE and 
incubated (10 min. 37.degree. C.) with 0 or 750 ng/ml CD59. In each case, 
the final concentration of Nonidet P-40 was less than 0.002%(v/v). After 
washing in ice-cold GVBE, 2.8.times.10.sup.8 of these cells were incubated 
(37.degree. C.) in a total volume of 100 .mu.l with 1 ng rb C8 plus 0-50 
ng of recombinant C9, serially diluted in Opti-MEM I. Hemolysis was 
determined after 30 minutes at 37.degree. C., with correction for 
nonspecific lysis, determined in the absence of C9. In each experiment, 
the inhibitory activity of CD59 towards each recombinant C9 construct was 
determined from the reduction in complement lysis of those cells 
reconstituted with CD59, versus the identically-treated cells omitting 
CD59, measured at the midpoint of the C9 titration (i.e., 50% hemolysis). 
In order to directly compare results obtained in experiments performed on 
different days, data for each recombinant C9 construct were normalized to 
results obtained in each experiment with hu C9. 
CD59 binding to hu C9 peptide 359-384. 
The specific binding of CD59 to hu C9-derived peptide 359-384 was measured 
by microtiter plate assay with biotin-CD59, according to modification of 
published methods of Chang, et al. (1994) and Husler, et al. (1995). 
Briefly, the BSA-peptide conjugate was adsorbed to 96 well polyvinyl 
microplates by overnight coating at 5 .mu.g/ml in 0.1M sodium bicarbonate, 
pH 8.5. After blocking with 1% (w/v) BSA, wells were washed and incubated 
(4 hrs., 37.degree. C.) with 0.5-1 , .mu.g/ml biotin-CD59. After washing, 
the bound biotin-CD59 was detected with Vectsstain.TM. (Vector Labs, 
Burlingame, Calif.), developed by addition of p-nitrophenyl phosphate (2 
mg/ml) and optical density recorded at 405 nm (VMAXMICROPLATE.TM. Reader, 
Molecular Devices, Inc.). In all experiments, correction was made for 
background adsorption of biotin-CD59 to BSA-coated wells (no peptide) and 
for nonspecific binding of biotin-CD59 to peptide, determined in the 
presence of a 20-fold excess of unlabeled CD59. As a positive control for 
specific binding, comparison was made in each experiment to wells coated 
with 2 .mu.g/ml hu C9. The capacity of monospecific antibody against hu C9 
peptide 359-384 to compete specific binding of CD59 was determined by 
prior incubation of the BSA-peptide-coated wells with antibody (2 hrs., 
0-100 .mu.g/ml LgG) before addition of biotin-CD59. 
Inhibition of MAC lysis by antibody against hu C9 peptide 359-384. 
The capacity of antibody against hu C9 peptide 359-384 to inhibit MAC was 
determined by hemolytic assay, using the chE target cells described above, 
omitting CD59. In these experiments, 0-1 mg/ml Fab of antibody against hu 
C9 peptide 359-384 (or, non-immune antibody control) was added with 
recombinant C9 (hu, rb, or chimeric), and complement-specific lysis 
determined. 
RESULTS 
C9 chimeras were constructed in which the segment of C9 corresponding to 
the putative CD59 binding site (residues 334-415 in hu C9; were 
interchanged between hu and rb C9. These chimeric proteins were then 
tested for hemolytic activity and for their sensitivity to inhibition by 
membrane CD59 (FIG. 1A and 1B). Substitution of hu C9 residues 334-415 
into rb C9 (chimera #1) resulted in a protein that was indistinguishable 
from hu C9 in its sensitivity to inhibition by CD59. Conversely, when this 
same segment of hu C9 was replaced by the corresponding rb C9 sequence 
(chimera #8), the resulting chimera was indistinguishable from rb C9 and 
virtually unaffected by the presence of membrane CD59. In these 
experiments, MAC was assembled using hu C5b67 and rb C8 so as to 
circumvent known inhibitory interaction of CD59 with hu C8 (Rollins, et 
al. J. Immunol. 146, 2345-2351 (1991), Ninomiya and Sims J. Biol. Chem. 
267, 13675-13680 (1992). 
As depicted in FIG. 2, the segment of hu C9 shown to bind CD59 is 
immediately C-terminal to the putative membrane-spanning domain of the 
protein, and corresponds to a segment of polypeptide exhibiting 
particularly low sequence conversation when hu C9 is aligned to C9 of rb 
or other non-primate species. The most prominent divergence of sequence 
occurs between two cysteines (Cys359-Cys384 in hu C9) that are conserved 
in the hu and rb proteins. In hu C9, these cysteines have been shown to 
form an intrachain disulfide bond (below), as reported by Schaller, et al. 
J. Protein Chem. 13, 472-473 (1994). 
In order to further localize the segment of hu C9 recognized by CD59 and to 
determine the specific contribution of residues spanning the Cys359/384 
disulfide, a series of hu/rb C9 chimeras was constructed by interchanging 
segments of corresponding hu and rb C9 sequences internal to residues 
334-415 . Each of these chimeric proteins was expressed and analyzed for 
MAC hemolytic function, and for sensitivity to inhibition by membrane 
CD59. All resulting hu/rb C9 chimeras were functionally active as 
determined by hemolytic titration against chE containing membrane C5b-8. 
As shown in FIG. 1, analysis of CD59-inhibitory activity towards each of 
these proteins revealed inhibition of MAC lytic activity by CD59 was 
unaffected by replacement of all residues N-terminal to Cys359 of hu C9 
with corresponding rb sequence (chimera #2), whereas replacement of all 
residues C-terminal to residue 358 of hu C9 with corresponding rb sequence 
(chimera #3) resulted in a protein indistinguishable from rb C9 and only 
weakly inhibited by CD59. Consistent with the results for chimeras #1-3, 
substitution of hu C9 residues 359-415 into the corresponding segment of 
otherwise rb C9 (chimera #4) resulted in a protein that was 
indistinguishable from hu C9, suggesting that this polypeptide segment of 
hu C9 (residues 359-415) contains the binding site for CD59. 
To further resolve the segment of hu C9 required for species-selective 
interaction with CD59, additional chimeras were constructed further 
truncating the segment of hu sequence substituted into rb C9 (chimera 
#5-7). Data for these chimeras revealed that whereas hu residues 359-391 
conferred full recognition by CD59 (chimera #5), hu C9 residues 392-415 
failed to confer any recognition by CD59 (chimera #5), hu C9 residues 
392-415 failed to confer any recognition by CD59 when inserted into an 
otherwise rb C9 (chimera #6). Truncation of the inserted segment of hu C9 
sequence from 359-391 (chimera #5) to 359-384 (chimera #7) was accompanied 
by a small but significant reduction in inhibition of MAC lytic activity 
by CD59. These results imply that CD59 directly interacts with the segment 
of hu C9 contained between residues 359-391, with the peptide segment 
spanning the intrachain Cys359/384 disulfide substantially contributing to 
this interaction. 
CD59's interaction with hu C9 was abrogated by replacement of sequence 
spanning this putative CD59 recognition domain with corresponding rb 
sequence (chimeras #8-12). Replacement of hu C9 residues 334-415 with 
corresponding rb sequence (chimera #8) completely eliminated hu-selective 
interaction with CD59, as anticipated for results obtained for the 
complementary construct, chimera #1. Nevertheless, when the segment of 
rb-derived sequence substituted into otherwise hu C9 was further 
truncated, the resulting chimeras (chimeras #9-12) retained a surprising 
degree of sensitivity to the inhibitory effects of CD59, characteristic of 
hu C9. Thus substitution of rb sequence for the residues internal to 
Cys359-384 of hu C9 (chimera #12) did not significantly diminish CD59's 
capacity to inhibit the lytic activity of C9, while C-terminal extension 
of the segment of rb sequence to residue 415 (chimera #9) did not 
completely eliminate interaction with CD59. Taken together with results 
for chimeras #1-5, these data indicate that whereas hu C9 residues 359-391 
alone are sufficient to confer recognition by CD59, segments of the 
polypeptide immediately flanking this segment significantly contribute to 
the extent to which this binding site is expressed. 
The Cys359/384 disulfide in hu C9 has recently been reported to be highly 
labile and subject to spontaneous reduction in the native protein, as 
reported Hatanaka, et al., Biochim. Biophys. Acta Protein Struct. Mol. 
Enzymol. 1209, 117-122 (1994). Since the data suggested that residues 
internal to Cys359/384 contribute in-large-part to species-selective 
recognition by CD59, the extent to which the CD59 recognition site in C9 
is affected by disruption of this bond was examined. Mutant hu C9 was 
expressed with Ala substitutions at Cys359 and Cys384 and tested for 
hemolytic activity and for sensitivity to inhibition by CD59. As revealed 
by data of FIG. 3, disruption of this disulfide bond did not significantly 
affect the hemolytic activity of the protein nor the capacity of CD59 to 
specifically inhibit C9 lytic activity. This suggests that the segment of 
hu C9 forming the CD59 binding site is either conformationally constrained 
independent of the Cys359-384 disulfide, or, that this binding site is 
expressed in the primary structure of hu C9, independent of protein 
folding. 
In order to confirm that the peptide segment spanning hu C9 359-384 can 
itself mediate interaction with CD59, this 26 residue peptide was 
synthesized, coupled to BSA, and analyzed for CD59 binding, using 
biotin-CD59 conjugate in a micro plate assay. As demonstrated by FIG. 4, 
biotin-CD59 specifically bound to C9 peptide 359-384, inhibited binding 
was inhibited by excess unlabeled CD59 or by antibody directed against the 
peptide. 
CD59 is known to bind to C9 after C9 incorporates into the C5b-9 complex, 
and through this interaction inhibit propagation of membrane-inserted C9 
polymer, limiting lytic activity of MAC. In order to confirm the 
importance of the peptide segment recognized by CD59 to MAC assembly, Fab 
of antibody raised against the hu C9 peptide 359-384 was tested for its 
capacity to inhibit the hemolytic activity of the hu C5b-9 complex, under 
the same condition used to evaluate the inhibitory function of CD59. As 
shown by the data of FIGS. 5 A-D, this Fab inhibited hemolytic activity of 
hu C9 (FIG. 5A) and C9 chimera #7 (representing rb C9 containing hu C9 
residues 359-384, FIG. 1, FIG. 5B), but had no effect on the hemolytic 
activity of either rb C9 (FIG. 5C) or chimera #12 (representing 
substitution of the corresponding segment of rb C9 residues into hu C9; 
FIG. 1, FIG. 5D). 
The experiments show that hu C9 residues 359-391 promote CD59 binding, and 
that this segment of hu C9 contributes to the species-selective regulation 
of MAC function, providing an initial clue to the structural motif(s) 
through which this inhibitor selectively regulates the lytic activity of 
hu C5b-9 complex. These data further indicate that the capacity of CD59 to 
optimally interact with this segment of hu C9 is significantly influenced 
by residues immediately C-terminal to this segment of the C9 polypeptide. 
Whereas the data establish that residues internal to Cys359-Cys384 
contribute to recognition by CD59, the disulfide bond between these two 
Cys is apparently not required either for maintenance of C9's hemolytic 
activity within MAC, or, for normal regulation of that activity by 
membrane CD59. These conclusions derived by Cys/Ala mutagenesis in 
recombinant hu C9 (FIG. 3) are consistent with previous reports 
indicating: (i) the intrinsic liability of the Cys 359-384 disulfide in C9 
purified from hu plasma, where spontaneous reduction of this bond did not 
appear to alter C9 hemolytic activity, and (ii) that a specific CD59 
binding site is retained in reduced and carboxymethylated hu C9, in hu 
C9-derived peptide fragments, and can be demonstrated for E. coli fusion 
proteins contains hu C9-derived sequence spanning residues 359-384. This 
suggests that the CD59 binding site expressed by this segment of hu C9 
reflects interactions between amino acid side chains that do not require 
formation of the Cys 359/Cys384 disulfide bond. 
As noted above, chimeras generated by substituting limited segments of hu 
C9 into rb C9 revealed that the segment of hu C9 between 359-384 uniquely 
conferred recognition by CD59, and that this interaction was enhanced by 
C-terminal extension of hu sequence to residue 391 (cf. Chimeras #1-7; 
FIG. 1). Surprisingly, chimeras generated by replacing these same segments 
of hu C9 with corresponding rb C9 sequence did not exhibit a complementary 
decrease in interaction with CD59, except when the segment of rb-derived 
sequence replaced in hu C9 residues spanning 334-415 (cf. Chimeras #8-12; 
FIG. 1). 
Modifications and variations will be obvious to those skilled in the art 
from the foregoing detailed description. Such modifications and variations 
are intended to come within the scope of the following claims. 
##STR1## 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 16 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2026 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CAGCATGTCAGCCTGCCGGAGCTTTGCAGTTGCAATCTGCATTTTAGAAATAAGCATCCT60 
CACAGCACAGTACACGACCAGTTATGACCCAGAGCTAACAGAAAGCAGTGGCTCTGCATC120 
ACACATAGACTGCAGAATGAGCCCCTGGAGTGAATGGTCACAATGCGATCCTTGTCTCAG180 
ACAAATGTTTCGTTCAAGAAGCATTGAGGTCTTTGGACAATTTAATGGGAAAAGATGCAC240 
CGACGCTGTGGGAGACAGACGACAGTGTGTGCCCACAGAGCCCTGTGAGGATGCTGAGGA300 
TGACTGCGGAAATGACTTTCAATGCAGTACAGGCAGATGCATAAAGATGCGACTTCGGTG360 
TAATGGTGACAATGACTGCGGAGACTTTTCAGATGAGGATGATTGTGAAAGTGAGCCCCG420 
TCCCCCCTGCAGAGACAGAGTGGTAGAAGAGTCTGAGCTGGCACGAACAGCAGGCTATGG480 
GATCAACATTTTAGGGATGGATCCCCTAAGCACACCTTTTGACAATGAGTTCTACAATGG540 
ACTCTGTAACCGGGATCGGGATGGAAACACTCTGACATACTACCGAAGACCTTGGAACGT600 
GGCTTCTTTGATCTATGAAACCAAAGGCGAGAAAAATTTCAGAACCGAACATTACGAAGA660 
ACAAATTGAAGCATTTAAAAGTATCATCCAAGAGAAGACATCAAATTTTAATGCAGCTAT720 
ATCTCTAAAATTTACACCCACTGAAACAAATAAAGCTGAACAATGTTGTGAGGAAACAGC780 
CTCCTCAATTTCTTTACATGGCAAGGGTAGTTTTCGGTTTTCATATTCCAAAAATGAAAC840 
TTACCAACTATTTTTGTCATATTCTTCAAAGAAGGAAAAAATGTTTCTGCATGTGAAAGG900 
AGAAATTCATCTGGGAAGATTTGTAATGAGAAATCGCGATGTTGTGCTCACAACAACTTT960 
TGTGGATGATATAAAAGCTTTGCCAACTACCTATGAAAAGGGAGAATATTTTGCCTTTTT1020 
GGAAACCTATGGAACTCACTACAGTAGCTCTGGGTCTCTAGGAGGACTCTATGAACTAAT1080 
ATATGTTTTGGATAAAGCTTCCATGAAGCGGAAAGGTGTTGAACTAAAAGACATAAAGAG1140 
ATGCCTTGGGTATCATCTGGATGTATCTCTGGCTTTCTCTGAAATCTCTGTTGGAGCTGA1200 
ATTTAATAAAGATGATTGTGTAAAGAGGGGAGAGGGTAGAGCTGTAAACATCACCAGTGA1260 
AAACCTCATAGATGATGTTGTTTCACTCATAAGAGGTGGAACCAGAAAATATGCATTTGA1320 
ACTGAAAGAAAAGCTTCTCCGAGGAACCGTGATTGATGTGACTGACTTTGTCAACTGGGC1380 
CTCTTCCATAAATGATGCTCCTGTTCTCATTAGTCAAAAACTGTCTCCTATATATAATCT1440 
GGTTCCAGTGAAAATGAAAAATGCACACCTAAAGAAACAAAACTTGGAAAGAGCCATTGA1500 
AGACTATATCAATGAATTTAGTGTAAGAAAATGCCACACATGCCAAAATGGAGGTACAGT1560 
GATTCTAATGGATGGAAAGTGTTTGTGTGCCTGCCCATTCAAATTTGAGGGAATTGCCTG1620 
TGAAATCAGTAAACAAAAAATTTCTGAAGGATTGCCAGCCCTAGAGTTCCCCAATGAAAA1680 
ATAGAGCTGTTGGCTTCTCTGAGCTCCAGTGGAAGAAGAAAACACTAGTACCTTCAGACT1740 
CCTACCCCTGAAGATAATCTTAGCTGCCAAGTAAATAGCAACATGCTTCATGAAAATCCT1800 
ACCAACCTCTGAAGTCTCTTCTCTCTTAGGTCTATAATTTTTTTTTTAATTTTTCTTCCT1860 
TAAACTCCTGTGATGTTTCCATTTTTTGTTCCCTAATGAGAAGTCAACAGTGAAATACGC1920 
CAGAACTGCTTTATCCCACGGAAAATGCCAATCTCTTCTAAAAAAAAACAAAATTAAATT1980 
AAAAACAGAATGTTGGTTTAAAAAACTTCAAAGAAAAAAAAAAAAA2026 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2034 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
CTCGTGAGCAGCATGGCCGCCAGCCACAGCTTCGCCTTTGTGGTCTGCGTTTTAGAAATC60 
GGTGCCCTGACGGCAGGACCCACTCCCAGCTATGTCCACGAGCCGATACAAAGGAGTGAC120 
CCTCTGCAGCCCATAGACTGCAGGATGAGCCCATGGAGTGAATGGTCGCACTGTGATCCT180 
TGTCTCAGGCAAATGTTTCGTTCAAGGAGCATCGAAGTCTTTGGACAATTTCATGGGAAA240 
AGTTGTGTGGATGCTCTGGGCGACAGGCGAGCGTGTATACCTACGGAGGCATGCGAAGAC300 
GCTGAGGAGGACTGTGAAAAAGACGAATTTCACTGTGGGACAGGCAGGTGCATAAAGAGG360 
CGACTGCTGTGTAATGGGGACAATGACTGCGGAGACTTTTCAGATGAGGATGACTGCGAA420 
ACGGAGCCCCGTCTTACCTGTCGCAACCGCGAGGTCCAAGAGTCGGAGCTGGCACGGACA480 
GCGGGCTATGGGATCAACATTTTAGGGATGGATCCCCTAGCCACACCTTTTGACAACGAG540 
TACTACCACGGACTCTGTGACCGTGTTTGGGATGGGAACACTTTGACACACTATCGAAAA600 
CCCTGGAATGTGGCTGTTTTGGCCTATGAAACAAAAATTGATAAAAATTTCAGAACTGAA660 
TACTATGAAGAACAGATGCAGGCATTCAAAAGTATCATTGAAGAAGAGACATCAAATTTT720 
AATGCAAATTTAGCTCTAAAATTTACACCCACCGAAGCAAAAGCAAGTAAGGCTGAAGAA780 
GCTTCTCCAAAAAACAAGTCTTTGGATGATAATGATAAAGGTTTCTCGAGTAAATTTCAA840 
TTTTCGTATTCCAAAAATGAAACTTACCAACTATTCTTGTCATATTCTTCACAGAAGGAA900 
AAAATGTTTCTGCTTGTGAAAGGAATAATTCAACTGGGAAGATTTGTGATGAAAAATCGG960 
GGTGTTATGCTGACAAATACCTTCTTGGATGATATAAAATCTCTGCCAACTACCTATGAA1020 
AAAGGAGAATATTTTGCATTTTTGGAAACCTATGGAACCCACTATAGTAGCTCTGGGTCT1080 
CTGGGAGGACGCTATGAGCTAATTTATGTTTTGGATAAAGCTTCCATGAAGGAGAAAGGG1140 
ATTGAGCTGAATGACATAAAGAAATGCCTTGGGTTTGACTTAGATTTATCTCTGAATATC1200 
CCTGGAAAATCTGCTGGGCTTTCGCTCACAGGACAAGCAAATAAAAACAATTGCTTAAAG1260 
AGTGGTCATGGTAATGCTGTAAACATCACCAGGGCTAACCTCATAGATGATGTGATTTCA1320 
CTCATAAGAGGAGGAACCCAAAAATTTGCGTTTGAATTGAAAGAAAAGCTTCTCACCAAA1380 
GCCAAGATGGTTGACGTGACGGACTTTATCAATTGGGCCTCTTCCTTAAGTGATGCTCCA1440 
GTGCTCATCAATCAAAAACTGTCCCCTATATATAATCTGATTCCTGTGAAAATAAAAGAT1500 
GCGCACCAAAAGAGACAGAATCTGGAGAGAGGAATTGAAGATTACATCAATGAATTCAGC1560 
ACGAAAAAGTGCTCCCCCTGCCAAAACGGAGGCACTGCACTTCTGATGGATGGCCAGTGT1620 
TTGTGTACCTGCCCGTTTATGTTCGAGGGGATTGCCTGTGAAATCTCCAAACGAAAACTG1680 
GCTTAAGGATTGCCAGCCCCCACCCCCACCCCCCAAAATGCAACTGTTGAGTTCCCTGAG1740 
CTCAAATGGAAGAAAAACAACACCAGGACCTTCCAATGTAAGATCCTGCCCTGCCTGGAG1800 
ATAGTCCTTGCTGGCACATGAAAAGCAACATGTTTCATGAAAACCCTACCAACCTCTGAA1860 
GCCTCGCTCTCTCTCTGGTCTGCAATGCCTGTTTTTCCCCATAAACCCCTGTAATGTTTC1920 
CATTTTTATTTAATGAAGAGACAGCCATGAGCTGTGCCAGAAGTGTTTTCTCCCACAGCC1980 
AATGCCAGCCTCTTGCTAATAAAAGAAAATAAAATTCAAAAAAAAAAAAAAAAA2034 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 82 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuTyrGluLeuIleTyrValLeuAspLysAlaSerMetLysArgLys 
151015 
GlyValGluLeuLysAspIleLysArgCysLeuGlyTyrHisLeuAsp 
202530 
ValSerLeuAlaPheSerGluIleSerValGlyAlaGluPheAsnLys 
354045 
AspAspCysValLysArgGlyGluGlyArgAlaValAsnIleThrSer 
505560 
GluAsnLeuIleAspAspValValSerLeuIleArgGlyGlyThrArg 
65707580 
LysTyr 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 86 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ArgTyrGluLeuIleTyrValLeuAspLysAlaSerMetLysGluLys 
151015 
GlyIleGluLeuAsnAspIleLysLysCysLeuGlyPheAspLeuAsp 
202530 
LeuSerLeuAsnIleProGlyLysSerAlaGlyLeuSerLeuThrGly 
354045 
GlnAlaAsnLysAsnAsnCysLeuLysSerGlyHisGlyAsnAlaVal 
505560 
AsnIleThrArgAlaAsnLeuIleAspAspValIleSerLeuIleArg 
65707580 
GlyGlyThrGlnLysPhe 
85 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 560 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
SerMetSerAlaCysArgSerPheAlaValAlaIleCysIleLeuGlu 
151015 
IleSerIleLeuThrAlaGlnTyrThrThrSerTyrAspProGluLeu 
202530 
ThrGluSerSerGlySerAlaSerHisIleAspCysArgMetSerPro 
354045 
TrpSerGluTrpSerGlnCysAspProCysLeuArgGlnMetPheArg 
505560 
SerArgSerIleGluValPheGlyGlnPheAsnGlyLysArgCysThr 
65707580 
AspAlaValGlyAspArgArgGlnCysValProThrGluProCysGlu 
859095 
AspAlaGluAspAspCysGlyAsnAspPheGlnCysSerThrGlyArg 
100105110 
CysIleLysMetArgLeuArgCysAsnGlyAspAsnAspCysGlyAsp 
115120125 
PheSerAspGluAspAspCysGluSerGluProArgProProCysArg 
130135140 
AspArgValValGluGluSerGluLeuAlaArgThrAlaGlyTyrGly 
145150155160 
IleAsnIleLeuGlyMetAspProLeuSerThrProPheAspAsnGlu 
165170175 
PheTyrAsnGlyLeuCysAsnArgAspArgAspGlyAsnThrLeuThr 
180185190 
TyrTyrArgArgProTrpAsnValAlaSerLeuIleTyrGluThrLys 
195200205 
GlyGluLysAsnPheArgThrGluHisTyrGluGluGlnIleGluAla 
210215220 
PheLysSerIleIleGlnGluLysThrSerAsnPheAsnAlaAlaIle 
225230235240 
SerLeuLysPheThrProThrGluThrAsnLysAlaGluGlnCysCys 
245250255 
GluGluThrAlaSerSerIleSerLeuHisGlyLysGlySerPheArg 
260265270 
PheSerTyrSerLysAsnGluThrTyrGlnLeuPheLeuSerTyrSer 
275280285 
SerLysLysGluLysMetPheLeuHisValLysGlyGluIleHisLeu 
290295300 
GlyArgPheValMetArgAsnArgAspValValLeuThrThrThrPhe 
305310315320 
ValAspAspIleLysAlaLeuProThrThrTyrGluLysGlyGluTyr 
325330335 
PheAlaPheLeuGluThrTyrGlyThrHisTyrSerSerSerGlySer 
340345350 
LeuGlyGlyLeuTyrGluLeuIleTyrValLeuAspLysAlaSerMet 
355360365 
LysArgLysGlyValGluLeuLysAspIleLysArgCysLeuGlyTyr 
370375380 
HisLeuAspValSerLeuAlaPheSerGluIleSerValGlyAlaGlu 
385390395400 
PheAsnLysAspAspCysValLysArgGlyGluGlyArgAlaValAsn 
405410415 
IleThrSerGluAsnLeuIleAspAspValValSerLeuIleArgGly 
420425430 
GlyThrArgLysTyrAlaPheGluLeuLysGluLysLeuLeuArgGly 
435440445 
ThrValIleAspValThrAspPheValAsnTrpAlaSerSerIleAsn 
450455460 
AspAlaProValLeuIleSerGlnLysLeuSerProIleTyrAsnLeu 
465470475480 
ValProValLysMetLysAsnAlaHisLeuLysLysGlnAsnLeuGlu 
485490495 
ArgAlaIleGluAspTyrIleAsnGluPheSerValArgLysCysHis 
500505510 
ThrCysGlnAsnGlyGlyThrValIleLeuMetAspGlyLysCysLeu 
515520525 
CysAlaCysProPheLysPheGluGlyIleAlaCysGluIleSerLys 
530535540 
GlnLysIleSerGluGlyLeuProAlaLeuGluPheProAsnGluLys 
545550555560 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
SerCysTrpLeuLeu 
15 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
AlaProValGluGluGluAsnThrSerThrPheArgLeuLeuProLeu 
151015 
LysIleIleLeuAlaAlaLys 
20 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
IleAlaThrCysPheMetLysIleLeuProThrSerGluValSerSer 
151015 
LeuLeuGlyLeu 
20 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
PhePhePhe 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
PhePhePheLeuLysLeuLeu 
15 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
CysPheHisPheLeuPheProAsnGluLysSerThrValLysTyrAla 
151015 
ArgThrAlaLeuSerHisGlyLysCysGlnSerLeuLeuLysLysAsn 
202530 
LysIleLysLeuLysThrGluCysTrpPheLysLysLeuGlnArgLys 
354045 
LysLysLys 
50 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 561 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
LeuValSerSerMetAlaAlaSerHisSerPheAlaPheValValCys 
151015 
ValLeuGluIleGlyAlaLeuThrAlaGlyProThrProSerTyrVal 
202530 
HisGluProIleGlnArgSerAspProLeuGlnProIleAspCysArg 
354045 
MetSerProTrpSerGluTrpSerHisCysAspProCysLeuArgGln 
505560 
MetPheArgSerArgSerIleGluValPheGlyGlnPheHisGlyLys 
65707580 
SerCysValAspAlaLeuGlyAspArgArgAlaCysIleProThrGlu 
859095 
AlaCysGluAspAlaGluGluAspCysGluLysAspGluPheHisCys 
100105110 
GlyThrGlyArgCysIleLysArgArgLeuLeuCysAsnGlyAspAsn 
115120125 
AspCysGlyAspPheSerAspGluAspAspCysGluThrGluProArg 
130135140 
LeuThrCysArgAsnArgGluValGlnGluSerGluLeuAlaArgThr 
145150155160 
AlaGlyTyrGlyIleAsnIleLeuGlyMetAspProLeuAlaThrPro 
165170175 
PheAspAsnGluTyrTyrHisGlyLeuCysAspArgValTrpAspGly 
180185190 
AsnThrLeuThrHisTyrArgLysProTrpAsnValAlaValLeuAla 
195200205 
TyrGluThrLysIleAspLysAsnPheArgThrGluTyrTyrGluGlu 
210215220 
GlnMetGlnAlaPheLysSerIleIleGluGluGluThrSerAsnPhe 
225230235240 
AsnAlaAsnLeuAlaLeuLysPheThrProThrGluAlaLysAlaSer 
245250255 
LysAlaGluGluAlaSerProLysAsnLysSerLeuAspAspAsnAsp 
260265270 
LysGlyPheSerSerLysPheGlnPheSerTyrSerLysAsnGluThr 
275280285 
TyrGlnLeuPheLeuSerTyrSerSerGlnLysGluLysMetPheLeu 
290295300 
LeuValLysGlyIleIleGlnLeuGlyArgPheValMetLysAsnArg 
305310315320 
GlyValMetLeuThrAsnThrPheLeuAspAspIleLysSerLeuPro 
325330335 
ThrThrTyrGluLysGlyGluTyrPheAlaPheLeuGluThrTyrGly 
340345350 
ThrHisTyrSerSerSerGlySerLeuGlyGlyArgTyrGluLeuIle 
355360365 
TyrValLeuAspLysAlaSerMetLysGluLysGlyIleGluLeuAsn 
370375380 
AspIleLysLysCysLeuGlyPheAspLeuAspLeuSerLeuAsnIle 
385390395400 
ProGlyLysSerAlaGlyLeuSerLeuThrGlyGlnAlaAsnLysAsn 
405410415 
AsnCysLeuLysSerGlyHisGlyAsnAlaValAsnIleThrArgAla 
420425430 
AsnLeuIleAspAspValIleSerLeuIleArgGlyGlyThrGlnLys 
435440445 
PheAlaPheGluLeuLysGluLysLeuLeuThrLysAlaLysMetVal 
450455460 
AspValThrAspPheIleAsnTrpAlaSerSerLeuSerAspAlaPro 
465470475480 
ValLeuIleAsnGlnLysLeuSerProIleTyrAsnLeuIleProVal 
485490495 
LysIleLysAspAlaHisGlnLysArgGlnAsnLeuGluArgGlyIle 
500505510 
GluAspTyrIleAsnGluPheSerThrLysLysCysSerProCysGln 
515520525 
AsnGlyGlyThrAlaLeuLeuMetAspGlyGlnCysLeuCysThrCys 
530535540 
ProPheMetPheGluGlyIleAlaCysGluIleSerLysArgLysLeu 
545550555560 
Ala 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 44 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GlyLeuProAlaProThrProThrProGlnAsnAlaThrValGluPhe 
151015 
ProGluLeuLysTrpLysLysAsnAsnThrArgThrPheGlnCysLys 
202530 
IleLeuProCysLeuGluIleValLeuAlaGlyThr 
3540 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
LysAlaThrCysPheMetLysThrLeuProThrSerGluAlaSerLeu 
151015 
SerLeuTrpSerAlaMetProValPheProHisLysProLeu 
202530 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
CysPheHisPheTyrLeuMetLysArgGlnPro 
1510 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
AlaValProGluValPheSerProThrAlaAsnAlaSerLeuLeuLeu 
151015 
IleLysGluAsnLysIleGlnLysLysLysLysLys 
2025 
__________________________________________________________________________