U.S. patent application number 12/192560 was filed with the patent office on 2010-11-11 for conjugates of soluble peptidic compounds with membrane-binding agents.
This patent application is currently assigned to AdProTech Limited. Invention is credited to Ian Dodd, Danuta Ewa Irena Mossakowkska, Richard Anthony Godwin SMITH.
Application Number | 20100286367 12/192560 |
Document ID | / |
Family ID | 10796956 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286367 |
Kind Code |
A1 |
SMITH; Richard Anthony Godwin ;
et al. |
November 11, 2010 |
CONJUGATES OF SOLUBLE PEPTIDIC COMPOUNDS WITH MEMBRANE-BINDING
AGENTS
Abstract
The present invention provides, among other things, soluble
derivatives of soluble polypeptides that incorporate membrane
binding elements. Methods of making these soluble derivatives, and
methods of using these soluble derivatives also are provided.
Inventors: |
SMITH; Richard Anthony Godwin;
(Hertfordshire, GB) ; Dodd; Ian; (Hertfordshire,
GB) ; Mossakowkska; Danuta Ewa Irena; (Essex,
GB) |
Correspondence
Address: |
Reddie & Grose;Perkins Coie LLP
607 Fourteenth Street, NW
Washington
DC
20005
US
|
Assignee: |
AdProTech Limited
Essex
GB
|
Family ID: |
10796956 |
Appl. No.: |
12/192560 |
Filed: |
August 15, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10742887 |
Dec 23, 2003 |
7655617 |
|
|
12192560 |
|
|
|
|
09612314 |
Jul 7, 2000 |
6713606 |
|
|
10742887 |
|
|
|
|
09214913 |
Mar 16, 1999 |
|
|
|
PCT/EP97/03715 |
Jul 8, 1997 |
|
|
|
09612314 |
|
|
|
|
Current U.S.
Class: |
530/326 ;
530/328; 530/329; 530/330; 530/331 |
Current CPC
Class: |
C07K 14/70596 20130101;
A61K 47/64 20170801; C07K 16/28 20130101; A61P 7/02 20180101; A61P
29/00 20180101; A61K 47/54 20170801; C07K 14/3153 20130101; C12Y
304/21073 20130101; C07C 323/59 20130101; C07C 323/41 20130101;
C12N 9/6462 20130101; A61K 38/00 20130101 |
Class at
Publication: |
530/326 ;
530/330; 530/329; 530/331; 530/328 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 5/10 20060101 C07K005/10; C07K 7/06 20060101
C07K007/06; C07K 5/08 20060101 C07K005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 15, 1996 |
GB |
9614871.3 |
Claims
1-49. (canceled)
50. A membrane localization reagent for directing a molecule to an
outer membrane of a cell, wherein the membrane localization reagent
is soluble and comprises: (a) at least one lipophilic binding
element comprising aliphatic acyl groups; (b) a hydrophilic peptide
binding element comprising basic amino acids, wherein the
hydrophilic binding element is bound to the lipophilic element; and
(c) a linker that covalently binds (i) the molecule to be directed
to the outer membrane of a cell to (ii) the hydrophilic peptide
binding element of the membrane localization reagent.
51. The membrane localization reagent of claim 50, wherein the
hydrophilic peptide binding element comprises lysine residues.
52. The membrane localization reagent of claim 51, wherein the
hydrophilic peptide binding element comprises three to ten lysine
residues.
53. The membrane localization reagent of claim 51, wherein the
hydrophilic peptide binding element comprises four to seven lysine
residues.
54. The membrane localization reagent of claim 50, wherein the
hydrophilic peptide binding element comprises arginine
residues.
55. The membrane localization reagent of claim 54, wherein the
hydrophilic peptide binding element comprises three to ten arginine
residues.
56. The membrane localization reagent of claim 55, wherein the
hydrophilic peptide binding element comprises four to seven
arginine residues.
57. A membrane localization reagent for directing a molecule to an
outer membrane of a cell, wherein the membrane localization reagent
is soluble and comprises a hydrophilic peptide binding element,
wherein the peptide comprises a sequence selected from the group
consisting of: TABLE-US-00038 (a) DGPKKKKKKSPSKSSG; (SEQ ID NO. 8)
(b) GSSKSPSKKKKKKPGD; (SEQ ID NO. 9) (c) SPSNETPKKKKKRFSFKKSG; (SEQ
ID NO. 10) (d) DGPKKKKKKSPSKSSK; (SEQ ID NO. 11) and (e)
SKDGKKKKKKSKTK. (SEQ ID NO. 12)
58. The membrane localization reagent of claim 57, wherein the
hydrophilic peptide binding element comprises GSSKSPSKKKKKKPGD (SEQ
ID NO. 9).
59. The membrane localization reagent of claim 50, wherein the
lipophilic binding element comprises 8 to 18 methylene units.
60. The membrane localization reagent of claim 59, wherein the
lipophilic binding element comprises 10 to 14 methylene units.
61. The membrane localization reagent of claim 58, wherein the
membrane localization reagent comprises a lipophilic binding
element, and wherein the lipophilic binding element comprises
myristoyl.
62. The membrane localization reagent of claim 60, wherein the
lipophilic binding element comprises myristoyl.
63. The membrane localization reagent according to claim 50,
wherein the linker is selected from the group consisting of a
cysteine residue; an N-haloacetyl group (where halo signifies
chlorine, bromine or iodine); a haloacetyl group (where halo
signifies chlorine, bromine or iodine) at an 6-amino group of a
lysine residue; a bond; an amide group at the C-terminus; an
N-terminal blocking group; and a fatty acid N-acyl group at the
N-terminus or at an E-amino group of a lysine residue.
64. The membrane localization reagent according to claim 50,
wherein the molecule is a therapeutic agent.
65. The soluble compound according to 50, wherein the linker is
selected from the group consisting of a cysteine residue; an
N-haloacetyl group (where halo signifies chlorine, bromine or
iodine); a haloacetyl group (where halo signifies chlorine, bromine
or iodine) at an .epsilon.-amino group of a lysine residue; a bond;
an amide group at the C-terminus; an N-terminal blocking group; and
a fatty acid N-acyl group at the N-terminus or at an
.epsilon.-amino group of a lysine residue.
66. A soluble derivative of a soluble polypeptide, wherein said
derivative consisting of a conjugate or fusion of the polypeptide
with a unit further consisting of two or more covalently linked
heterologous membrane binding elements which elements are not all
identical, which are capable of interacting independently with
components of cellular or artificial membranes exposed to
extracellular fluids and which are selected from the group
consisting of: a) a membrane-insertive hydrophobic group selected
from aliphatic acyl groups with between 8 and 18 methylene units
and aliphatic amines with between 8 and 18 methylene units; b) a
membrane-associative charged peptide consisting of between 3 and 25
amino acids of which no less than 2 and no more than 12 are basic
residues; and c) a membrane component ligand further consisting of
a peptide containing between 3 and 13 amino acids derived from
known ligands for membrane components such as transmembrane
proteins and identified by screening of chemical, bacteriophage, or
other displayed libraries against specific membrane-derived
molecular targets.
67. A soluble derivative of a soluble polypeptide according to
claim 66, wherein the basic residues comprise lysine.
68. A soluble derivative of a soluble polypeptide according to
claim 66, wherein the membrane-associative charged peptide
comprises basic amino acids; and other amino acids selected from
the group consisting of Serine, Threonine, Glycine, Proline,
Alanine, Cysteine, Aspartic Acid, Glutamic Acid, Asparagine, and
Glutamine.
69. A soluble derivative of a soluble polypeptide according to
claim 66, wherein the membrane-associative charged peptide
comprises lysine.
Description
[0001] This invention relates to polypeptide derivatives, their use
in therapy and methods and intermediates for their production.
[0002] Essentially all protein drugs are administered as solutions
and function in vivo in the solution phase. In biochemistry and
pharmacology, however, a large number of control and mediator
proteins are associated with or function within or on the plasma
membranes of cells. Except for soluble, truncated versions of one
class of these molecules, no membrane-associated proteins have been
developed as therapeutic agents. There are two main reasons for
this situation. Firstly, overexpression of proteins that are
retained in the membranes of the producer cells is limited by the
the low capacity of membranes for proteins and often by the toxic
effects of retention when expression is intrinsically efficient.
Secondly, extraction of these proteins from membranes requires
detergents or organic solvents, often results in inactivation of
the protein, leads to difficulties in achieving the high purity
needed for drug use and usually gives a product which is hard to
formulate for intravenous administration. In addition, retention of
very hydrophobic membrane anchoring elements may cause proteins to
associate strongly with lipid-binding proteins in blood when
administered intravenously thus preventing access to cell
membranes.
[0003] Soluble, truncated versions of membrane-associated proteins
overcome the production difficulties associated with full length
proteins. However such truncated molecules lack the membrane
binding capability and specificity of the full length proteins
which properties may be advantageous or even essential to the
desired therapeutic activity.
[0004] The main classes of interaction of proteins with membranes
can be summarised as follows: [0005] 1. Direct and specific
interactions with phospholipid head groups or with other
hydrophilic regions of complex lipids or indirectly with proteins
already inserted in the membrane. The latter may include all the
types of intrinsic membrane protein noted below and such
interactions are usually with extracellular domains or sequence
loops of the membrane proteins; [0006] 2. Through anchoring by a
single hydrophobic transmembrane helical region near the terminus
of the protein. These regions commonly present a hydrophobic face
around the entire circumference of the helix cylinder and transfer
of this structure to the hydrophilic environment of bulk water is
energetically unfavourable. [0007] 3. Further anchoring is often
provided by a short sequence of generally cationic aminoacids at
the cytoplasmic side of the membrane, C-terminal to the
transmembrane helix: [0008] 4. Through the use of multiple
(normally 2-12 and commonly 4,7 and 10) transmembrane regions which
are usually predicted to be helical or near-helical. Although these
regions are normally hydrophobic overall, they frequently show some
amphipathic behaviour--an outer hydrophobic face and an inner more
hydrophilic one being identifiable within a helix bundle located in
the lipid bilayer; [0009] 5. Through postranslationally linked
phosphatidyl inositol moeities (GPI-anchors). These are generated
by a specific biosynthetic pathway which recognises and removes a
specific stretch of C-terminal aminoacids and creates a
membrane-associating diacyl glycerol unit linked via a hydrophilic
carbohydrate spacer to the polypeptide; [0010] 6. In a related
process, single fatty acid groups such as myristoyl, palmitoyl or
prenyl may be attached postranslationally to one or more sites in a
protein (usually at N- or C-termini). Again, amino acids (such as
the C-terminal CAAX box in Ras proteins) may be removed.
[0011] Artificial membranes are considered to be lipid complexes
that mimic the basic properties of the cell membrane, i.e., a lipid
vacuole with an aqueous interior and a surface chemistry that
resembles the cell membrane. The artificial membrane typically
contains phospholipids or mimics thereof and may be unilemellar or
bilemellar and the outer surface will contain charged groups
similar to the choline groups of the most abundant phospholipid.
The prototype artificial membrane is known as a liposome and the
technologies for the construction of liposomes including the
incorporation of therapeutically useful agents into them is well
known to those in the an. Liposomes have been evaluated in a number
of disease states and liposomes containing the anti-fungal
Amphotericin are commercially available. In addition,
proteoliposomes have been described. For example, the use of
immunoliposomes encapsulating amphotericin B has been reported to
be of benefit in the treatment of experimental fungal infections in
animal models (e.g. Hospenthal, D. et al (1989) J. Med. Microbiol.
30 193-197; Dromer, F. et al (1990) Antimicrob. Agents Chemother.
34 2055-2060).
[0012] Mimics of natural or artificial membranes are often related
in structure and will mimic one or more properties of the membrane.
One such example is the provision of an artificial surface having
pendant groups which mimic the phospholipid zwitterionic groups
which are found on the outside of cell surfaces. For example
WO92/06719 (Biocompatibles Limited) discloses natural and synthetic
phospholipids which may be coated on an artificial surface, e.g. a
device which, in use, will come into contact with
protein-containing or biological fluids, to provide improved
biocompatibility and haemocompatibility and WO 94/16749 discloses
additional zwitterionic groups that may be used to improve
biocompatibility in a similar way.
[0013] The present invention provides a soluble derivative of a
soluble polypeptide, said derivative comprising two or more
heterologous membrane binding elements with low membrane affinity
covalently associated with the polypeptide which elements are
capable of interacting, independently and with thermodynamic
additivity, with components of cellular or artificial membranes
exposed to extracellular fluids.
[0014] By `heterologous` is meant that the elements are not found
in the native full length protein from which a soluble protein may
be derived.
[0015] By `soluble polypeptide` is meant a truncated derivative of
a full length protein which lacks its natural membrane binding
capability, and/or a polypeptide which has a solubility level in
aqueous media of >100 .mu.g/ml.
[0016] By `membrane binding element with low membrane affinity` is
meant that the element has only moderate affinity for membranes,
that is a dissociation constant greater than 0.1 .mu.M, preferably
1 .mu.M-1 mM. The elements preferably have a size <5 kDa.
[0017] The derivative should incorporate sufficient elements with
low affinities for membrane components to result in a derivative
with a high (preferably 0.01-10 nM dissociation constant) affinity
for specific membranes. The elements combine so as to create an
overall high affinity for the particular target membrane but the
combination lacks such high affinity for other proteins for which
single elements may be (low-affinity) ligands.
[0018] The elements should be chosen so as to retain useful
solubility in pharmaceutical formulation media, preferably >100
.mu.g/ml. Preferably at least one element is hydrophilic.
[0019] The invention thus promotes localisation of a therapeutic
protein at cellular membranes and thereby provides one or more of
several biologically significant effects with potential therapeutic
advantages including:
[0020] Potency: If the protein is a receptor and an agonist or
antagonist activity is localised on the same surface as the
receptor itself, an increase in effective concentration may result
from the reduction in the diffusional degrees of freedom.
[0021] Pharmacokinetics and dosing frequency: Interaction of a
derivatised protein with long-lived cell types or serum proteins
would be expected to prolong the plasma residence time of the
protein and produce a depot effect through deposition on cell
surfaces.
[0022] Specificity: Many clinically important pathological
processes are associated with specific cell types and tissues (for
example the vascular endothelium and the recruitment thereto of
neutrophils bearing the sialyl Lewis' antigen to ELAM-1, see
below). Hence targeting the modified protein to regions of membrane
containing pathology-associated membrane markers may improve the
therapeutic ratio of the protein targeted.
[0023] The derivatives of the invention may be used in association
with artificial membranes or mimics thereof to allow delivery of
the therapeutic protein to sites where it will provide therapeutic
benefit. For example, polypeptides associated with liposomes formed
by contacting liposomes with a derivative of the invention may be
more stable than the free polypeptide. The liposome may incorporate
a therapeutic agent, for example an antiflammatory or cytotoxic
agent. The polypeptide derivative of the invention may thus be used
to target the therapeutic agent. When the polypeptide is itself a
therapeutic agent, the liposome incorporated therapeutic agent may
be used to enhance further the efficacy or tolerability of the
therapy.
[0024] Association of derivatives of the invention with mimics of
cell membranes may be used to concentrate the therapeutic protein
at sites where therapeutically useful concentrations of
underivatised protein might be difficult to achieve. For example,
indwelling medical devices coated with mimics of the phospholipid
zwitterionic groups which are found on the outside of cell
surfaces, such as those disclosed in WO92/06719 and WO 94/16749,
may be additionally treated with derivatives of the invention. For
example complement inhibitors derivatised in accordance with the
invention could be incorporated into the outer surface of
indwelling catheters or hip replacements or heart valves in order
to minimise development of inflammation associated with these
operations.
[0025] It will be appreciated that all associations of heterologous
amino acid sequences with a polypeptide which is a soluble
derivative of a human protein will need to be assessed for
potential immunogenicity, particularly where the amino acid
sequence is not derived from a human protein. The problem can be
minimised by using sequences as close as possible to known human
ones and through computation of secondary structure and
antigenicity indices.
[0026] Examples of protein therapeutic agents which may be modified
according to the invention include but are not restricted to the
following:
TABLE-US-00001 Therapeutic Base Protein Cell Target Application
IL-4 Y124D mutein B-cells Anti-allergy (IL-4 antagonist)
Plasminogen activators Erythrocytes, Prevention of venous e.g.
Prourokinase, vascular endothelium thrombosis streptokinase,
tissue-type plasminogen activator, reteplase Leptin Choroid plexus,
Weight loss (agonist) Hypothalamus Complement inhibitors* Vascular
endothelium, Ischaemic injury, Myocytes, transplantation,
Erythrocytes, inflammation Lymphocytes scFv antibody against
Eosinophils Asthma, cytokines (IL-1, IL-, allergic disease IL-5,
IL-6) Protein C Vascular endothelium Prevention of venous
thrombosis Antibodies against CD4, Lymphocytes Immunosuppression
B7/CD28, CD3/TCR, CD11b(CR3) Interferon-.beta. and Lymphocytes
Immunomodulation, derivatives multiple sclerosis *Complement
regulatory proteins e.g.: CR1 (CD35); DAF (CD55); MCP (CD46); CD59;
Factor H; and C4 binding protein; and hybrids or muteins thereof
such as CR1-CD59 (S. G. El Feki and D. T. Fearon Molecular
Immunology 33 (supp 1). p 57, 1996), MCP-DAF (P. J. Higgins et al,
J. Immunology. 158, 2872-2881, 1997) and soluble CR1 polypeptide
fragments.
[0027] The derivative preferably comprises two to eight, more
preferably two to four membrane binding elements.
[0028] Membrane binding elements are preferably selected from:
fatty acid derivatives such as fatty acyl groups; basic amino acid
sequences; ligands of known integral membrane proteins; sequences
derived from the complementarity-determining region of monoclonal
antibodies raised against epitopes of membrane proteins; membrane
binding sequences identified through screening of random chemical
or peptide libraries.
[0029] The selection of suitable combination of membrane binding
elements will be guided by the nature of the target cell membrane
or components thereof.
[0030] Suitable fatty acid derivatives include myristoyl (12
methylene units) which is insufficiently large or hydrophobic to
permit high affinity binding to membranes. Studies with
myristoylated peptides (eg R. M. Peitzsch & S. McLaughlin,
Biochemistry, 32, 10436-10443, 1993)) have shown that they have
effective dissociation constants with model lipid systems of
.about.10.sup.-4 M and around 10 of the 12 methylene groups are
buried in the lipid bilayer. Thus, aliphatic acyl groups with about
8 to 18 methylene units, preferably 10-14, are suitable membrane
binding elements. Other examples of suitable fatty acid derivatives
include long-chain (8-18, preferably 10-14 methylene) aliphatic
amines and thiols, steroid and farnesyl derivatives.
[0031] Membrane binding has been found to be associated with
limited (single-site) modification with fatty acyl groups when
combined with a cluster of basic aminoacids in the protein sequence
which may interact with acidic phospholipid head groups and provide
the additional energy to target membrane binding. This combination
of effects has been termed the `myristoyl-electrostatic switch` (S.
McLaughlin and A. Aderem, TIBS, 20, 272-276, 1994; J. F. Hancock et
al, Cell. 63, 133-139, 1990). Thus, a further example of suitable
membrane binding elements are basic aminoacid sequences such as
those found in proteins such as Ras and MARCKS (myristoylated
alanine-rich C-kinase substrate, P. J. Blackshear, J. Biol. Chem.,
268, 1501-1504, 1993) which mediate the electrostatic `switch`
through reversible phosphorylation of serine residues within the
sequence and a concomitant neutralisation of the net positive
charge. Such sequences include but are not restricted to
consecutive sequences of Lysine and Arginine such as (Lys)n where n
is from 3 to 10, preferably 4 to 7.
[0032] Suitable examples of amino acid sequences comprising basic
amino acids include:
TABLE-US-00002 i) DGPKKKKKKSPSKSSG ii) GSSKSPSKKKKKKPGD iii)
SPSNETPKKKKKRFSFKKSG iv) DGPKKKKKKSPSKSSK v) SKDGKKKKKKSKTK
(N-terminus on left)
[0033] Sequences i) to v) are examples of electrostatic switch
sequences.
[0034] Examples of amino acid sequences derived from ligands of
known integral membrane proteins include RGD-containing peptides
such as GRGDSP which are ligands for the .alpha..sub.db.beta..sub.3
integrin of human platelet membranes. Another example is
DGPSEILRGDFSS derived from human fibrinogen alpha chain, which
binds to the GpIIb/IIIa membrane protein in platelets.
[0035] Further examples of such sequences include those known to be
involved in interactions between membrane proteins such as
receptors and the major histocompatibility complex. An example of
such a membrane protein ligand is the sequence GNEQSFRVDLRTLLRYA
which has been shown to bind to the major histocompatibility
complex class 1 protein (MHC-1) with moderate affinity (L. Olsson
et al, Proc. Natl. Acad. Sci. USA. 91, 9086-909, 1994).
[0036] Yet further examples of such sequences employ a membrane
insertive address specific for T-cells. Such sequence is derived
from the known interaction of the transmembrane helix of the T-cell
antigen receptor with CD3 (Nature Medicine 3, 84-88, 1997).
Examples are peptides containing the sequence GFRILLLKV such
as:
TABLE-US-00003 SAAPSSGFRILLLKV AAPSVIGFRILLLKVAG
[0037] An example of a ligand for an integral membrane protein is
the carbohydrate ligand Sialyl Lewis' which has been identified as
a ligand for the integral membrane protein ELAM-1 (M. L. Phillips
et al, Science, 250, 1130-1132, 1990 & G. Walz et al, Ibid,
250, 1132-1135, 1990).
[0038] Sequences derived from the complementarity-determining
regions of monoclonal antibodies raised against epitopes within
membrane proteins (see, for example, J. W. Smith et al, J. Biol.
Chem. 270, 30486-30490, 1995) are also suitable membrane binding
elements, as are binding sequences from random chemical libraries
such as those generated in a phage display format and selected by
biopanning operations in vitro (G. F. Smith and J. K. Scott,
Methods in Enzymology, 217H, 228-257, 1993) or in vivo (R.
Pasqualini & E. Ruoslahti, Nature, 380, 364-366, 1996).
[0039] Optionally, conditional dissociation from the membrane may
be incorporated into derivatives of the invention using mechanisms
such as pH sensitivity (electrostatic switches), regulation through
metal ion binding (using endogenous Ca.sup.2+, Zn.sup.2+ and
incorporation of ion binding sites in membrane binding elements)
and protease cleavage (e.g plasminolysis of lysine-rich membrane
binding sequences to release and activate prourokinase)
[0040] Preferred derivatives of this invention have the following
structure:
[P]-{L-[W]}.sub.n--X
in which: [0041] P is the soluble polypeptide, [0042] each L is
independently a flexible linker group, [0043] each W is
independently a peptidic membrane binding element, [0044] n is an
integer of 1 or more and [0045] X is a peptidic or non-peptidic
membrane-binding entity which may be covalently linked to any
W.
[0046] Peptidic membrane binding elements are preferably 8 to 20
amino acids long and elements W are preferably located sequentially
either at the N or C terminus of the soluble polypeptide. The amino
acid sequences are linked to one another and to the soluble peptide
by linker groups which are preferably selected from hydrophilic
and/or flexible aminoacid sequences of 4 to 20 aminoacids; linear
hydrophilic synthetic polymers; and chemical bridging groups.
[0047] Peptide linkages may be made chemically or biosynthetically
by expression of appropriate coding DNA sequences. Non peptide
linkages may be made chemically or enzymatically by
post-translational modification.
[0048] The polypeptide portion of the derivatives of the invention
may be prepared by expression in suitable hosts of modified genes
encoding the soluble polypeptide of interest plus one or more
peptidic membrane binding elements and optional residues such as
cysteine to introduce linking groups to facilitate post
translational derivatisation with additional membrane binding
elements.
[0049] In a further aspect, therefore, the invention provides a
process for preparing a derivative according to the invention which
process comprises expressing DNA encoding the polypeptide portion
of said derivative in a recombinant host cell and recovering the
product and thereafter post translationally modifying the
polypeptide to chemically introduce membrane binding elements.
[0050] In particular, the recombinant aspect of the process may
comprise the steps of:
[0051] i) preparing a replicable expression vector capable, in a
host cell, of expressing a DNA polymer comprising a nucleotide
sequence that encodes said polypeptide portion;
[0052] ii) transforming a host cell with said vector,
[0053] iii) culturing said transformed host cell under conditions
permitting expression of said DNA polymer to produce said
polypeptide; and
[0054] iv) recovering said polypeptide.
[0055] Where the polypeptide portion is novel, the DNA polymer
comprising a nucleotide sequence that encodes the polypeptide
portion as well as the polypeptide portion itself and S-derivatives
thereof, also form part of the invention. In particular the
invention provides a polypeptide portion of a derivative of the
invention comprising the soluble peptide linked by a peptide bond
to one peptidic membrane binding element and/or including a
C-terminal cysteine, and DNA polymers encoding the polypeptide
portion.
[0056] The recombinant process of the invention may be performed by
conventional recombinant techniques such as described in Sambrook a
al., Molecular Cloning: A laboratory manual 2nd Edition. Cold
Spring Harbor Laboratory Press (1989) and DNA Cloning vols I, II
and III (D. M. Glover ed., IRL Press Ltd).
[0057] The invention also provides a process for preparing the DNA
polymer by the condensation of appropriate mono-, di- or oligomeric
nucleotide units.
[0058] The preparation may be carried out chemically,
enzymatically, or by a combination of the two methods, in vitro or
in vivo as appropriate. Thus, the DNA polymer may be prepared by
the enzymatic ligation of appropriate DNA fragments, by
conventional methods such as those described by D. M. Roberts et
al., in Biochemistry 1985, 24, 5090-5098.
[0059] The DNA fragments may be obtained by digestion of DNA
containing the required sequences of nucleotides with appropriate
restriction enzymes, by chemical synthesis, by enzymatic
polymerisation, or by a combination of these methods.
[0060] Digestion with restriction enzymes may be performed in an
appropriate buffer at a temperature of 20.degree.-70.degree. C.,
generally in a volume of 50 .mu.l or less with 0.1-10 .mu.g
DNA.
[0061] Enzymatic polymerisation of DNA may be carried out in vitro
using a DNA polymerase such as DNA polymerase 1 (Klenow fragment)
in an appropriate buffer containing the nucleoside triphosphates
dATP, dCTP, dGTP and dTTP as required at a temperature of
10.degree.-37.degree. C., generally in a volume of 50 .mu.l or
less.
[0062] Enzymatic ligation of DNA fragments may be carried out using
a DNA ligase such as T4 DNA ligase in an appropriate buffer at a
temperature of 4.degree. C. to 37.degree. C., generally in a volume
of 50 .mu.l or less.
[0063] The chemical synthesis of the DNA polymer or fragments may
be carried out by conventional phosphotriester, phosphite or
phosphoramidite chemistry, using solid phase techniques such as
those described in `Chemical and Enzymatic Synthesis of Gene
Fragments--A Laboratory Manual` (ed. H. G. Gassen and A. Lang).
Verlag Chemie, Weinheim (1982), or in other scientific
publications, for example M. J. Gait, H. W. D. Matthes M. Singh, B.
S. Sproat and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243;
B. S. Sproat and W. Bannwarth, Tetrahedron Letters, 1983, 24, 5771;
M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21,
719; M. D. Matteucci and M. H. Caruthers, Journal of the American
Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of
the American Chemical Society, 1983, 105, 661; N. D. Sinha, J.
Biemat, J. McMannus and H. Koester, Nucleic Acids Research, 1984,
12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801.
Preferably an automated DNA synthesiser (for example, Applied
Biosystems 381A Synthesiser) is employed.
[0064] The DNA polymer is preferably prepared by ligating two or
more DNA molecules which together comprise a DNA sequence encoding
the polypeptide.
[0065] The DNA molecules may be obtained by the digestion with
suitable restriction enzymes of vectors carrying the required
coding sequences.
[0066] The precise structure of the DNA molecules and the way in
which they are obtained depends upon the structure of the desired
product. The design of a suitable strategy for the construction of
the DNA molecule coding for the polypeptide is a routine matter for
the skilled worker in the art.
[0067] In particular, consideration may be given to the codon usage
of the particular host cell. The codons may be optimised for high
level expression in E. coli using the principles set out in
Devereux et al., (1984) Nucl. Acid Res., 12, 387.
[0068] The expression of the DNA polymer encoding the polypeptide
in a recombinant host cell may be carried out by means of a
replicable expression vector capable, in the host cell, of
expressing the DNA polymer. Novel expression vectors also form part
of the invention.
[0069] The replicable expression vector may be prepared in
accordance with the invention, by cleaving a vector compatible with
the host cell to provide a linear DNA segment having an intact
replicon, and combining said linear segment with one or more DNA
molecules which, together with said linear segment, encode the
polypeptide, under ligating conditions.
[0070] The ligation of the linear segment and more than one DNA
molecule may be carried out simultaneously or sequentially as
desired.
[0071] Thus, the DNA polymer may be preformed or formed during the
construction of the vector, as desired. The choice of vector will
be determined in part by the host cell, which may be prokaryotic,
such as E. coli, or eukaryotic, such as mouse C127, mouse myeloma,
chinese hamster ovary, fungi e.g. filamentous fungi or unicellular
`yeast` or an insect cell such as Drosophila. The host cell may
also be in a transgenic animal. Suitable vectors include plasmids,
bacteriophages, cosmids and recombinant viruses derived from, for
example, baculoviruses or vaccinia.
[0072] The DNA polymer may be assembled into vectors designed for
isolation of stable transformed mammalian cell lines expressing the
fragment e.g. bovine papillomavirus vectors in mouse C127 cells, or
amplified vectors in chinese hamster ovary cells (DNA Cloning Vol.
II D. M. Glover ed. IRL Press 1985; Kaufman, R. J. et al.,
Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G. N.
and Hamer, D. H. Proceedings of the National Academy of Sciences
(USA) 80, 397-401, 1983; Goeddel, D. V. et al., European Patent
Application No. 0093619, 1983).
[0073] The preparation of the replicable expression vector may be
carried out conventionally with appropriate enzymes for
restriction, polymerisation and ligation of the DNA, by procedures
described in, for example, Sambrook et al., cited above.
Polymerisation and ligation may be performed as described above for
the preparation of the DNA polymer. Digestion with restriction
enzymes may be performed in an appropriate buffer at a temperature
of 20.degree.-70.degree. C., generally in a volume of 50 .mu.l or
less with 0.1-10 .mu.g DNA.
[0074] The recombinant host cell is prepared, in accordance with
the invention, by transforming a host cell with a replicable
expression vector of the invention under transforming conditions.
Suitable transforming conditions are conventional and are described
in, for example, Sambrook e: al., cited above, or "DNA Cloning"
Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
[0075] The choice of transforming conditions is determined by the
host cell. Thus, a bacterial host such as E. coli, may be treated
with a solution of CaCl.sub.2 (Cohen et al., Proc. Nat. Acad. Sci.,
1973, 69, 2110) or with a solution comprising a mixture of RbCl.
MnCl.sub.2, potassium acetate and glycerol, and then with
3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol or by
electroporation as for example described by Bio-Rad Laboratories,
Richmond, Calif., USA, manufacturers of an electroporator.
Mammalian cells in culture may be transformed by calcium
co-precipitation of the vector DNA onto the cells or by using
cationic liposomes.
[0076] The invention also extends to a host cell transformed with a
replicable expression vector of the invention.
[0077] Culturing the transformed host cell under conditions
permitting expression of the DNA polymer is carried out
conventionally, as described in, for example, Sambrook et al., and
"DNA Cloning" cited above. Thus, preferably the cell is supplied
with nutrient and cultured at a temperature below 45.degree. C.
[0078] The protein product is recovered by conventional methods
according to the host cell. Thus, where the host cell is bacterial
such as E. coli and the protein is expressed intracellularly, it
may be lysed physically, chemically or enzymatically and the
protein product isolated from the resulting lysate. Where the host
cell is mammalian, the product is usually isolated from the
nutrient medium.
[0079] Where the host cell is bacterial, such as E. coli, the
product obtained from the culture may require folding for optimum
functional activity. This is most likely if the protein is
expressed as inclusion bodies. There are a number of aspects of the
isolation and folding process that are regarded as important. In
particular, the polypeptide is preferably partially purified before
folding, in order to minimise formation of aggregates with
contaminating proteins and minimise misfolding of the polypeptide.
Thus, the removal of contaminating E. coli proteins by specifically
isolating the inclusion bodies and the subsequent additional
purification prior to folding are important aspects of the
procedure.
[0080] The folding process is carried out in such a way as to
minimise aggregation of intermediate-folded states of the
polypeptide. Thus, careful consideration needs to be given to,
among others, the salt type and concentration, temperature, protein
concentration, redox buffer concentrations and duration of folding.
The exact condition for any given polypeptide generally cannot be
predicted and must be determined by experiment.
[0081] There are numerous methods available for the folding of
proteins from inclusion bodies and these are known to the skilled
worker in this field. The methods generally involve breaking all
the disulphide bonds in the inclusion body, for example with 50 mM
2-mercaptoethanol, in the presence of a high concentration of
denaturant such as 8M urea or 6M guanidine hydrochloride. The next
step is to remove these agents to allow folding of the proteins to
occur. Formation of the disulphide bridges requires an oxidising
environment and this may be provided in a number of ways, for
example by air, or by incorporating a suitable redox system, for
example a mixture of reduced and oxidised glutathione.
[0082] Preferably, the inclusion body is solubilised using 8M urea,
in the presence of mercaptoethanol, and protein is folded, after
initial removal of contaminating proteins, by addition of cold
buffer. Suitable buffers may be identified using the techniques
described in I. Dodd et al, `Perspectives in Protein Engineering
and Complementary Technologies`, Mayflower Publications, 66-69,
1995. A suitable buffer for many of the SCR constructs described
herein is 20 mM ethanolamine containing 1 mM reduced glutathione
and 0.5 mM oxidised glutathione. The folding is preferably carried
out at a temperature in the range 1 to 5.degree. C. over a period
of 1 to 4 days.
[0083] If any precipitation or aggregation is observed, the
aggregated protein can be removed in a number of ways, for example
by centrifugation or by treatment with precipitants such as
ammonium sulphate. Where either of these procedures are adopted,
monomeric polypeptide is the major soluble product.
[0084] If the bacterial cell secretes the protein, folding is not
usually necessary.
[0085] The polypeptide portion of the derivative of the invention
may include a C-terminal cysteine to facilitate post translational
modification. A soluble polypeptide including a C-terminal cysteine
also forms part of the invention. Expression in a bacterial system
is preferred for proteins of moderate size (up to -70 kDa) and with
<.about.8 disulphide bridges. More complex proteins for which a
free terminal Cys could cause refolding or stability problems may
require stable expression in mammalian cell lines (especially CHO).
This will also be needed if a carbohydrate membrane binding element
is to be introduced post-translationally. The use of insect cells
infected with recombinant baculovirus encoding the polypeptide
portion is also a useful general method for preparing more complex
proteins and will be preferred when it is desired to carry out
certain post-translational processes (such as palmitoylation)
biosynthetically (see for example, M. J. Page et al J. Biol. Chem.
264, 19147-19154, 1989)
[0086] A preferred method of handling proteins C-terminally
derivatised with cysteine is as a mixed disulphide with
mercaptoethanol or glutathione or as the 2-nitro, 5-carboxyphenyl
thio-derivative as generally described below in Methods.
[0087] Peptide membrane binding elements may be prepared using
standard solid state synthesis such as the Merrifield method and
this method can be adapted to incorporate required non-peptide
membrane binding elements such as N-acyl groups derived from
myristic or palmitic acids at the N terminus of the peptide. In
addition activation of an amino acid residue for subsequent linkage
to a protein can be achieved during chemical synthesis of such
membrane binding elements. Examples of such activations include
formation of the mixed 2-pyridyl disulphide with a cysteine thiol
or incorporation of an N-haloacetyl group. Both of these groups are
capable of reaction with free thiols, through disulphide
interchange and alkylation, respectively. Peptides can optionally
be prepared as the C-terminal amide and/or with a conventional
N-terminal blocking group such as acetyl.
[0088] The invention also provides a peptidic membrane binding
element comprising one or more derivatisations selected from:
[0089] a terminal cysteine residue optionally activated at the
thiol group; [0090] an N-haloacetyl group (where halo signifies
chlorine, bromine or iodine) located at the N-terminus of the the
peptide or at an .epsilon.-amino group of a lysine residue; [0091]
an amide group at the C-terminus; [0092] an N-terminal blocking
group; and [0093] a fatty acid N-acyl group at the N-terminus or at
an E-amino group of a lysine residue.
[0094] Chemical bridging groups and reagents suitable for their
formation include those described in EP0109653, EP0152736,
EP0155388 and EP0284413, incorporated herein by reference. The
bridging group is generally of the formula:
-A-R-B- (I)
[0095] in which each of A and B, which may be the same or
different, represents --CO--, --C(.dbd.NH.sub.2.sup.+)--,
maleimido, --S-- or a bond and R is a bond or a linking group
containing one or more --(CH.sub.2)-- or meta-, ortho- or
para-disubstituted phenyl units, preferably ortho or para,
optionally together with a hydrophilic portion.
[0096] Where the polypeptide portion of the derivative of the
invention and a peptidic membrane binding element both include a
C-terminal cysteine the chemical bridging group will take the form
--S--S--. The bridge is generated by conventional disulphide
exchange chemistry, by activating a thiol on one polypeptide and
reacting the activated thiol with a free thiol on the other
polypeptide. Such activation procedures make use of disulphides
which form stable thiolase anions upon cleavage of the S--S linkage
and include reagents such as 2,2' dithiopyridine and
5,5'-dithio(2-nitrobenzoic acid, DTNB) which form intermediate,
mixed disulphides capable of further reaction with thiols to give
stable disulphide linkages.
[0097] R may include moieties which interact with water to maintain
the water solubility of the linkage and suitable moieties include
--CO--NH--, --CO--NMe-, --S--S--, --CH(OH)--, --SO.sub.2--,
--CO.sub.2--, --(CH.sub.2CH.sub.2--O).sub.m-- and --CH(COOH)--
where m is an integer of 2 or more, or linear hydrophilic polymers
such as polyethylene glycol, polypropylene glycol, polyglycine,
polyalanine or polysarcosine.
[0098] Other examples of R include --(CH.sub.2).sub.r--,
--(CH.sub.2).sub.p--S--S--(CH.sub.2).sub.q-- and
--(CH.sub.2).sub.p--CH(OH)--CH(OH)--(CH.sub.2).sub.q--, in which r
is an integer of at least 2, preferably at least 4 and p and q are
independently integers of at least 2.
[0099] In a further aspect R may take the form --U--V--W-- where U
is (CH.sub.2).sub.2CONH(CH.sub.2).sub.n in which n is an integer of
3 to 8, V is O, S, NR.sub.a or NR.sub.a--NR.sub.a where each
R.sub.a is H or C.sub.1-6 alkyl, NH--O or O--NH, and W is benzyl
substituted at the 2- or 4-position by the group B. In a preferred
embodiment R is (CH.sub.2).sub.2CONH(CH.sub.2).sub.nNH-(4-phenyl)
where n is an integer of 3 to 8. The bridging group of formula (I)
may be derived from a linking agent of formula (II):
X--R.sub.1--Y (II)
[0100] in which R.sub.1 is a bond or a linking group and X and Y
are functional groups reactable with surface amino acid groups,
preferably a lysine or cysteine group, the N-terminal amino group,
a catalytic serine hydroxyl or a protein attachment group, and X,
R.sub.1-- and Y are chosen so as to generate the required bridging
group -A-R--B--.
[0101] Preferred agents are those where X and Y are different,
known as heterobifunctional agents. Each end of the agent molecule
is reacted in turn with each polypeptide to be linked in separate
reactions. Examples of heterobifunctional agents of formula (II)
include:
[0102] N-succinimidyl 3-(2-pyridyldithio)propionate
[0103] succinimidyl 4-(N-maleimido)caproate
[0104] 3-(2-pyridyl)methyl propionimidate hydrochloride
[0105] 4'-amidinophenyl
4-N-[2-N-(3-[2-pyridyldithio]ethykarbonyl)aminoethyl]aminobenioate
hydrochloride.
[0106] Other suitable agents are disclosed in EP0109653, EP0152736,
EP0155388 and EP0284413, in particular those of formula (II) in
EP0155388 and (III) in EP0284413 incorporated herein by
reference.
[0107] In each case Y is capable of reacting with a thiol group on
a polypeptide, which may be a native thiol or one introduced as a
protein attachment group.
[0108] The protein attachment group is a functionality derived by
modification of a polypeptide or protein with a reagent specific
for one or more amino acid side chains, and which contains a group
capable of reacting with a cleavable section on the other
polypeptide. An example of a protein attachment group is a thiol
group. An example of a cleavable section is a disulphide bond.
Alternatively the cleavable section may comprise an .alpha., .beta.
dihydroxy function.
[0109] As an example, the introduction of a free thiol function by
reaction of a polypeptide with 2-iminothiolane, N-succinimidyl
3-(2-pyridyldithio)propionate (with subsequent reduction) or
N-acetyl homocysteine thiolactone will permit coupling of the
protein attachment group with a thiol-reactive Y structure.
Alternatively the protein attachment group can contain a
thiol-reactive entity such as the 6-maleimidohexyl group or a
2-pyridyl-dithio group which can react with a free thiol in X.
Preferably, the protein attachment group is derived from protein
modifying agents such as 2-iminothiolane that react with lysine
.epsilon.-amino groups in proteins.
[0110] When X represents a group capable of reacting directly with
the amino acid side chain of a protein, it is preferably an
N-succinimidyl group. When X represents a group capable of reacting
with a protein attachment group, it is preferably a pyridylthio
group. When X represents a group capable of reacting with a
catalytic serine hydroxyl it is preferably an 4-amidinophenyl ester
group optionally substituted by one or more electron withdrawing
groups which increases the reactivity of the ester, of the kind
contained in the compounds of formula (II) in EP0155388 and (III)
in EP0284413.
[0111] In the above processes, modification of a polypeptide to
introduce a protein attachment group is preferably carried out in
aqueous buffered media at a pH between 3.0 and 9.0 depending on the
reagent used. For a preferred reagent, 2-iminothiolane, the pH is
preferably 6.5-8.5. The concentration of polypeptide is preferably
high (>10 mg/ml) and the modifying reagent is used in a moderate
(1.1- to 5-fold) molar excess, depending on the reactivity of the
reagent. The temperature and duration of reaction are preferably in
the range 0.degree.-40.degree. C. and 10 minutes to 7 days. The
extent of modification of the polypeptide may be determined by
assaying for attachment groups introduced.
[0112] Such assays may be standard protein chemical techniques such
as titration with 5,5'-dithiobis-(2-nitrobenzoic acid). Preferably,
0.5-3.0 moles of protein attachment group will be introduced on
average per mole of polypeptide. The modified polypeptide may be
separated from excess modifying agents by standard techniques such
as dialysis, ultrafiltration, gel filtration and solvent or salt
precipitation. The intermediate material may be stored in frozen
solution or lyophilised.
[0113] Where the linking agent of formula (II) contains an amidino
phenyl ester group X the agent is preferably first reacted with a
polypeptide enzyme via the group X and the reaction is preferably
carried out under the conditions described for the introduction of
blocking groups in European Published Patent Application No.
0,009,879. Having been freed of excess reagent by suitable
techniques such as high performance size exclusion chromatography
or diafiltration, the acylated enzyme may then be reacted with the
other polypeptide at a ratio of approximately 1:1 in a
non-nucleophilic buffer at pH 7.0-8.0 and 0.degree.-30.degree. C.
for up to 6 h. However, it is preferable to conduct the coupling
below 10.degree. C. (preferably 0.degree.-4.degree. C.) in order to
minimise the hydrolysis of the acylated enzyme.
[0114] Where a protein attachment group is introduced in this way,
the bridging group (I) will be formed from a reaction of the
linking agent an and the protein attachment group.
[0115] The polypeptides to be linked are reacted separately with
the linking agent or the reagent for introducing a protein
attachment group by typically adding an excess of the reagent to
the polypeptide, usually in a neutral or moderately alkaline
buffer, and after reaction removing low molecular weight materials
by gel filtration or dialysis. The precise conditions of pH,
temperature, buffer and reaction time will depend on the nature of
the reagent used and the polypeptide to be modified. The
polypeptide linkage reaction is preferably carried out by mixing
the modified polypeptides in neutral buffer in an equimolar ratio.
Other reaction conditions e.g. time and temperature, should be
chosen to obtain the desired degree of linkage. If thiol exchange
reactions are involved, the reaction should preferably be carried
out under an atmosphere of nitrogen. Preferably, UV-active products
are produced (eg from the release of pyridine 2-thione from
2-pyridyl dithio derivatives) so that coupling can be
monitored.
[0116] After the linkage reaction, the polypeptide conjugate can be
isolated by a number of chromatographic procedures such as gel
filtration, ion-exchange chromatography, affinity chromatography or
hydrophobic interaction chromatography. These procedures my be
either low pressure or high performance variants.
[0117] The conjugate may be characterised by a number of techniques
including low pressure or high performance gel filtration, SDS
polyacrylamide gel electrophoresis or isoelectric focussing.
[0118] Membrane binding elements which are fatty acid derivatives
are attached post translationally to a peptidic membrane binding
element, preferably at the terminus of the polypetide chain.
Preferably, where the recombinant polypeptide portion of the
derivative of the invention contains the peptidic membrane binding
element, it has a unique cysteine for coupling to the fatty acid
derivative. Where the recombinant polypeptide has a cysteine
residue, a thiol-derivative of the fatty acid is added to the
refolded recombinant protein at a late stage in purification (but
not necessarily the final stage) and at a reagent concentration
preferably below the critical micelle concentration. One of the
fatty acid derivative and the recombinant peptide will have the
thiol group activated as described above for thiol interchange
reactions. The fatty acid derivative is preferably a C.sub.10-20
fatty acyl derivative of an aminoC.sub.2-6alkane thiol (optionally
C-substituted) such as N-(2-myristoyl)aminoethanethiol or
N-myristoyl L-cysteine and forms part of the invention.
[0119] Suitable examples of hydrophilic synthetic polymers include
polyethyleneglycol (PEG), preferably .alpha.,.omega. functionalised
derivatives, more preferably .alpha.-amino, .omega.-carboxy-PEG of
molecular weight between 400 and 5000 daltons which are linked to
the polypeptide for example by solid-phase synthesis methods (amino
group derivatisation) or by thiol-interchange chemistry.
[0120] Membrane binding elements derived from ligands of known
integral membrane proteins, either amino acid sequences or
carbohydrates may be generated by post-translational modification
using the glycosylation pathways of eukaryotic cells targeted to
N-linked glycosylation sites in the peptide sequence.
[0121] Convenient generic final stage purification strategies are
hydrophobic interaction chromatography (HIC) on C2-C8 media and
cation exchange chromatography for separation of derivatised and
underivatised proteins into which a hydrophobic-electrostatic
switch combination has been inserted.
[0122] The derivatives of this invention are preferably
administered as pharmaceutical compositions.
[0123] Accordingly, the present invention also provides a
pharmaceutical composition comprising a derivative of the invention
in combination with a pharmaceutically acceptable carrier.
[0124] The compositions according to the invention may be
formulated in accordance with routine procedures for administration
by any route, such as oral, topical, parenteral, sublingual or
transdermal or by inhalation. The compositions may be in the form
of tablets, capsules, powders, granules, lozenges, creams or liquid
preparations, such as oral or sterile parenteral solutions or
suspensions or in the form of a spray, aerosol or other
conventional method for inhalation.
[0125] The topical formulations of the present invention may be
presented as, for instance, ointments, creams or lotions, eye
ointments and eye or ear drops, impregnated dressings and aerosols,
and may contain appropriate conventional additives such as
preservatives, solvents to assist drug penetration and emollients
in ointments and creams.
[0126] The formulations may also contain compatible conventional
carriers, such as cream or ointment bases and ethanol or oleyl
alcohol for lotions. Such carriers may be present as from about 1%
up to about 98% of the formulation. More usually they will form up
to about 80% of the formulation.
[0127] Tablets and capsules for oral administration may be in unit
dose presentation form, and may contain conventional excipients
such as binding agents, for example syrup, acacia, gelatin,
sorbitol, tragacanth, or polyvinylpyrollidone; fillers, for example
lactose, sugar, maize-starch, calcium phosphate, sorbitol or
glycine; tabletting lubricants, for example magnesium stearate,
talc, polyethylene glycol or silica; disintegrants, for example
potato starch; or acceptable wetting agents such as sodium lauryl
sulphate. Tablets may also contain agents for the stablisation of
polypeptide drugs against proteolysis and absorbtion-enhancing
agents for macromolecules. The tablets may be coated according to
methods well known in normal pharmaceutical practice.
[0128] Suppositories will contain conventional suppository bases,
e.g. cocoa-butter or other glyceride.
[0129] For parenteral administration, fluid unit dosage forms are
prepared utilizing the compound and a sterile vehicle, water being
preferred. The compound, depending on the vehicle and concentration
used is dissolved in the vehicle. In preparing solutions the
compound can be dissolved in water for injection and filter
sterilised before filling into a suitable vial or ampoule and
sealing.
[0130] Parenteral formulations may include sustained-release
systems such as encapsulation within microspheres of biodegradable
polymers such as poly-lactic co-glycolic acid.
[0131] Advantageously, agents such as a local anaesthetic,
preservative and buffering agents can be dissolved in the vehicle.
To enhance the stability, the composition can be frozen after
filling into the vial and the water removed under vacuum. The dry
lyophilized powder is then sealed in the vial and an accompanying
vial of water for injection may be supplied to reconstitute the
liquid prior to use. Advantageously, a surfactant or wetting agent
is included in the composition to facilitate uniform distribution
of the compound.
[0132] Compositions of this invention may also suitably be
presented for administration to the respiratory tract as a snuff or
an aerosol or solution for a nebulizer, or as a microfine powder
for insufflation, alone or in combination with an inert carrier
such as lactose. In such a case the particles of active compound
suitably have diameters of less than 50 microns, preferably less
than 10 microns for example diameters in the range of 1-50 microns,
1-10 microns or 1-5 microns. Where appropriate, small amounts of
anti-asthmatics and bronchodilators, for example sympathomimetic
amines such as isoprenaline, isoetharine, salbutamol, phenylephrine
and ephedrine; xanthine derivatives such as theophylline and
aminophylline and corticosteroids such as prednisolone and adrenal
stimulants such as ACTH may be included.
[0133] Microfine powder formulations may suitably be administered
in an aerosol as a metered dose or by means of a suitable
breath-activated device.
[0134] Suitable metered dose aerosol formulations comprise
conventional propellants, cosolvents, such as ethanol, surfactants
such as oleyl alcohol, lubricants such as oleyl alcohol, desiccants
such as calcium sulphate and density modifiers such as sodium
chloride.
[0135] Suitable solutions for a nebulizer are isotonic sterilised
solutions, optionally buffered, at for example between pH 4-7,
containing up to 20 mg ml-.sup.1 of compound but more generally 0.1
to 10 mg ml-.sup.1, for use with standard nebulisation
equipment.
[0136] The quantity of material administered will depend upon the
potency of the derivative and the nature of the complaint be
decided according to the circumstances by the physician supervising
treatment. However, in general, an effective amount of the
polypeptide for the treatment of a disease or disorder is in the
dose range of 0.01-100 mg/kg per day, preferably 0.1 mg-10 mg/kg
per day, administered in up to five doses or by infusion.
[0137] No adverse toxicological effects are indicated with the
compounds of the invention within the above described dosage
range.
[0138] The invention also provides a derivative of the invention
for use as a medicament.
[0139] The invention further provides a method of treatment of
disorders amenable to treatment by a soluble peptide which
comprises administering a soluble derivative of said soluble
peptide according to the invention, and the use of a derivative of
the invention for the preparation of a medicament for treatment of
such disorders.
[0140] In one preferred aspect the present invention relates to
derivatives for use in the therapy of disorders involving
complement activity and various inflammatory and immune
disorders.
[0141] In this preferred aspect the soluble polypeptide which is
derivatised in accordance with the invention is a soluble
complement inhibitor such as a soluble CR1 polypeptide
fragment.
[0142] Constituting about 10% of the globulins in normal serum, the
complement system is composed of many different proteins that are
important in the immune system's response to foreign antigens. The
complement system becomes activated when its primary components are
cleaved and the products alone or with other proteins, activate
additional complement proteins resulting in a proteolytic cascade.
Activation of the complement system leads to a variety of responses
including increased vascular permeability, chemotaxis of phagocytic
cells, activation of inflammatory cells, opsonization of foreign
particles, direct killing of cells and tissue damage. Activation of
the complement system may be triggered by antigen-antibody
complexes (the classical pathway) or, for example, by
lipopolysaccharides present in cell walls of pathogenic bacteria
(the alternative pathway).
[0143] Complement receptor type 1 (CR1) has been shown to be
present on the membranes of erythrocytes, monocytes/macrophages,
granulocytes. B cells, some T cells, splenic follicular dendritic
cells, and glomerular podocytes. CR1 binds to the complement
components C3b and C4b and has also been referred to as the C3b/C4b
receptor. The structural organisation and primary sequence of one
allotype of CR1 is known (Klickstein et al., 1987, J. Exp. Med.
165:1095-1112, Klickstein et al., 1988, J. Exp. Med. 168:1699-1717;
Hourcade et al., 1988, J. Exp. Med. 168:1255-1270, WO 89/09220, WO
91/05047). It is composed of 30 short consensus repeats (SCRs) that
each contain around 60-70 amino acids. In each SCR, around 29 of
the average 65 amino acids are conserved. Each SCR has been
proposed to form a three dimensional triple loop structure through
disulphide linkages with the third and first and the fourth and
second half-cystines in disulphide bonds. CR1 is further arranged
as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a
leader sequence, the CR1 molecule consists of the N-terminal LHR-A,
the next two repeats, LHR-B and LHR-C, and the most C-terminal
LHR-D followed by 2 additional SCRs, a 25 residue putative
transmembrane region and a 43 residue cytoplasmic tail.
[0144] Based on the mature CR1 molecule having a predicted
N-terminal glutamine residue, hereinafter designated as residue 1,
the first four SCR domains of LHR-A are defined herein as
consisting of residues 2-58, 63-120, 125-191 and 197-252,
respectively, of mature CR1.
[0145] Several soluble fragments of CR1 have been generated via
recombinant DNA procedures by eliminating the transmembrane region
from the DNAs being expressed (WO 89/09220, WO 91/05047). The
soluble CR1 fragments were functionally active, bound C3b and/or
C4b and demonstrated Factor I cofactor activity depending upon the
regions they contained. Such constructs inhibited in vitro
complement-related functions such as neutrophil oxidative burst,
complement mediated hem olysis, and C3a and C5a production. A
particular soluble construct, sCR1/pBSCR1c, also demonstrated in
vivo activity in a reversed passive Arthus reaction (WO 89/09220,
WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250), suppressed
post-ischemic myocardial inflammation and necrosis (WO 89/09220, WO
91/05047; Weisman et al., Science. 1990, 249:146-1511; Dupe, R. et
al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extended
survival rates following transplantation (Pruitt & Bollinger,
1991, J. Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52;
868). Furthermore, co-formulation of sCR1/pBSCR1c with
p-anisoylated human plasminogen-streptokinase-activator complex
(APSAC) resulted in similar anti-haemolytic activity as sCR1 alone,
indicating that the combination of the complement inhibitor sCR1
with a thrombolytic agent was feasible (WO 91/05047).
[0146] The soluble CR1 polypeptide fragment encoded by
sCR1/pBSCR1c, residues 1-1929 of CR1, may be derivatised in
accordance with the invention.
[0147] Soluble polypeptides corresponding to part of CR1 have been
found to possess functional complement inhibitory, including
anti-haemolytic, activity. WO94/00571 discloses soluble
polypeptides comprising, in sequence, one to four short consensus
repeats (SCR) selected from SCR 1, 2, 3 and 4 of long homologous
repeat A (LHR-A) as the only structurally and functionally intact
SCR domains of CR1 and including at least SCR3.
[0148] In preferred aspects, the polypeptide comprises, in
sequence, SCR 1, 2, 3 and 4 of LHR-A or SCR 1, 2 and 3 of LHR-A as
the only structurally and functionally intact SCR domains of
CR1.
[0149] In one aspect, the polypeptides may be represented
symbolically as follows:
NH.sub.2--V.sup.1--SCR1--W.sup.1--SCR2--X.sup.1--SCR3--Y.sup.1--OH
(A)
[0150] in which SCR1 represents residues 2-58 of mature CR1, SCR2
represents residues 63-120 of mature CR1, SCR3 represents residues
125-191 of mature CR1, and V.sup.1, W.sup.1, X.sup.1 and Y.sup.1
represent bonds or short linking sequences of amino acids,
preferably 1 to 5 residues in length and which are preferably
derived from native interdomain sequences in CR1.
[0151] In a preferred embodiment of formula (I), W.sup.1, X.sup.1
and Y.sup.1 represent residues 59-62, 121-124 and 192-196,
respectively, of mature CR1 and V.sup.1 represents residue 1 of
mature CR1 optionally linked via its N-terminus to methionine.
[0152] In another aspect the polypeptides may be represented
symbolically as follows:
NH.sub.2--V.sup.2--SCR1--W.sup.2--SCR2--X.sup.2--SCR3--Y.sup.2--SCR4--Z.-
sup.2OH (B)
[0153] in which SCR1, SCR2 and SCR3 are as hereinbefore defined,
SCR4 represents residues 197-252 of mature CR1 and V.sup.2,
W.sup.2, X.sup.2, Y.sup.2 and Z.sup.2 represents bonds or short
linking sequences of amino acids, preferably 1 to 5 residues in
length and which are preferably derived from native interdomain
sequences in CR1.
[0154] In preferred embodiments of formula (U), W.sup.2, X.sup.2,
Y.sup.2 and Z.sup.2 represent residues 59-62, 121-124, 192-196, and
residues 253 respectively, of mature CR1 and V.sup.2 represents
residue 1 of mature CR1 optionally linked via its N-terminus to
methionine.
[0155] In one particular embodiment of formula (B) arginine 235 is
replaced by histidine.
[0156] In the preferred embodiment of formula (B), residue 235 is
arginine.
[0157] In one further aspect, the polypeptide may be represented
symbolically as follows:
NH.sub.2--X.sup.3--SCR3--Y.sup.3--OH (C)
[0158] in which SCR3 is as hereinbefore defined and X.sup.3 and
Y.sup.3 represent bonds or short linking sequences of amino acids,
preferably 1 to 5 residues in length and which are preferably
derived from native interdomain sequences in CR1.
[0159] In a preferred embodiment of formula (C) X.sup.3 represents
amino acids 122-124 of mature CR1 optionally linked to methionine
at its N-terminus and Y.sup.4 represents amino acids 192-196 of
mature CR1.
[0160] In another further aspect, the polypeptide may be
represented symbolically as follows:
NH.sub.2--X.sup.4--SCR3--Y.sup.4--SCR4--Z.sup.4--OH (D)
[0161] in which SCR3 and SCR4 are as hereinbefore defined and
X.sup.4, Y.sup.4 and Z.sup.4 represent bonds or short linking
sequences of amino acids, preferably 1 to 5 residues in length and
which are preferably derived from native interdomain sequences in
CR1.
[0162] In a preferred embodiment of formula (D) X.sup.4 represents
amino acids 122-124 of mature CR1 optionally linked to methionine
at its N-terminus and Y.sup.4 and Z.sup.4 represent amino acids
192-196 and 253 respectively of mature CR1.
[0163] The soluble CR1 polypeptide is derivatised in accordance
with the invention by any convenient strategy from those outlined
above. In a preferred embodiment the soluble CR1 polypeptide
consists of residues 1-196 of CR1 and with an N-terminal methionine
and the derivative comprises a myristoyl group and one or more
polypeptides sequence selected from
TABLE-US-00004 DGPKKKKKKSPSKSSGC GSSKSPSKKKKKKPGDC
CDGPKKKKKKSPSKSSK SKDGKKKKKKSKTKC CSAAPSSGFRILLLKV
AAPSVIGFRILLLKVAGC and DGPSEILRGDFSSC (N-terminus on left).
[0164] The soluble complement inhibitor, such as a soluble CR1
polypeptide, derivative of this invention is useful in the
treatment of many complement-mediated or complement-related
diseases and disorders including, but not limited to, those listed
below.
[0165] Disease and Disorders Involving Complement
[0166] Neurological Disorders
[0167] multiple sclerosis
[0168] stroke
[0169] Guillain Barre Syndrome
[0170] traumatic brain injury
[0171] Parkinson's disease
[0172] allergic encephalitis
[0173] Alzheimer's disease
[0174] Disorders of Inappropriate or Undesirable Complement
Activation
[0175] haemodialysis complications
[0176] hyperacute allograft rejection
[0177] xenograft rejection
[0178] corneal graft rejection
[0179] interleukin-2 induced toxicity during IL-2 therapy
[0180] paroxysmal nocturnal haemoglobinuria
[0181] Inflammatory Disorders
[0182] inflammation of autoimmune diseases
[0183] Crohn's Disease
[0184] adult respiratory distress syndrome
[0185] thermal injury including burns or frostbite
[0186] uveitis
[0187] psoriasis
[0188] asthma
[0189] acute pancreatitis
[0190] Post-Ischemic Reperfusion Conditions
[0191] myocardial infarction
[0192] balloon angioplasty
[0193] atherosclerosis (cholesterol-induced) & restenosis
[0194] hypertension
[0195] post-pump syndrome in cardiopulmonary bypass or renal
haemodialysis
[0196] renal ischemia
[0197] intestinal ischaemia
[0198] Infectious Diseases or Sepsis
[0199] multiple organ failure
[0200] septic shock
[0201] Immune Complex Disorders and Autoimmune Diseases
[0202] rheumatoid arthritis
[0203] systemic lupus erythematosus (SLE)
[0204] SLE nephritis
[0205] proliferative nephritis
[0206] glomerulonephritis
[0207] haemolytic anemia
[0208] myasthenia gravis
[0209] Reproductive Disorders
[0210] antibody- or complement-mediated infertility
[0211] Wound Healing
[0212] The present invention is also directed to a pharmaceutical
composition for treating a disease or disorder associated with
inflammation or inappropriate complement activation comprising a
therapeutically effective amount of a soluble complement inhibitor,
such as a soluble CR1 polypeptide, derivative of this invention,
and a pharmaceutically acceptable carrier or excipient.
[0213] The present invention also provides a method of treating a
disease or disorder associated with inflammation or inappropriate
complement activation comprising administering to a subject in need
of such treatment a therapeutically effective amount of a soluble
complement inhibitor, such as a soluble CR1 polypeptide, derivative
of this invention.
[0214] In the above methods, the subject is preferably a human.
[0215] Further provided is the use of a soluble complement
inhibitor, such as a soluble CR1 polypeptide, derivative of this
invention in the manufacture of a medicament for the treatment of a
disease or disorder associated with inflammation or inappropriate
complement activation.
[0216] In order to inhibit complement activation and, at the same
time; provide thrombolytic therapy, the present invention provides
compositions which further comprise a therapeutically active amount
of a thrombolytic agent. An effective amount of a thrombolytic
agent is in the dose range of 0.01-10 mg/kg; preferably 0.1-5
mg/kg. Preferred thrombolytic agents include, but are not limited
to, streptokinase, human tissue type plasminogen activator and
urokinase molecules and derivatives, fragments or conjugates
thereof. The thrombolytic agents may comprise one or more chains
that may be fused or reversibly linked to other agents to form
hybrid molecules (EP-A-0297882 and EP 155387), such as, for
example, urokinase linked to plasmin (EP-A-0152736), a fibrinolytic
enzyme linked to a water-soluble polymer (EP-A-0183503). The
thrombolytic agents may also comprise muteins of plasminogen
activators (EP-A-0207589). In a preferred embodiment, the
thrombolytic agent may comprise a reversibly blocked in vitro
fibrinolytic enzyme as described in U.S. Pat. No. 4,285,932. A most
preferred enzyme is the p-anisoyl plasminogen-streptokinase
activator complex, anistreplase as described in U.S. Pat. No.
4,808,405 (Monk et al., 1987, Drugs 34:25-49).
[0217] Routes of administration for the individual or combined
therapeutic compositions of the present invention include standard
routes, such as, for example, intravenous infusion or bolus
injection. Active complement inhibitors and thrombolytic agents may
be administered together or sequentially, in any order.
[0218] The present invention also provides a method for treating a
thrombotic condition, in particular acute myocardial infarction, in
a human or non-human animal. This method comprises administering to
a human or animal in need of this treatment an effective amount of
a soluble complement inhibitor, such as a soluble CR1 polypeptide,
derivative according to this invention and an effective amount of a
thrombolytic agent.
[0219] Also provided is the use of a soluble complement inhibitor,
such as a soluble CR1 polypeptide, derivative of this invention and
a thrombolytic agent in the manufacture of a medicament for the
treatment of a thrombotic condition in a human or animal. Such
methods and uses may be carried out as described in WO
91/05047.
[0220] This invention further provides a method for treating adult
respiratory distress syndrome (ARDS) in a human or non-human
animal. This method comprises administering to the patient an
effective amount of a soluble complement inhibitor, such as a
soluble CR1 polypeptide, derivative according to this
invention.
[0221] The invention also provides a method of delaying hyperacute
allograft or hyperacute xenograft rejection in a human or non-human
animal which receives a transplant by administering an effective
amount of a soluble complement inhibitor, such as a soluble CR1
polypeptide, derivative according to this invention. Such
administration may be to the patient or by application to the
transplant prior to implantation.
[0222] The invention yet further provides a method of treating
wounds in a human or non-human animal by administering by either
topical or parenteral e.g. intravenous routes, an effective amount
of a soluble complement inhibitor, such as a soluble CR1
polypeptide derivative according to this invention.
[0223] In another preferred aspect the soluble polypeptide is a
thrombolytic agent such as prourokinase, streptokinase, tissue-type
plasminogen activator or reteplase and the derivative of the
invention is useful in the treatment of thrombotic disorders such
as acute myocardial infarction. The invention thus provides a
pharmaceutical composition for treating thrombotic disorders
comprising a therapeutically effective amount of a derivative of a
thrombolytic agent according to the invention, and a
pharmaceutically acceptable carrier or excipient. The invention
further provides a method of treatment of thrombotic disorders by
administering an effective amount of a derivative of a thrombolytic
agent according to the invention, and the use of such derivative in
the preparation of a medicament for the treatment of thrombotic
disorders.
[0224] The following Methods and Examples illustrate the
invention.
GENERAL METHODS USED IN EXAMPLES
(i) DNA Cleavage
[0225] Cleavage of DNA by restriction endonucleases was carried out
according to the manufacturer's instructions using supplied
buffers. Double digests were carried out simultaneously if the
buffer conditions were suitable for both enzymes. Otherwise double
digests were carried out sequentially where the enzyme requiring
the lowest salt condition was added first to the digest. Once the
digest was complete the salt concentration was altered and the
second enzyme added.
(ii) DNA Ligation
[0226] Ligations were carried out using T4 DNA ligase purchased
from Promega, as described in Sambrook et al, (1989) Molecular
Cloning: A Laboratory Manual 2nd Edition. Cold Spring Harbour
Laboratory Press.
(iii) Plasmid Isolation
[0227] Plasmid isolation was carried out by the alkaline lysis
method described in Sambrook et al, (1989) Molecular Cloning: A
Laboratory Manual 2nd Edition. Cold Spring Harbour Laboratory Press
or by one of two commercially available kits: the Promega
Wizard.TM. Plus Minipreps or Qiagen Plasmid Maxi kit according to
the manufacturer's instructions.
(iv) DNA Fragment Isolation
[0228] DNA fragments were excised from agarose gels and DNA
extracted using one of three commercially available kits: the QIAEX
gel extraction kit or Qiaquick gel extraction kit (QIAGEN Inc.,
USA), or GeneClean (Bio 101 Inc, USA) according to the
manufacturer's instructions.
(v) Introduction of DNA into E. Coli
[0229] Plasmids were transformed into E. coli BL21(DE3) (Studier
and Moffat, (1986), J. Mol. Biol 189:113), E. coli BL21 (DE3)
pLys-S or BLR (DE3) pLys-S that had been made competent using
calcium chloride as described in Sambrook et at, (1989). Cell lines
were purchased as frozen competent cultures from Stratagene. E.
coli JM109 was purchased as a frozen competent culture from
Promega.
(vi) DNA Sequencing
[0230] Plasmid DNA was sequenced on a Vistra DNA Labstation 625.
The sequencing chemistry was performed using Amersham
International's Thermo Sequenase fluorescent dye-terminator cycle
sequencing kit' (RPN 2435), in conjunction with their `FMP
fluorescent dye-terminator precipitation kit` (RPN 2433) according
to the manufacturer's instructions.
[0231] The sequences produced by the above procedure were analysed
by a Perkin Elmer ABI Prism 377 DNA Sequencer. This is an
electrophoretic technique using 36 cm.times.0.2 mm 4% acrylamide
gels, the fluorescentiy labeled DNA fragments being detected by a
charge coupled device camera according to the manufacturer's
instructions.
(vii) Production of Oligonucleotides
[0232] Oligonucleotides were purchased from Cruachem.
(viii) pBROC413
[0233] The plasmid pT7-7 [Tabor, S (1990), Current Protocols in
Molecular Biology, F. A. Ausubel, Brent, R. E. Kingston, D. D.
Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds.] pp.
16.2.1-16.2.11, Greene Publishing and Wiley-Interscience, New
York.] contains DNA corresponding to nucleotides 2065-4362 of
pBR322 and like pBR322 can be mobilized by a conjugative plasmid in
the presence of a third plasmid Co1K. A mobility protein encoded by
Co1K acts on the nic site at nucleotide 2254 of pBR322 initiating
mobilization from this point. pT7-7 was digested with LspI and
BglII and the protruding 5' ends filled in with the Kienow fragment
of DNA PolymeraseI. The plasmid DNA fragment was purified by
agarose gel electrophoresis, the blunt ends ligated together and
transformed into E. coli DH1 by electroporation using a Bio-Rad
Gene Pulser and following the manufacturers recommended conditions.
The resultant plasmid pBROC413 was identified by restriction enzyme
analysis of plasmid DNA.
[0234] The deletion in pBROC413 from the LspI site immediately
upstream of the f 10 promoter to the BglII site at nucleotide 434
of pT7-7 deletes the DNA corresponding to nucleotides 2065-2297 of
pBR322. The nic site and adjacent sequences are therefore deleted
making pBROC413 non mobilizable.
(ix) Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
(SDS PAGE)
[0235] SDS PAGE was carried out generally using the Novex system
(British Biotechnology) according to the manufacturer's
instructions. Prepacked gels of 4-20% acrylamide were used. Samples
for electrophoresis, including protein molecular weight standards
(for example LMW Kit, Pharmacia or Novex Mark 12) were usually
diluted in 1% (w/v) SDS--containing buffer (with or without 5%
(v/v) 2-mercaptoethanol), and left at room temperature for about 10
to 30 min before application to the gel.
(x) Reduction of Disulphides and Modification of Thiols in
Proteins
[0236] There are a number of methods used for achieving the title
goals. The reason it may be necessary to carry out selective
reduction of disulphides is that during the isolation and
purification of multi-thiol proteins, in particular during
refolding of fully denatured multi-thiol proteins, inappropriate
disulphide pairing can occur. In addition, even if correct
disulphide paring does occur, it is possible that a free cysteine
in the protein may become blocked, for example with glutathione.
These derivatives are generally quite stable. In order to make them
more reactive, for example for subsequent conjugation to another
functional group, they need to be selectively reduced, with for
example dithiothreitol (DTT) or Tris (2-carboxyethyl) phosphine.HCl
(TCEP) then optionally modified with a function which is moderately
unstable. An example of the latter is Ellman reagent (DTNB) which
gives a mixed disulphide. In the case where treatment with DTNB is
omitted, careful attention to experimental design is necessary to
ensure that dimerisation of the free thiol-containing protein is
minimised. Reference to the term `selectively reduced` above means
that reaction conditions eg. duration, temperature, molar ratios of
reactants have to be carefully controlled so that reduction of
disulphide bridges within the natural architecture of the protein
is minimised. All the reagents are commercially available eg. from
Sigma or Pierce.
[0237] The following general examples illustrate the type of
conditions that may be used and that are useful for the generation
of free thiols and their optional modification. The specific
reaction conditions to achieve optimal thiol reduction and/or
modification are ideally determined for each protein batch.
[0238] TCEP may be prepared as a 20 mM solution in 50 mM Hepes
(approx. pH 4.5) and may be stored at -40 degrees C. DTT may be
prepared at 10 mM in sodium phosphate pH 7.0 and may be stored at
-40 degrees C. DTNB may be prepared at 10 mM in sodium phosphate pH
7.0 and may be stored at -40 degrees C. All of the above reagents
are typically used at molar equivalence or molar excess over
protein concentration, the precise concentrations ideally
identified experimentally. The duration and the temperature of the
reaction are similarly determined experimentally. Generally the
duration would be in the range 1 to 24 hours and the temperature
would be in the range 2 to 30 degrees C. Excess reagent may be
conveniently removed by buffer exchange, for example using Sephadex
G25 or Sephadex G50. A suitable buffer is 0.1M sodium phosphate
pH7.0 or the solution may be left untreated.
EXAMPLES
Example 1
Preparation of N-(Myristoyl) 2-aminoethane thiol (MAET)
[0239] Myristoyl chloride (1.0 mmol)was added with vigorous
stirring to ice-cooled dry pyridine (1.0 ml), and followed
immediately by N-hydroxysuccinimide (1.5 mmol). The mixture was
stirred for 4 h at ambient temperature (-23.degree. C.).
2-aminoethanethiol free base (1.1 mmol) was added as solid to the
mixture and allowed to react for 6 h at ambient temperature,
followed by 3 days at 4.degree. C. The product was treated with
water (5 ml), stirred for 1 h at ambient and filtered, washing with
cold water. The white solid was dissolved in dimethylsulphoxide and
reprecipitated with water and then vacuum dried over phosphorous
pentoxide. The final yield was 0.21 g (-70%). Thiol titration using
Ellman's reagent indicated that the product contained -45% free
thiol.
Example 2
Synthesis of Myristoyl/Electrostatic Switch Peptide Reagent 1
(MSWP-1) (SEQ ID NO: 27)
TABLE-US-00005 [0240]
N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-
Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-Thiopyridyl)Cys- NH.sub.2
[0241] The peptide:
TABLE-US-00006 (SEQ ID NO: 5)
Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-
Lys-Pro-Gly-Asp-Cys-NH.sub.2
was prepared using solid phase synthesis via the general Fmoc/tBu
strategy developed by Sheppard and Atherton (E. Atherton and R. C.
Sheppard, Solid Phase Synthesis, IRL Press, Oxford, 1989).
Kieselguhr-supported polydimethylacrylamide resin (Macrosorb 100)
was used as the solid support and was derivatised with ethylene
diamine.
[0242] Coupling reactions were carried out using N-.alpha.-Fmoc
protected reagents pre-activated with
N,N'-diisopropylcarbodiimide/N-hydroxybenzotriazole (in 4-fold
molar excess) with bromophenol blue monitoring. Fmoc cleavages used
20% piperidine in DMF. Reactions to assemble the peptide chain were
carried out by repeated cycles of coupling and deprotection
including the attachment of the modified Rink linkage reagent
(p-[R,S)-.alpha.-[1-(9H-fluoreny-9-yl-methoxyformamido]2,4
dimethoxybenzyl]-phenoxyacetic acid) designed to yield a C-terminal
amide on final cleavage. The side chain functionalities of the
individual amino-acids were protected as follows:
TABLE-US-00007 Ser (tButyl), Lys (Boc), Asp (O-tButyl), Cys
(Trityl).
[0243] On completion of the peptide assembly and with the peptide
still attached to the resin, the myristoyl group was attached to
the amino group of the N terminal glycine by direct coupling of
myristic acid by the same activation procedure. This modified
peptide was then cleaved from the resin and the side-chain
protecting groups removed at the same time by treatment with
trifluoracetic acid containing 2.5% water and 2.5% triisopropyl
silane.
[0244] The crude product was treated with 2,2' dithiopyridine in
0.01M ammonium acetate solution at pH 8-9 for approx. 2 h, then
acidified with acetic acid and purified by preparative high
performance liquid chromatography (HPLC) in 0.1% trifluoracetic
acid (TFA)/water and 0.1% TFA/acetonitiile as gradient component.
After lyophilisation, the peptide was a white amorphous powder,
soluble to at least 10 mg/ml in dimethylsulphoxide. Fast atom
bombardment mass spectrometry gave main peaks at m/e 2107.8, 2129.7
and 2145.8, corresponding to the monoprotonated, monosodiated and
monopotassiated molecular ions of the peptide. The 2-thiopyridyl
content of the peptide was measured by dissolving it to around 0.03
mM to 0.2 mM in 0.1M Sodium Borate pH 8.0 and reducing by addition
of dithiothreitol to 5 mM. The change in optical density at 343 nm
was used to calculate the amount of pyridine 2-thione released
using an extinction coefficient at this wavelength of 8080
cm.sup.-1 M.sup.-1. This indicated that the peptide content was
approximately 60% of the dry weight.
Example 3
Synthesis of Myristoyl/Electrostatic Switch Peptide Reagent 2
(MSWP-2) (SEQ ID NO: 28)
TABLE-US-00008 [0245] N-acetyl-Cys (2-thiopyridyl)
Asp-Gly-Pro-Lys-Lys- Lys-Lys-Lys-Lys-Ser-Pro-Ser
Lys-Ser-Ser-Lys-(.epsilon.-N- (Myristoyl))-NH.sub.2
[0246] The peptide:
TABLE-US-00009 (SEQ ID NO: 18)
Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Pro-
Ser-Lys-Ser-Ser-Lys-NH.sub.2
was prepared by solid-phase synthesis using the general method
described in Example 2 and with the following variations:
[0247] a. The C-terminal lysine was protected by alkylation with
the 4-methyl trityl (MTT) group; all other lysines were N-e
protected with the t-Boc group
[0248] b. MTT was removed with 1% v/v trifluoracetic acid in
dichloromethane and the resulting unique free amino group
derivatised with myristic acid prior to deprotection of the other
lysines (as described in Example 2)
[0249] The N-terminus was acetylated with acetic anhydride upon
completion of the peptide chain assembly. Generation of the
2-pyridyldithiocysteine moiety was by reation of the deprotected
peptide with 2,2'-dithiopyridine as described above. The product
was purified as described in Example 2. Fast-atom bombardment mass
spectrometry gave a molecular ion peak at 2221.3 (cf 2220.3 for the
monoprotonated theoretical mass).
Amino-Acid Analysis:
TABLE-US-00010 [0250] Asx Ser Gly Pro Theory: 1.0 4.0 1.0 2.0 Found
0.97 3.53 1.15 1.88 (Asx = Asn or Asp)
Amino-acid analysis indicated a net peptide content by weight of
68.7%. The 2-pyridyl disulphide content was approximately 60% by
weight using the method of Example 2.
Example 4
Synthesis of Myristoyl/Electrostatic Switch Peptide Reagent 3
(MSWP-3) (SEQ ID NO: 29)
TABLE-US-00011 [0251]
N-(Myristoyl)-Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-
Lys-Ser-Lys-Thr-Lys-(S-2-Thiopyridyl)Cys-NH.sub.2
[0252] The peptide:
TABLE-US-00012 (SEQ ID NO: 19)
Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys- Thr-Lys-Cys
was prepared using the general solid-phase synthesis protocol of
Example 2. Myristoylation, C-terminal amidadon and derivatisation
of the Cys residue were performed as described in Example 2. After
purification, mass spectrometry gave the major peak at 2040.5,
corresponding to a monoprotonated form (Theory: 2039.5)
Amino-Acid Analysis:
TABLE-US-00013 [0253] Asx Ser Gly Thr Lys Theory: 1 2 1 1 9 Found:
1.02 2.04 1.14 1.06 8.85
The peptide content was about 56% by weight
Example 5
Synthesis of T-Cell Targeting Peptide Reagent 1 (TCTP-1) (SEQ ID
NO: 30)
TABLE-US-00014 [0254] N-acetyl-(2-thiopyridyl)Cys
Ser-Ala-Ala-Pro-Ser- Ser-Gly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val-
CONH(CH.sub.2).sub.9CH.sub.3
The peptide
TABLE-US-00015 (SEQ ID NO: 20)
Cys-Ser-Ala-Ala-Pro-Ser-Ser-Gly-Phe-Arg-Ile-Leu-
Leu-Leu-Lys-Val
was prepared using the general solid-phase methodology of Example 2
and N-acetylated as in Example 3. The C-terminus was derivatised
using n-decylamine in place of the Rink reagent. Mass spectrometry
of the purified peptide gave a major peak at 1952.3 corresponding
to a monoprotonated molecular ion (Theory: 1951.1.) An ion at
1843.3 was also observed, this is believed to correspond to loss of
the thiopyridyl group in the spectrophotometer.
Amino-Acid Analysis:
TABLE-US-00016 [0255] Ser Gly Arg Ala Pro Val Ile Phe Leu Lys
Theory: 3 1 1 2 1 1 1 1 3 1 Found: 2.95 1.10 1.10 2.11 1.04 0.60
0.92 1.00 3.03 1.03
The peptide content by weight was 53%
Example 6
Expression and Isolation of [SCR1-3]-Cys (SEQ ID NO: 6)
[0256] (a) Construction of plasmid pDB1030 encoding [SCR
1-3]-Cys
[0257] The plasmid coding for SCR1-3 of LHR-A of CR1, pDB1013-5
(patent application WO 94/00571) was digested with restriction
endonucleases EcoRI and HindIII and the 2.2 kB plasmid band was
isolated from an agarose gel using a Qiagen Qiaex DNA extraction
kit according to the manufacturer's instructions. This is fragment
1. A second batch of pDB1013-5 was digested with BanI and EcoRI and
the 196 bp band was extracted from agarose as above. This is
fragment 2. Two oligonucleotides, SEQ ID No.1 and SEQ ID No.2, were
annealed to give a final DNA concentration of 100 pmoles/ul. The
annealed oligo has a BanI/EcoRI overhang and duplicates the
sequence at the 3' end of pDB1013-5 but in addition contains a
codon coding for cysteine just before the stop codon. This is
fragment 3.
[0258] Fragments 1,2 and 3 were ligated with T4 DNA ligase in a
single reaction to give pDB1030. The ligated plasmid was
transformed into competent E. coli JM109 purchased from Promega.
Resulting colonies were analysed by restriction endonuclease
digestion and DNA sequencing confirmed that the encoded amino acid
sequence of SCR(1-3) (SEQ ID No.27 of WO 94/00571) had been altered
by a single C-terminal cysteine residue to give SEQ ID No.6.
(b) Expression of [SCR1-3]-Cys from pDB1030
[0259] pDB1030 was transformed into calcium chloride competent E.
coli BL21(DE3) and resultant colonies were isolated and checked for
plasmid content. To express protein from pDB1030 in E. coli
BL21(DE3), a single colony was inoculated into 10 ml LB-phosphate
media (20 g/L tryptone, 15 g/L yeast extract, 0.8 g/L NaCl, 0.2 g/L
Na.sub.2HPO.sub.4, 0.1 g/L KH.sub.2PO.sub.4) containing 50 ug/ml
ampicillin. The culture was grown for 6 hours at 37.degree. C., 230
r.p.m. before being used to inoculate 100 ml of the same media
containing 50 ug/ml ampicillin. Growth was under the same
conditions overnight 25 ml of each culture were then used to
inoculate 600 ml of the same media with 50 ug/ml ampicillin in 3 L
erlenmeyer flasks. Cells were grown to an OD of 0.8-1.0 at
A.sub.600 nm. IPTG (isopropyl B-D galactopyranoside) was added to a
final concentration of 1 mM and cells allowed to continue growth
for a further 3-4 hours before harvesting by centrifugation at 8000
g/10 min. Pellet from 2 L of culture was stored at -80.degree.
C.
(c) Isolation, Refolding, Purification and Formulation of
[SCR1-3]-Cys
[0260] The methods described are essentially those detailed in Dodd
I. et al (1995) Protein Expression and Purification 6 727-736.
i) Isolation of Solubilised Inclusion Bodies
[0261] The frozen cell pellet of E. coli BL21(DE3) (pDB1030) was
resuspended in 50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM PMSF pH 8.0
at a ratio of 33 ml for each litre of culture pellet. The
suspension was transferred to a glass beaker surrounded by ice and
sonicated (Heat systems--Ultrasonics W380; 50.times.50% pulse,
pulse time=5 sec.) for typically 3-6 minutes. The disrupted pellet
was then frozen and stored at -80.degree. C. Approx. 2 weeks later
the sonicate was thawed and centrifuged at approx. 8000 g for 20
min. The pellet was resuspended in 20 mM Tris/8M urea/1 mM EDTA/50
mM 2-mercaptoethanol pH 8.5 (200 ml) at room temperature by
vigorous swirling, then left for 1 h at room temperature followed
by overnight at 4.degree. C.
ii) Initial Purification Using SP-Sepharose
[0262] To the viscous solution was added SP-Sepharose FF (approx.
30 g wet weight) that had been water washed and suction-dried. The
mixture was swirled vigorously and left static for 1-2 h at room
temperature. The supernatant was decanted, sampled and discarded.
The remaining slurry was resuspended to a uniform suspension and
poured into a glass jacket and allowed to settle into a packed bed.
The column was equilibrated with 0.02M Tris/8M urea/0.05M
2-mercaptoethanol/0.001 M EDTA pH 8.5 at 4.degree. C. When the
A.sub.280 of the eluate bad stabilised at baseline, the buffer was
changed to equilibration buffer additionally containing 1M NaCl. A
single A.sub.280 peak was eluted by the 1M NaCl-containing buffer;
the volume was approx. 50 ml. The protein concentration of the
solution was estimated by A.sub.280 determination, using a molar
extinction coefficient of 25000 cm.sup.-1 of a sample that had been
buffer-exchanged (Sephadex G25) into-50 mM formic acid. This showed
the product had a protein concentration of 1.6 mg/ml. The solution
was stored at -40.degree. C.
iii) Folding and Further Processing
[0263] 25 ml of the SP-Sepharose-purified product was added
gradually over a 1 min period to 780 ml freshly prepared, cold
0.02M ethanolamine/1 mM EDTA with continuous swirling, and left
static for 1 h/4.degree. C. Reduced glutathione (GSH) was added to
1 mM and oxidised glutathione (GSSG) was added to 0.5 mM. The
solution was clear and was left static approx 2-3.degree. C. for 3
d. The solution was then ultraffitered using a YM10 membrane to a
final retentate volume of about 35 ml; the retentate was slightly
cloudy and had the appearance of a translucent solution. It was
stored for 12 days at 4.degree. C. It was then spun at 30 000 g for
15 mins and the supernatant mixed with 9 vol. 0.1M
NaH.sub.2PO.sub.4/1M (NH.sub.4).sub.2SO.sub.4 pH 7.0 (Buffer A) at
room temperature and immediately centrifuged at 3000 rpm for 15
min. The supernatant was ultrafiltered (YM10) to about 4 ml and
then buffer-exchanged into 0.1M sodium phosphate pH 7.0 (5.0 ml);
this solution had a protein concentration of 1.7 mg/ml by A280
analysis. It was treated with dithio bis nitrobenzoic acid (DTNB)
(8-fold molar excess) for 30 min at room temperature. Free thiol
content based on A412 measurement and an extinction coefficient
(for the free thionitrobenzoate ion) of 13 600 was 6 uM equivalent
to only about 10% derivatisation to give Product A. The majority of
the product was believed to be [SCR1-3]-Cys where the free
C-terminal thiol was blocked by reaction with glutathione or
2-mercaptoethanol during the refolding stage.
(d) Alternative Method for Isolation, Refolding, Purification and
Formulation of [SCR1-3]-Cys
[0264] The method was similar to that described above, except that
it more closely followed the procedures described in Dodd et al (op
cit.). Notably, the ultraffitered retentate post refolding was
immediately treated with ammonium sulphate followed by
clarification by centrifugation and Butyl Toyopearl chromatography.
The resulting A280-absorbing fractions that eluted at about 0.2 to
0.4M ammonium sulphate were pooled and regarded as Product B.
Starting with a nominal 100 mg of fully reduced SCR1-3/cys, Product
B contained 17 mg. The product contained one major species by
non-reduced SDS PAGE with an estimated purity of >90% and an
apparent molecular weight of 21 000. On the basis of studies with
similarly produced preparations it was believed to be the
S-glutathione and/or S-mercaptoethanol derivatised form of the
parent protein, although at least some batches produced in a
similar way or stored for a period of time might exist as the free
cysteine variant. The product also contained a polypeptide with an
apparent molecular weight of about 40 000 On the basis of studies
with similar batches of protein enriched in this species it was
identified as the dimer of [SCR1-3]-Cys.
Example 7
Expression and Isolation of SCR1-3/Switch Fusion (SEQ ID NO: 7)
TABLE-US-00017 [0265] H.sub.2N-[SCR
1-3]-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-
Lys-Ser-Pro-Ser-Lys-Ser-Ser-Gly-Cys-OH
(a) Construction of Plasmid pDB1031 Encoding SCR1-3/Switch
[0266] Fragment 1 and fragment 2 of pDB1013-5 were the same as
Example 6 above. Two oligonucleotides, SEQ ID No.3 and SEQ ID No.
4, prepared by Cruachem were annealed to give a final DNA
concentration of 100 pmoles/ul. The annealed oligo has an
BanI/EcoRI overhang and duplicates the sequence at the 3' end of
pDB1013-5 but in addition contains 17 additional codons coding for
DGPKKKKKKSPSKSSGC just before the stop codon. This is fragment
4.
[0267] Fragments 1, 2 and 4 were ligated with T4 DNA ligase in a
single reaction to give pDB1031. The ligated plasmid was transfomed
into competent E. coli JM109. Resulting colonies were analysed by
restriction endonuclease digestion and DNA sequencing confirmed
that the encoded amino acid sequence of SCR1-3 (SEQ ID 27 of WO
94/00571) had been altered by C terminal addition of amino acids
DGPKKKKKKSPSKSSGC to give SEQ ID NO: 7.
(b) Expression of SCR1-3/Switch from pDB1031
[0268] pDB1031 was transformed into calcium chloride competent E.
coli BL21(DE3) and resultant colonies were isolated and checked for
plasmid content. To express protein from pDB1031 in E. coli
BL21(DE3), a single colony was inoculated into 10 ml LB-phosphate
media (20g/L tryptone, 15 g/L yeast extract, 0.8 g/L NaCl, 0.2 g/L
Na.sub.2HPO.sub.4, 0.1 g/L KH.sub.2PO.sub.4) containing 50 ug/ml
ampicillin. The culture was grown for 6 hours at 37.degree. C., 230
r.p.m. before being used to inoculate 100 ml of the same media
containing 50 ug/ml ampicillin. Growth was under the same
conditions overnight. 25 ml of each culture were then used to
inoculate 600 ml of the same media with 50 ug/ml ampicillin in 3 L
erlenmeyer flasks. Cells were grown to an OD of 0.8-1.0 at
A.sub.600 nm. IPTG (isopropyl B-D galactopyranoside) was added to a
final concentration of 1 mM and cells allowed to continue growth
for a further 3-4 hours before harvesting by centrifugation at 8000
g/10 min. The cell pellet was frozen at -40 degrees C.
(c) Isolation, Refolding, Purification and Formulation of
SCR1-3/Switch
[0269] The methods described are essentially those detailed in Dodd
I. et al (1995) Protein Expression and Purification 6 727-736, with
some modifications.
i) Isolation of Solubilised Inclusion Bodies
[0270] The frozen cell pellet of E. coli BL21(DE3) (pDB1031) was
thawed and resuspended in 50 mM Tris/50 mM NaCl/1 mM EDTA/0.1 mM
PMSF pH 8.0 at a ratio of 33 ml for each litre of culture pellet.
The suspension was transferred to a glass beaker surrounded by ice
and sonicated (Heat systems--Ultrasonics W380; 50.times.50% pulse.
pulse time=5 sec.) for typically 3-6 minutes. The disrupted pellet
was then frozen and stored at -80.degree. C. Approx. 1 d later the
sonicate was thawed and centrifuged at approx. 8000 g for 20 min.
The pellet was resuspended in 20 mM Tris/8M urea/1 mM EDTA/50 mM
2-mercaptoethanol pH 8.5 (240 ml) at room temperature by vigorous
swirling, then left for 1 h at room temperature followed by 5 days
at 4.degree. C.
ii) Preliminary Purification Using SP-Sepharose
[0271] To the viscous solution was added SP-Sepharose FF (approx.
30 g wet weight) that had been water washed and suction dried. The
mixture was swirled vigorously and left static for approx. 2 h at
room temperature. The supernatant was decanted, sampled and
discarded. The remaining slurry was resuspended to a uniform
suspension and poured into a glass jacket and allowed to settle
into a packed bed. The column was equilibrated with 0.02M Tris/8M
urea/0.05M 2-mercaptoethanol/0.001 M EDTA pH 8.5 at 4.degree. C.
When the A.sub.280 of the eluate had stabilised at baseline, the
buffer was changed to equilibration buffer additionally containing
1M NaCl. A single A.sub.280 peak was eluted by the 1M
NaCl-containing buffer, the volume was approx. 50 ml. The protein
concentration of the solution was estimated by A.sub.280
determination, using a molar extinction coefficient of 25000
cm.sup.-1, of a sample that had been buffer-exchanged (Sephadex
G25) into 50 mM formic acid. This showed the product had a protein
concentration of 2.8 mg/ml. Analysis by SDS PAGE/stain showed a
major band (approx 80%) at about 23 000 Da. The solution was stored
at -40.degree. C.
iii) Folding and Further Processing
[0272] 14 ml of the SP-Sepharose-purified product was added
gradually over a 1 min period to 430 ml freshly prepared, cold
0.05M Hepes/2 M sodium chloride/1 mM EDTA pH 8.0 with continuous
swirling, and left static for 1 h/4.degree. C. Reduced glutathione
(GSH) was added to 1 mM and oxidised glutathione (GSSG) was added
to 0.5 mM. The solution was clear and was left static approx
2-3.degree. C. for 3 d. The solution was then ultrafiltered using a
YM10 membrane to a final retentate volume of about 34 ml; the
retentate was slightly cloudy. It was then spun at 25 000g for 15
mins and the supernatant buffer-exchanged into 0.1M sodium
phosphate pH 7.0 (46 ml). This fraction contained 2 mg of protein
on the basis of an A280 determination. The solution was mixed with
DTNB (20 mM; 0.65 ml) for 20 min at 4 degrees C. and then
ultrafiltered to 2.4 ml. This retentate was buffer-exchanged into
0.1M sodium phosphate pH 7.0 (3.0 ml) and stored at -40 degrees C.
Absorbance measurements at 412 nm on the solution prior to
ultrafiltration suggested 25% derivatisation with DTNB.
(d) Alternative Isolation, Refolding, Purification and Formulation
of SCR1-3/Switch
[0273] The method was similar to that described in (c) above,
except that following the ultrafiltration step after refolding it
more closely followed the procedures described in Dodd et al (op
cit.). Notably, the ultrafiltered retentate post refolding was
immediately treated with ammonium sulphate followed by
clarification by centrifugation and Butyl Sepharose chromatography.
The resulting A280-absorbing fractions that eluted at about 0.2 to
0.4M ammonium sulphate were pooled and regarded as initial product.
Additional treatment with TCEP essentially as above, followed by
DTNB yielded a final product at 10 uM final protein concentration.
The final product contained one major species by non-reduced SDS
PAGE with an estimated purity of >90% and an apparent molecular
weight of 23 000 and contained about 2 moles TNB per mole of
protein.
Example 8
Preparation of [SCR1-3]-Cys-S-S4MSWP-1] (SEQ ID NO: 8)
[0274] ##STR00001## [0275] (a) Product A of Example 6(c) (1.5 ml)
was treated with dithiothreitol (30 ul of 0.5M in water, final
concentration 10 mM) for 60 min at 4.degree. C. to give the free
peptide SEQ ID NO 6. The yellow solution was gel filtered at
4.degree. C. on a small column of Sephadex G-25 (PD-10, Pharmacia)
into 0.05M Hepes.HCl buffer pH 7.5 (3.0 ml). The slightly cloudy
solution was mixed with a solution of MSWP-1 (Example 2) (3.8 mM
dithiopyridyl equivalents, 150 ul) to a final concentration of 0.18
mM (-8 molar equivalents). The mixture was held for 2 h on ice and
then gel filtered as before but using 2 PD10 columns (1.6 ml
applied, 3.2 ml eluted). The final eluate was not cloudy and was
stored frozen at -70.degree. C. in aliquots of 0.4 ml. [0276] (b)
[SCR1-3]-Cys protein product B described in Example 6(d) (1.5 ml;
31 uM protein) was mixed with TCEP (20 mM; 0.007 ml) and incubated
at room temperature for 23 h to give the free protein SEQ ID NO: 6.
MSWP-1 (Example 2) (10 mM; 0.093 ml) was added and the solution
incubated for a further 4 h. 0.75 ml of the final solution was
buffer-exchanged into 50 mM formic acid and aliquots left in
solution or lyophilised. The product was >80% pure by SDS PAGE
and had an apparent molecular weight of 23 000, clearly shifted
from the original parent molecular weight of 21 000. The
lyophilisate was easily soluble in 50 mM formic acid at an
estimated protein concentration of 2 mg/ml. [0277] (c) [SCR1-3]-Cys
protein product B described in Example 6(d) (21.6 ml; 31 uM
protein) was mixed with TCEP (20 mM; 0.1 ml) and incubated at room
temperature for 22 h to give the free protein SEQ ID NO 6. MSWP-1
(20 mM in 0.1M sodium phosphate pH 7.0; 0.67 ml) was added and the
solution incubated for a further 4 h. All 22 ml was
buffer-exchanged into 50 mM formic acid using Sephadex G50 (Vt 160
ml). Three A280 peaks were obtained. The first one, eluting at
volume 56-106 ml, was the title compound according to SDS PAGE
analysis. The fraction was aliquoted and aliquots stored at -40
degrees C. or lyophilised. Amino acid analysis of the
pre-lyophilisation solution indicated a protein concentration of
0.42 mg/ml. A280 (1 cm path length) was 0.44. C8 reverse phase HPLC
and SDS PAGE both indicated a purity of approx 80%. The latter
technique showed the major band had an apparent molecular weight of
23 000; clearly shifted from the original parent molecular weight
of 21 000; on reduction the 23 000 band shifted to two bands with
molecular weights of approx 21 000 and approx 5 000. The
lyophilisate was easily soluble in 50 mM formic acid or in PBS `A`
(Dulbecco) at a protein concentration of 6 mg/ml. [0278] (d)
[SCRI-3)-Cys-S--S-[MSWP-1] from (c) was divided into 0.3 ml
aliquots and freeze-dried. Individual aliquots were resolubilised
in 50 mM formic acid (0.3 ml or 0.039 ml).
Example 9
Preparation of [SCR1-3/Switch Fusion]Disulphide Linked to [MAET]
(SEQ ID NO: 31)
##STR00002##
[0280] Title compound can be synthesised using TNB-activated
SCR1-3/switch (SEQ ID NO: 7) prepared as in Example 7(d). The
TNB-activated SCR1-3/switch is mixed with a molar excess of MAET
(Example 1), which might be typically made up at 2.0 mg/ml in DMSO,
equivalent to about 3 mM free thiol. Typical reaction conditions
would be 1 to 4 hours at room temperature or overnight at 4 degrees
C. using a protein concentration of 1 to 100 uM. The reaction may
be monitored by checking the generation of yellow colour, which is
caused by the release of free TNB ion. Once the reaction is
complete the solution may be buffer exchanged into a suitable
buffer, for example 0.1M sodium phosphate pH 7.0, and stored at -40
degrees C. until required.
Example 10
Preparation of [SCR1-3/Switch Fusion] Disulphide Linked to
[MSWP.sub.--1] (SEQ ID NO: 9)
##STR00003##
[0281] Method (a)
[0282] 0.02 ml of MSWP-1 (Example 2, 10 mM in 0.1M sodium phosphate
pH 7.0) was mixed with 0.005 ml of TCEP (20 mM in 50 mM Hepes) and
left for 10 min at room temperature. The resultant solution was
Solution A containing the myristoylated peptide of SEQ ID NO: 5.
TNB-activated SCR1-3/switch (SEQ ID NO: 7) prepared in a similar
way to that described in Example 7(c) (0.3 ml; 15 uM in 0.1M sodium
phosphate pH 7.0) was mixed with 0.0056 ml of Solution A to give a
theoretical MSWP-SH molar excess of five-fold over protein. The
mixture was left for 4 h at room temperature followed by 18 h at 4
degrees C. Analysis by SDS PAGE followed by protein staining
indicated one major band at apparent M.sub.r 23K, corresponding to
unreacted protein, and a minor band at apparent M.sub.r 26K,
corresponding to title protein.
Method (b)
[0283] TNB-activated SCR1-3/switch product (SEQ ID NO: 7) (10 uM;
0.43 ml) prepared in asimilar way to that described in Example 7(d)
was mixed with TCEP (5 mM; 0.0026 ml) and incubated for 17 h at
room temperature to yield the free fusion protein SEQ ID NO: 7.
MSWP-1 (10 mM; 0.0086 ml) was added and incubation was continued
for a further 4 h. Small particles or crystals were present in the
solution, but it was otherwise clear. The particulate solution was
buffer-exchanged into 50 mM formic acid (1.0 ml), aliquoted and
frozen. Analysis by SDS PAGE under non-reducing conditions showed a
number of bands, which included a species with an apparent
Molecular weight of 25 000--the target species.
Example 11
Preparation of [CR1: 1-1929-Cys-S--S-[MSWP-1] (SEQ ID NO: 10)
##STR00004##
[0285] Human complement receptor 1 (CR1, CD35) is a known regulator
of complement activation which has been produced in a recombinant
soluble form containing all of the extracellular SCR domains of a
major natural allotype (Fearon et al, WO 89/09220, WO 91/05047).
This form (sCR1) has been expressed as an active protein in Chinese
Hamster Ovary (CHO) cells. Mutagenesis of the DNA sequence
immediately downstream of the codon for Cys-1924 is performed to
generate a new C-terminal cysteine residue.
[0286] A suitable example of a modified terminus of the cDNA
sequence of sCR1 is as follows:
TABLE-US-00018 (5909) Bal I (5914) .....CCT CTG GCC AAA TGT ACC TCT
CGT GCA CAT TGC TGA
The codon Asp-1930 in CR1 is replaced by that for a Cysteine
(followed by a stop codon to generate a soluble protein) through
ligation of a modified oligonucleotide to the unique BalI
restriction endonuclease site at position 5914 (numbering from
Fearon et al, 1989, 1991).
[0287] Expression of this modified cDNA in CHO cells and isolation
of the product by standard chromatographic procedures generates a
modified sCR1 protein which can be treated as in Example 8(a), (b)
or (c) to couple it to MSWP-1 (Example 2) to yield the tide
compound.
Example 12
Preparation of [SCR1-3]-Cys-S--S-[MSWP-2] (SEQ ID No. 11)
##STR00005##
[0289] [SCR1-3]-Cys protein (SEQ ID NO: 6) prepared in a similar
way to that described in Example 6(d) (46 uM protein; 0.20 ml) was
mixed with TCEP (5 mM; 0.0054 ml) and incubated at room temperature
for approx. 20 h. 0.05 ml of this solution was mixed with 0.025 ml
of 0.1M ethanolamine and 0.003 ml of MSWP-2 (see Example 3; 5 mM in
DMSO;); the solution was incubated for a further 3 h at room
temperature. SDS PAGE analysis showed the major band in the
preparation had an apparent molecular weight of 23 000, clearly
shifted from the original parent molecular weight of 21 000. The
purity of the target protein was estimated from the SDS PAGE gel to
be approx. 80%.
Example 13
Preparation of [SCR1-3]-Cys-S--S-[MSWP-3] (SEQ ID No. 12)
##STR00006##
[0291] [SCR1-3]-Cys protein (SEQ ID NO: 6) prepared in a similar
way to that described in Example 6(d) (46 uM protein; 0.10 ml) was
mixed with TCEP (5 mM; 0.0037 ml) and incubated at room temperature
for approx. 18 h. 0.01 ml of 0.5M ethanolamine was added. 0.03 ml
of this 0.11 ml solution was mixed with 0.0032 ml of MSWP-3 (see
Example 4; 2 mM in 0.1M sodium phosphate pH 7.0); the solution was
incubated for a further 3 h at room temperature. SDS PAGE analysis
showed the major band in the preparation had an apparent molecular
weight of 23 000, clearly shifted from the original parent
molecular weight of 21 000. The purity of the target protein was
estimated from the SDS PAGE gel to be approx 80%.
Example 14
Preparation of [SCR1-3]-Cys-S--S-[TCPT-1] (SEQ ID No. 13)
##STR00007##
[0293] [SCR1-3]-Cys protein prepared in a similar way to that
described in Example b(d) (46 uM protein; 0.08 ml) was mixed with
TCEP (5 mM; 0.0029 ml) and incubated at room temperature for
approx. 18 h. 0.008 ml of 0.5M ethanolamine was added. 0.04 ml of
this 0.088 ml solution was mixed with 0.0029 ml of TCPT-1 (see
Example 5; 2.9 mM in DMSO). The TCPT-1 was added in 6 aliquots over
a 2 h period to minimise aggregation. The solution was incubated
for a further 2 h at room temperature. The final appearance of the
mixture was one of a colloidal suspension and centrifugation at
2000 g for 1 min showed that the target protein was
compartmentalised in the precipitate. SDS PAGE analysis showed the
major band in the preparation had an apparent molecular weight of
about 23 000, clearly shifted from the original parent molecular
weight of 21 000. The purity of the target protein was estimated
from the SDS PAGE gel to be approx 80%.
Example 15
Preparation of a Rabbit Anti-(Human Erythrocyte Membrane)
Antibody--[MSWP-1] Conjugate (RAEM-MSWP-1) (SEQ ID NO: 32)
##STR00008##
[0295] Rabbit polyclonal antihuman erythrocyte membrane) (RAEM)
antiserum (Dako, Denmark, 13 mg/ml, 0.25 ml) was diluted to 1.0 ml
with 50 mM sodium phosphate 0.1M sodium chloride pH 7.4 (PBS) and
treated with 30 ul of 100 mM 2-iminothiolane in PBS (freshly
dissolved) for 30 min at 25.degree. C. These conditions have been
shown (R. A. G. Smith & R. Cassels, Fibrinolysis, 2,189-195,
1988) to introduce an average of 2-3 free thiol groups per molecule
of immunoglobulin G.
[0296] The product was purified by gel permeation chromatography on
a small disposable column of Sephadex G-25m (PD-10, Pharmacia,
Stockholm, Sweden) at 4.degree. C. 23 ml of the product (total
volume 3.0 ml, theoretical protein concentration -6.1 uM) was
treated with MSWP-1 (Example 2, 0.125 ml of 5 mM solution in
dimethyl sulphoxide, final cont .about.240 uM) and incubated at
25.degree. C. for 30 min. The product was gel-filtered on a PD10
column as above to give 3.0 ml of a solution .about.5 uM in
protein. This was stored frozen at -70.degree. C.
Example 16
Preparation of a Conjugate of Streptokinase and MSWP-1 (SEQ ID No
21)
##STR00009##
[0298] Streptokinase (SK) stock solution (Behringwerke, Marburg,
Germany, 12.8 mg/ml, 271 uM, 2.5 ml) was gel filtered using a PD10
column into 3.2 ml of PBS buffer (see Example 15) containing 0.01%
w/v Tween 80 [PST buffer]. Freshly made up 2-iminothiolane (64 ul
of 100 mM) was added and the mixture incubated at 25.degree. C. for
1 h. The product was gel filtered in 2.times.1.6 ml batches into
2.times.3.0 ml PST at 4.degree. C. on two PD10 columns. This
solution was stored in aliquots of 13 ml at -75.degree. C.
[0299] Titration of the product with Ellman's reagent (0.1 mM in
0.5 ml 0.1M Triethanolamine.HCl pH 8.0) showed that it contained
approximately 0.3 mM free thiol groups. This corresponds to an
average of 3-3.5 thiol groups per molecule of SK. The stock
thiolated SK solution (2.times.0.5 ml) was processed by modifying
one aliquot with MSWP-1 (32 ul of 5 mM stock in DMSO), incubated 1
h at 25.degree. C. and gel filtered (PD10 column) into 3.0 ml PST
at 4.degree. C. A control aliquot was processed in parallel without
exposure to MSWP-1. Both products contained -0.8 mg/ml protein
based on an extinction coefficient of 0.76 (mg/ml).sup.-1 at 280 nm
for SK and were stored at -75.degree. C.
Example 17
Reversible Linkage of MSWP-1 to the Active Centre of Human
Tissue-Type Plasminogen Activator (SEQ ID No 22)
##STR00010##
[0301] The thiol-reactive acyl-enzyme
4-N-[2-N-(3-[2-pyridyldithio]-ethylcarbonyl)aminoethyl]aminobenzoyl--[Ser-
-478] human tissue-type plasminogen activator [PDAEB.fwdarw.tPA]
was prepared by the method of Smith and Cassels (Fibrinolysis, 2,
189-195, 1988). Tissue plasminogen activator (Actilyse, Boehringer
Ingelheim, Germany, approx 2 mg) was dissolved in the PST buffer of
Example 16 (1.0 ml) and treated with 25 ul of a 20 mM solution of
4'-amidinophenyl
4-N-[2-N-(3-[2-pyridyldithio]-ethylcarbonyl)aminoethyl]aminobenzoate
hydrochloride (S. B. Kalindjian & R. A. G. Smith, Biochem. J.
248, 409-413, 1987) in dimethylsulphoxide. The mixture was
incubated for 1 h at 25.degree. C. and stored frozen at -80.degree.
C. It was reduced by addition of dithiothreitol (5 ul of 0.5M in
water) for 30 min at 0.degree. C. followed by buffer-exchange into
PST buffer (3.0 ml) as described in Example 16. The product was
divided immediately into a retained sample (0.6 ml) and a reaction
sample (2.4 ml) which was mixed with MSWP-1 (Example 2, 100 ul of a
5 mM solution in dimethylsulphoxide) and incubated for 90 min on
ice. The product was buffer-exchanged as above into 3.2 ml PST and
stored in aliquots at -196.degree. C.
Example 18
Expression and Purification of [SCR1-3]-Cys (SEQ ID 6) from a
Fermentation Run
[0302] (a) Fermentation of E. Coli Harbouring the Plasmid
pDB1030
[0303] A frozen stock of E. coli harbouring the plasmid pDB1030 was
initially prepared by plating the culture out onto LB agar plus
ampicillin at 100 .mu.g/ml. 1 ml aliquots were preserved in a 10%
glycerol/PBS cryopreservative and stored under liquid nitrogen. A 1
ml vial was thawed and was used to inoculate 100 ml LB.sup.Amp100
primary seed medium (Difco Bactotryptone, 10 gl.sup.-1; Difco yeast
extract, 5 gl.sup.-1; sodium chloride, 5 gl.sup.-1; pH
pre-sterilisation 7.4) in a 500 ml flask. The primary seed stage
was incubated at 37.degree. C. for 3 hours before transfer to the
second seed stage, also 100 ml LB.sup.Amp100 per 500 ml flask using
a 1% inoculum. Following incubation as above for a further 4 hours
a 1% inoculum was transferred to the tertiary seed stage, 10 litres
LB.sup.Amp100 in a 15 litre Biolafitte fermenter. The 10 litres
tertiary seed medium was sterilised insitu for 45 minutes at
121.degree. C. before inoculation. Following incubation for 14.5
hours, the tertiary seed was transferred to the final stage
fermenter as a 6% inoculum. Incubation conditions for the seed
stage were as follows: airflow at 101 min.sup.-1 (1.0 vvm),
temperature 37.degree. C., agitation at 400 rpm (1.9 ms.sup.-1) and
overpressure 0.2 bar. 300 litres Tryptone phosphate
medium.sup.Amp100 (Difco Bactotryptone, 20 gl.sup.-1; Difco yeast
extract, 15 gl.sup.-1; sodium chloride, 8 gl.sup.-1; disodium
hydrogen orthophosphate, 2 gl.sup.-1; potassium dihydrogen
orthophosphate, 1 gl.sup.-1; Dow Corning 1520 antifoam, 0.1
gl.sup.-1; pH pre-sterilisation 7.4) was sterilised in situ for 30
minutes at 121.degree. C. in a 450 L Bioengineering fermenter. The
fermenter was inoculated with 20 litre inoculum from the tertiary
seed stage and incubated under the following conditions: airflow
450 L min.sup.-1 (1.5 vvm), temperature 37.degree. C., agitation
230 rpm (1.5 ms.sup.-1) and overpressure 0.5 bar. After an
OD.sub.350 mm of 3.5 was obtained, 1 mM IPTG was added. Harvest
followed after continued incubation for 2 hours. A cell slurry was
recovered after primary centrifugation through a Westfalia CSA19
(two discharges). The cells were further spun at 4700 rpm (7000 g)
for 30 minutes in a Sorvall RC3B centrifuge. The total cell yield
(wet weight) was 2.98 Kg and was stored at -80 degrees C. in
approx. 600 g lots.
(b) Isolation of Inclusion Bodies and Purification of
[SCR1-3]-Cys
[0304] Inclusion bodies from 100 g (wet weight) cell pellet were
isolated and solubilised essentially as described in Example 6. The
purification of target protein from resolubilised inclusion bodies
was also as described in Example 6 with some modifications. The
major ones were:
[0305] 1. The use of Macroprep High S (Biorad) instead of
S-Sepharose. 200 g of matrix was used for 100 g of cell pellet that
had been sonicated. 1.4 g of approx. 60% pure target protein was
produced in the solubflised and partially purified fractionon
bodies.
[0306] 2. Refolding of a 100 mg sample of the partially purified
protein was carried out by diluting the fully denatured protein (2
mg/ml) 100-fold in cold 60 mM ethanolamine/1 mM EDTA, followed by
addition of the glutathione redox couple.
[0307] The product of the above process was capable of being
modified with MSWP-1 (Example 2) in a way similar to that described
in Example 8.
Example 19
Expression and Isolation of [SCR1-3(delN195-K196)]TNANKSLSSISCQT
(SEQ ID NO: 14)
[0308] (a) Construction of Plasmid pBC04-29 Encoding
[SCR1-3(delN195-K196)]TNANKSLSSISCQT
[0309] Plasmid pBC04-29 was constructed from plasmid pDB1013-5
encoding SCR1-3 of LHR-A of CR1 (patent application WO 94100571) by
QuickChange site directed mutagenesis (Stratagene) according to the
manufacturers protocols. Two complementary oligonucleotides (SEQ ID
No 15 and SEQ ID No 16 were used to generate a novel restriction
site (silent) at G186/P187 and a C terminal cysteine. In the event
the reaction produced a frame-shift mutation at position N195. In
the resulting sequence the C terminal amino acids N195 and K196 are
replaced with a 14 amino acid peptide TNANKSLSSISCQT. Fortuitously,
this sequence contains an internal cysteine close to the C
terminus, preceeded by a spacer sequence of 11 amino acids.
(b) Expression of Plasmid pBC04-29 Encoding
[SCR1-3(delN195-K196)]TNANKSLSSISCQT in E. Coli
[0310] pBC04-29 was transformed into competent E. coli
BL21(DE3)pLys-S and resultant colonies were isolated and checked
for plasmid content. A single colony was inoculated into 10 ml LB
medium (10 g/L bactotryptone, 5 g/L yeast extract, 10 g/L NaCl)
containing 50 ug/ml ampicillin. The culture was grown for 6-18
hours at 37.degree. C., 230 r.p.m. before being used to inoculate 1
litre of the same medium containing 50 ug/ml ampicillin at a
dilution of 1 in 100 in 4 L erlenmeyer flasks. Cells were grown to
an OD of 0.8-1.0 at A.sub.600 nm. IPTG (isopropyl B-D
galactopyranoside) was added to a final concentration of 1 mM and
cells allowed to continue growth for a further 3-4 hours or
overnight before harvesting by centrifugation at 8000 g/10 min.
Pellet from 1 L of culture was stored at -80.degree. C.
(c) Isolation and Purification of
[SCR1-3(delN195-K196)]TNANKSLSSISCQT
[0311] The methods are essentially those detailed in Dodd I. et al
(1995) Protein Expression and Purification 6 727-736, subsequently
modified as described in Example 18. Briefly, the cell pellet from
1 L of culture from (b) was resuspended in buffer, sonicated and
the inclusion bodies isolated by centrifugation. The inclusion
bodies were resolubilised in 100 ml of fully reducing buffer and
target protein purified on Macroprep High S (30 g wet weight).
Product (27 ml at nominal 1.5 mg/ml) that eluted from the column in
the 1M NaCl-containing buffer was refolded by dilution into 2.5 L
cold 60 mM ethanolamine/1 mM EDTA, with the glutathione redox
agents added at 1 h. After 3 d at 4 degrees C. the solution was
ultrafiltered using a YM10 membrane and the retentate was treated
with ammonium sulphate, centrifuged and the supernatant purified on
Butyl Toyopearl 650M (bed volume 53 ml). A single A280 peak was
eluted by the decreasing ammonium sulphate gradient. SDS PAGE under
non-reducing conditions followed by protein staining revealed a
major polypeptide with an apparent molecular weight of 22 000,
believed to be the target protein. One of the contaminating
polypeptides had an apparent molecular weight of about 40 000,
which was identified as the dimer of the monomeric form of the
target by comparison with adjacent markers of [SCR1-3]-Cys. The
product had an estimated protein concentration of 30 uM.
Example 20
Preparation of
[SCR1-3(delN195-K196)]TNANKSLSSISC-(--S--S-[MSWP-1])QT (SEQ ID No.
17)
##STR00011##
[0313] [SCR1-3(delN195-K196)]TNANKSLSSISCQT prepared as described
in Example 19 (approx. 30 uM protein; 0.1 ml) was mixed with TCEP
(5 mM in 50 mM. Hepes pH 4.5; 0.0072 ml) and incubated at room
temperature (22 degrees C.) for 15 h. 0.05 ml of this solution was
mixed with 0.005 ml of 0.5M ethanolamine and 0.003 ml of 7 mM
MSWP-1 (see Example 2); the solution was incubated for a further 4
h at room temperature. SDS PAGE analysis showed a major band in the
preparation had an apparent molecular weight of 25 000, clearly
shifted from the original parent molecular weight of 23 000.
Example 21
Preparation of [SCR1-3]GPSEILRGDFSSC (SEQ ID No. 23)
[0314] (a) Construction of Plasmid pBC04-31 Encoding
[SCR1-3]DGPSEILRGDFSSC
[0315] Plasmid pBC04-31 was constructed using plasmid pBC04-29
(described in Example 19) and a synthetic oligonucleotide pair (SEQ
ID No. 25 and SEQ ID No. 26). pBC04-29 was digested with the
restriction enzymes HindIII and ApaI and the large fragment (2170
bp) isolated. The two oligonucleotides were annealed by warming to
>90.degree. C. and slowly cooling to room temperature and
ligated with the DNA fragment. The ligated DNA was transformed into
competent E. coli XLI-Blue. Colonies were analyzed for plasmids in
which the oligonucleotides had been inserted by looking for the
presence of a novel AvaI site at position 2733. On digestion with
AvaI pBC04-31 yielded fragments of 2311 and 495 bp. DNA from
positive clones was used to transform the expression strains. The
oligonucleotides inserted added the peptide sequence DGPSEILRGDFSSC
to the C terminus of SCR1-3 and also repaired the frame-shift error
seen in pBC04-29.
(b) Expression, Isolation and Purification of
[SCR1-3]DGPSEILRGDFSSC
[0316] Expression, isolation and purification of
[SCR1-3]DGPSEILRGDFSSC is carried out using pBC04-31 by procedures
generally described in Example 6.
Example 22
Preparation of [SCR1-3]DGPSEILRGDFSSC--(--S--S-[MSWP-1]) [SEQ ID
No. 24)
##STR00012##
[0318] [SCR1-3] DGPSEILRGDFSSC protein prepared in a similar way to
that described in Example 21 is reacted with MSWP-1 as described in
Example 8 to give the tide compound.
Example 23
Preparation of [SCR1-3] AAPSVIGFRILLLKVAGC (SEQ ID No. 33)
[0319] (a) Construction of Plasmid pBC04-34 Encoding [SCR1.3]
AAPSVIGFRILLLKVAGC
[0320] Plasmid pBC04-34 was constructed using plasmid pBC04-29
(described in Example 19) and a synthetic oligonucleotide pair (SEQ
ID No. 34 and SEQ ID No. 35). pBC04-29 was digested with the
restriction enzymes HindIII and ApaI and the large fragment (2170
bp) isolated. The two oligonucleotides were annealed by warming to
>90.degree. C. and slowly cooling to room temperature and were
ligated with the DNA fragment. The ligated DNA was transformed into
competent E. coli XLI-Blue. Colonies were analyzed for plasmids in
which the oligonucleotides had been inserted by looking for an
increase in size of the NdeI/HindIII fragment by 59 base pairs. The
presence of the cysteine codon was determined by the presence of a
DdeI site at position 2781. pBC04-34 digested with DdeI yielded
diagnostic bands of 481 and 109 bp DNA from positive clones was
used to transform the expression strains (see next section). The
oligonucleotides inserted added the peptide sequence
AAPSVIGFRILLLKVAGC to the C terminus of SCR1-3 and also repaired
the frame-shift error seen in pBC04-29.
(b) Expression, Isolation and Purification of
[SCR1-3]APSVIGFRILLLKVAGC
[0321] Expression isolation and purification of [SCR 1-3]
APSVIGFRILLLKVAGC is carried out using pBC04-34 by procedures
generally described in Example 6.
Example 24
Preparation of [SCR1-3]AAPSVIGFRILLLKVAGC--(--S--S-[MSWP-1]) (SEQ
ID No. 36)
##STR00013##
[0323] [SCR1-3]AAPSVIGFRILLLKVAGC protein prepared in a similar way
to that described in Example 23 is reacted with MSWP-1 as described
in Example 8.
Biological Activity
(I) Anti-Complement Activity Measured by the Classical
Pathway-Mediated Haemolysis of Sheep Erythrocytes
[0324] (i) Functional activity of complement inhibitors was
assessed by measuring the inhibition of complement-mediated lysis
of sheep erythrocytes sensitized with rabbit antibodies (Diamedix
Corporation, Miami, USA). The assay is designed to be specific for
the classical pathway of complement activation. Human serum diluted
1:500 or 1:400 (final concentration in assay mixture) in 0.1 M
Hepes/0.15 M NaCl/0.1% gelatin pH 7.4 was used as a source of
complement. The serum was prepared from a pool of volunteers
essentially as described in Dacie & Lewis, 1975. Briefly, blood
was warmed to 37.degree. C. for 5 minutes, the clot removed and the
remaining serum clarified by centrifugation. The serum fraction was
split into small aliquots and stored at -196.degree. C. or
-80.degree. C. Aliquots were thawed as required and diluted in the
Hepes buffer immediately before use.
[0325] Inhibition of complement-mediated lysis of sensitized sheep
erythrocytes was measured using a standard haemolytic assay using a
v-bottom microtitre plate format as follows:
[0326] 50 .mu.l of a range of concentrations of inhibitor diluted
in Hepes buffer were mixed with 50 .mu.l of the diluted serum and
100 .mu.l of sensitized sheep erythrocytes and then incubated for 1
hour at 37.degree. C. Samples were spun at 1600 rpm at ambient
temperature for 3 minutes before transferring 150 .mu.l of
supernatant to a flat bottom microfilm plate and determining the
absorption at 405 or 410 nm. Maximum lysis (Amax) was determined by
incubating serum with erythrocytes in the absence of any inhibitor.
Background lysis (Ao) was determined by incubating erythrocytes in
the absence of any serum or inhibitor. Inhibition was expressed as
a fraction of the total cell lysis such that IH50 represents the
concentration of inhibitor required to give 50% inhibition of
lysis.
% inhibition=1-[(A-Ao)/(Amax-Ao)]
Results
TABLE-US-00019 [0327] Compound IH50 WO94/00571 0.2-0.3 ug/ml [10-15
nM](1) SEQ ID NO 27 Example 6* 0.65 ug/ml [30 nM] (mean of two) (2)
Example 7* 0.3-1.0 ug/ml [15-50 nM] (n = 3) Example 8a 0.014 ug/ml
[0.6 nM] Example 8b <0.001 ug/ml [<0.04 nM] Example 8c 0.001
ug/ml [0.043 nM] {close oversize parenthesis} (3) Example 8d.sup.+
[0.06 nM] Example 10a 0.02 ug/ml [0.8 nM] Example 10b ~0.01 ug/ml
[~0.4 nM] Example 12 ~0.0016 ug/ml [0.07 nM] Example 13 ~0.009
ug/ml [0.4 nM] Example 14 ~1.1 ug/ml [50 nM] Example 19 [4 nM] *As
2-mercaptoethanol/glutathione derivatives .sup.+Assay of the two
solutions and the original pre-lyophilisation solution from Example
8d. Other IH.sub.50 values generated for similar batches include:
(1) 15 nM (2) 8 nM, 5 nM, 8 nM, 4 nM (3) 0.3 nM, 0.2 nM, 0.07 nM,
0.06 nM, 0.2 nM, 0.4 nM, 0.5 nM, 0.6 nM.
[0328] The above data show that: [0329] 1. The complement
inhibitory activities of the `base` protein (SCR1-3 of human
complement receptor 1 of WO94/00571) and its derivatives with
either an additional C-terminal cysteine (SCR1-3/cys, Example 6) or
a single cationic `switch` sequence (SCR1-3/switch, Example 7) are
similar. [0330] 2. However, incorporation of two membrane binding
elements (electrostatic switch and myristoyl) by addition of
MSWPs-1, 2 or 3 (which contain both elements) to SCR1-3/cys or
three membrane binding elements by addition of the MSWP-1 to the
SCR1-3/switch construct results in products which are significantly
more potent (-20-200.times.) than the base or single membrane
binding element proteins. The use of TCTP-1 which is targeted to
membrane elements found in CD3-positive cells and not to
erythrocyte membranes gave a conjugate of similar potency to SCR1-3
derivatives with no or single membrane addresses. Thus, the
increases in potency in an assay which depends on an erythrocyte
membrane event (cytolysis by the membrane attack complex of
complement) can be attributed to membrane targeting of the
cytolysis inhibitor proteins by the combination of two membrane
binding elements.
(ii) Assay of Anti-Complement Activity in the Classical Pathway
Haemolytic Assay: Activity in the Sera of Domestic Pig, Guinea Pig,
Rat and Marmoset.
[0331] The activity of [SCR1-3]-Cys-S--S-[MSWP-1) was examined in
the classical pathway haemolytic assay using the sera of pig,
guinea pig, rat or marmoset. The methodology was essentially as
described in (I) with minor modifications, for example small
changes to the concentration of serum used.
[SCR1-3]-Cys-S--S-[MSWP-1] was prepared essentially as described in
Example 8c. The IH50 values for the different sera were: pig, 0.2
nM; guinea pig, 0.3 nM; rat, 0.4 nM; marmoset, 0.2 nM. These
results show that [SCR1-3]-Cys-S--S-[MSWP-1] is capable of
inhibiting classical pathway complement inhibition in the sera of a
variety of animal species.
(II) Anti-Complement Activity Measured by Alternative
Pathway-Mediated Haemolysis of Guinea Pig Erythrocytes
[0332] Functional activity of complement inhibitors was assessed by
measuring the inhibition of complement mediated lysis of guinea pig
erythrocytes essentially as described by Scesney, S. M. et al
(1996) J. Immunol. 26 1729-1735. The assay is designed to be
specific for the alternative pathway of complement activation.
Human serum prepared from a pool of volunteers essentially as
described in Dacie & Lewis, 1975 was used as the source of
complement. Briefly, blood was warmed to 37.degree. C. for 5
minutes, the clot removed and the remaining serum clarified by
centrifugation. The serum fraction was split into small aliquots
and stored at -196.degree. C. or -80.degree. C. Aliquots were
thawed as required and diluted in 0.1 M Hepes/0.15 M NaCl/0.1%
gelatin/8 mM EGTA/5 mM MgCl.sub.2 pH 7.4 (buffer A) immediately
before use. Guinea pig erythrocytes were prepared from-guinea pig
whole blood collected into EDTA-coated tubes as follows. The blood
was spun at 1600 rpm for 5 min and the erythrocyte pellet washed 3
times with 0.1 M Hepes/0.15 M NaCl/0.1% gelatin pH 7.4 until the
supernatant of the spin was essentially colourless. The
erythrocytes were finally resuspended to the original volume of
blood used and were stored at +4 degrees C. They were used within 2
weeks.
[0333] 50 .mu.l of a range of concentrations of inhibitor diluted
in buffer A in a v-bottom microlitre plate were mixed with, first,
100 .mu.l of serum that had been diluted 1:3 and second, 50 .mu.l
of guinea pig erythrocytes (diluted 1:49 in buffer A) and incubated
for 1 hour at 37.degree. C. The plate was spun at 1600 rpm for 3
minutes before transferring 150 .mu.l of each supernatant to a flat
bottom microlitre plate and determining the absorption at 405 nm,
which reflects the amount of lysis in each test solution. Maximum
lysis (Amax) was determined by incubating serum with erythrocytes
in the abience of any inhibitor. Background lysis (Ao) was
determined by incubating erythrocytes in the absence of any serum
or inhibitor. The final dilution of serum used in the assay did
absorb at 405 nm but the level of absorbance (approx 10% of Amax)
was considered to have a neglible affect on the overall assay
results and it was ignored in the calculations. Inhibition was
expressed as a fraction of the total cell lysis such that IH150
represents the concentration of inhibitor required to give 50%
inhibition of lysis.
% inhibition=1-[(A-A.sub.0)/(Amax-Ao)]
Results
[0334] Two aliquots (one lyophilized and resolubilized in a neutral
buffer, the other not lyophilized) of a single batch of of
[SCR1-3]-Cys-S--S-[MSWP-1] prepared in a similar way to that
described in Example 8 (c) were tested in the haemolytic assay. The
IH50 values for the compounds were:
TABLE-US-00020 [SCR1-3]-Cys-S-S-[MSWP-1] (not lyoph) 310 nM
[SCR1-3]-Cys-S-S-[MSWP-1] (lyoph) 480 nM
[0335] The result shows that [SCR1-3]-Cys-S--S-[MSWP-1] exhibited
activity against the alternative pathway of the complement system
and that lyophilization and subsequent resolubilization of the
protein had no affect (within experimental error) on the biological
activity of the protein.
(III) Plasminogen Activator Assay
[0336] (i) SK-related molecules from Example 16 were assayed using
a plasminogen activation assay. A solution of purified human
Lys.sub.77-Plasminogen (1 uM in PST buffer containing 25%v/v
glycerol [PSTG buffer], 0.5 ml) was incubated with thiolated SK
(final concentration 0.1 to 1.07 nM) for 1 h at 25.degree. C. An
aliquot of this mixture (10 ul) was incubated with 1.0 mM of the
plasmin substrate S-2251 (H-D-Val-Leu-Lys-p-nitroanilide,
KabiVitrum, Stockholm, Sweden) in 0.1M Triethanolamine HCl pH 8.0
(0.5 ml) at 25.degree. C. The release of p-nitroaniline was
monitored continously at 405 nm. Under these conditions, one
substrate unit (SU) of plasmin activity is defined as the amount of
enzyme giving an increase in optical density at 405 nm of 0.001
min.sup.-1. Under these conditions thiolated SK (1 nM) generated
plasmin at a nearly linear of 4225 SU/mL
[0337] SK-MSWP-1 conjugate was diluted 1:100 in PSTG buffer and
5-50 ul aliquots tested in the plasminogen activation assay. The
stock preparation was found to contain approximately 2.9 uM
functional SK.
[0338] (ii) The potential activity of the acyl-enzyme preparations
of Example 17 was estimated by dilution 25-50 fold into PST buffer
and incubation for 2 h at 37.degree. C., followed by assay using 2
mM S-2288 (H-D-Ile-Pro-Arg-p-nitroanilide 2HCl) under the same
conditions used in (i) above. Under these conditions, the potential
activity of the reduced PDAEB.fwdarw.tPA preparation was 2760 SU/ml
and that of the MSWP-1/PDAEB.fwdarw.tPA conjugate 535 SU/ml.
(IV) Erythrocyte Binding Assays
(I) Erythrocyte Aggregation Test for Modified and Unmodified Rabbit
Anti-(Human Erythrocyte Membrane) Antibody.
[0339] Human pooled erythrocytes (Ortho A2, Raritan, New Jersey, 3%
v/v, 50 ul) were added to microtitre plates wells and either
unmodified rabbit anti-(human erythrocyte membrane) antibody [RAEM]
or RAEM-MSWP1 conjugate from Example 15 added at concentrations
expressed relative to undiluted stock RAEM. Cells were agitated at
.about.100 rpm for 40 min at 25.degree. C. 5 ul was removed from
each well and examined by light microscopy at .times.20
magnification. A visual scoring scale was used as follows:
[0340] - No clumping, cells moving freely relative to each
other.
[0341] + Small clumps (<10 cells)
[0342] ++ Larger clumps (100 plus cells)
[0343] +++ Very large visible aggregates
Results
TABLE-US-00021 [0344] Controls (n = 6) - RAEM-MSWP1 1/3900 +/- RAEM
1/1100 - RAEM-MSWP1 1/1000 +/- RAEM 1/600 - RAEM 1/350 +/-
RAEM-MSWP1 1/357 +++ RAEM 1/50 ++ RAEM-MSWP1 1/62 +++
Conclusion
[0345] The antibody preparation modified to contain a
membrane-binding unit was more effective at inducing aggregation of
cells because binding to the cell membrane through MSWP1 allowed a
higher effective concentration of bridging antibody on the membrane
surface.
(ii) Binding of 125-Iodine-[SCR1-3]-Cys-S--S-[MSWP-1) to Human
Erythrocytes
[0346] [SCR1-3]-Cys-S--S-[MSWP-1] (2 mg/ml in PBS; 0.25 ml) was
mixed with 0.5 mCi of 125-Iodine (Amersham) in the presence of 9
nmoles potassium iodide following the Iodogen procedure and
reagents (Pierce and Warriner (UK) Ltd.). The labelling was allowed
to proceed for 20 min at room temperature, the reaction was
quenched with 0.1 ml of 1M potassium iodide and the solution
buffer-exchanged into PBS/0.1% albumin. Citrated blood collected
from a healthy volunteer was used as a source of human
erythrocytes. Blood (0.2 ml) was mixed with 10 microlitres of
appropriately diluted 125-Iodine-[SCR1-3]-Cys-S--S-[MSWP-1] (final
concentration 700 pM) and incubated for 30 min at 37 degrees C. The
erythrocytes were then isolated by three repeat washings in
PBS/centrifugation steps and samples counted in a Wallac 1470
Wizard gamma counter. The results were as follows:
TABLE-US-00022 cpm 1st wash 3 600 000 1st pellet 140 000 2nd wash
52 000 3rd wash 6 500 final pellet 26 000
[0347] Using values of 5.times.10.sup.9 erythrocytes per ml of
blood and and a specific radioactivity of 2.7.times.10.sup.7
cpm/nmole for the [SCR1-3]-Cys-S--S-[MSWP-1] it was calculated that
about 600 molecules of [SCR1-3]-Cys-S--S-[MSWP-1] bound per cell
(the value for `final pellet`).
(iii) Binding of Fluorescein-Labelled-[SCR1-3]-Cys-S--S-[MSWP-1] to
Human Erythrocytes
[0348] [SCR1-3]-cys (prepared in a similar way to that described in
Example 18) (45 uM.1.0 mg/ml in 0.1M sodium phosphate. approx. 0.2M
ammonium sulphate pH 7.0) was partially reduced by incubation at
25.degree. C. for 18 h by the addition of a 4-molar excess of
Tris(2-carboxyethyl)phosphine (TCEP; Pierce & Warriner (UK)
Ltd.). The solution was buffer exchanged into 50 mM Hepes pH7.0;
post buffer exchange the protein concentration was 22 uM. The
partially reduced [SCR1-3]-cys was incubated with a 4-fold molar
excess of 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl
ester (Molecular Probes Inc., USA) and incubated for 1 h at
4.degree. C. The excess fluorescent label was removed by buffer
exchange of the protein solution into 50 mM Hepes pH7.0.
Fluorescein-[SCR1-3]-cys-S--S-[MSWP-1] was synthesized by adding
MSWP-1 (Example 2) to give a five-fold molar excess over
fluorescein labelled protein and was incubated for 4 h at
25.degree. C. The solution was buffered exchanged into PBS and this
solution was used for the microscopy studies.
[0349] [SCR1-3], 10 mg/ml in 50 mM formic acid, was mixed in a 1:10
ratio with 50 mM NaHCO.sub.3 pH8.5; the pH of the solution was
adjusted with NaOH to pH9.5. The fluorescein was extracted from
Celite-fluorescein isothiocyante (Celite:fluorescein; 1:10, Sigma)
by DMSO in a 1:4 (w/v) ratio. The fluorescein-DMSO solution was
added to the protein solution in a 1:14 ratio and incubated for 1 h
at RT. Excess label was removed by gel filteration into PBS
containing 0.01% Tween-80 and this solution was used for microscopy
studies.
[0350] Citrated blood was collected from a healthy volunteer and
the erythrocytes isolated, washed in PBS and diluted 250-fold
compared to the original blood volume. 0.05 ml of erythrocytes were
incubated with 2 uM fluorescein-[SCR1-31-cys-S--S-[MSWP-1] or 2 uM
fluorescein-[SCR1-3] and incubated for 30 min at 37.degree. C. An
eight microlitre sample of each incubation was mounted on a slide
and viewed on an inverted confocal microscope (Biorad). The cells
incubated with fluorescein-[SCR1-3] showed no specific staining
whereas with those incubated with
fluorescein-[SCR1-3]-cys-S--S-[MSWP-1] staining appeared diffusely
over the cell surface and also intensely stained patches were
visible on the cell membrane. No labelling was seen
intracellularly.
(iv) Binding of MSWP-1/PDAEB.fwdarw.tPA to Human Erythrocytes
[0351] Human trypsinized and glutaraldehyde-treated red blood cells
(1.0 ml of a 4% suspension) was pelleted by low-speed
centrifugation and resuspended in a total volume of 0.5 ml PST
containing either no additions or approximately 270 SU of either
reduced PDEAB.fwdarw.tPA or MSWP-1/PDAEB.fwdarw.tPA conjugate of
Example 17. The mixtures were incubated by gentle rolling for 5 min
at 23.degree. C. and then the cells were pelleted by centrifugation
followed by two washes with 1.0 ml PST buffer. Finally, the cells
were suspended in 0.5 ml PST and incubated at 37.degree. C. Samples
of the supernatant (100 ul) were removed after pelleting. Assay
using S-2288 (as above) showed that after 2 h, approximately 7% of
the applied t-PA activity was present in the supernatant of cells
exposed to MSWP-1/PDAEB.fwdarw.tpA whereas only-2.8% was present in
the supernatant of cells exposed to reduced PDAEB.fwdarw.IPA alone.
No t-PA amidolytic activity was detected in controls.
[0352] This experiment suggests that reversible linkage of the
active site of t-PA to MSWP-1 increases the tendency of this enzyme
to bind to red blood cells.
(v) Localization of SK-MSWP-1 Conjugate on the Surface of Human
Erythrocytes
[0353] A stabilized preparation of human erythrocytes (trypsinized,
glutaraldehyde-treated, Sigma, Gillingham, UK, 4% v/v, 0.4 ml) was
pelleted by centrifugation (.about.2000 g/2 min) and resuspended in
0.4 ml PST buffer with either 0.1 uM thiolated SK or 0.1 uM
SK-MSWP-1 from Example 16.
[0354] The suspensions were incubated for 30 min at 37.degree. C.
and then washed by two cycles of centrifugation and resuspension in
PST buffer. Finally, they were resuspended in PSTG buffer (0.4 ml)
containing 1 uM plasminogen and incubated and assayed for plasmin
as described above.
[0355] The control thiolated-SK generated plasmin at a rate of 522
SU/ml, while the SK-MSWP-1 conjugate produced 6184 SU/ml. The
latter activity corresponds to around 2100 thiolated SK
molecules/cell.
(vi) Binding of [SCR1-3]-Cys-S--S-[MSWP-1] to Human Erythrocyte
Membranes
[0356] 4.times.2.0 ml of trypsinized, glutaraldehyde-treated human
erythrocytes (Sigma, R0127) were centrifuged for 2 min at about
3000 rpm. The supernatants were discarded and the cells resuspended
in phosphate/saline/Tween (0.01%) (PST) (1 ml per tube) and
[SCR1-3]-Cys-S--S-[MSWP-1] of Example 8 was added to a final
concentration of 20 ug/ml to three of the tubes. The mixtures were
then incubated at 37 degrees C. for 30 min., then washed five times
by repeat centrifugation and washing in PST. The cells were finally
suspended in 1 ml PST and were held at 4 degrees C.
[0357] The ability of these cells to inhibit complement-mediated
lysis of sheep erythrocytes was measured using the standard
classical pathway complement inhibition assay described in (I)
above. The human erythrocytes were added to the assay at four
different dilutions, followed by the human serum and then the sheep
red blood cells and incubation at 37 degrees C. as usual. The %
inhibition data are shown below.
TABLE-US-00023 human erythrocytes, human erythrocytes, treated with
Final dilution untreated [SCR1-3]-Cys-S-S-[MSWP1] 1/4 22% 62% 1/16
-8% 88% 1/256 5% 74% 1/2500 -7% 51%
[0358] Thus the percentage inhibition for the
[SCR1-3]-Cys-S--S-[MSWP-1]-treated cells at maximum dilution was
significantly higher than the untreated cells at 1/4 dilution. The
[SCR1-3]-Cys-S--S-[MSWP-1]-treated cells, therefore, contained at
least 600-fold more complement inhibitory activity than the
untreated cells, even though the cells had been washed extensively
to remove any non-bound [SCR1-3]-Cys-S--S-[MSWP-1].
[0359] The following methods and examples further illustrate
aspects of the invention.
[0360] Introduction of DNA into E. coli
[0361] Plasmids were transformed into E. coli XL1-Blue
(Stratagene), HMS174(DE3) (Novagen, UK) or UT5600(DE3) (see below)
that had been made competent using calcium chloride as described in
Sambrook et al, (op.cit.). UT5600 was purchased from New England
Biolabs (#801-I) and was converted to a DE3 lysogen. UT5600 was
isolated as a mutant of K12 strain RW193 (itself derived from
AB1515) which was insensitive to colicin-B (McIntosh et al. (1979)
J. Bact. 137 p 653). It was not initially known that ompT had been
lost, but further work by the same group showed that protein a (now
OmpT) was lacking (Earhart et al (1979) FEMS Micro Letts 6 p 277).
The nature of the mutation was determined to be a large deletion
(Elish et al (1988) J Gen Micro 134 1355).
[0362] DNA Sequencing
[0363] DNA sequencing was contracted out to Lark (Saffron Walden,
Essex UK) or MWG (Milton Keynes, UK).
[0364] Production of Oligonucleotides
[0365] Oligonucleotides were purchased from Cruachem (UK) or
Genosys-Sigma (Pampisford, Cambridgeshire UK)
[0366] Polymerase Chain Reaction Amplification of DNA
[0367] Purified DNA or DNA fragments from ligation reactions or DNA
fragments excised and purified from agarose gels were amplified by
PCR from two primers complementary to the 5' ends of the DNA
fragment. Approximately 0.1-1 mg of DNA was mixed with commercially
available buffers for PCR amplification such as 10 mM Tris pH 8.3
(at 25.degree. C.), 50 mM KCl, 0.1% gelatin; MgCl.sub.2
concentrations were varied from 1.5 mM to 6 mM to find a suitable
concentration for each reaction. Oligonucleotide primers were added
to a final concentration of 2 mM; each dNTP was added to a final
concentration of 0.2 mM. 1 unit of Taq DNA polymerase was then
added to the reaction mixture (purchased from a commercial source,
e.g. Gibco). The final reaction volume varied from 20 ml to 100 ml,
which was overlayed with mineral oil to prevent evaporation.
Thermal Research. A typical example of conditions used was
94.degree. C. for 5 minute, 55.degree. C. for 1 minute, and
72.degree. C. for 2 minutes; however, the optimal temperatures for
cycling can be determined empirically by workers skilled in the
art. The DNA fragment was amplified by repeating this temperature
cycle for a number of times, typically 30 times.
pET15b Vector for DAF Expression
[0368] The pET15b expression vector is a T7 promotor based vector
available commercially through Novagen (Wisconsin, USA). Briefly,
the vector carries the following features which make it a useful
vehicle for the expression of heterologous proteins in E. coli; a
selectable antibiotic marker (.beta.-lactamase) conferring
ampicillin resistance, a copy of the lad gene providing lac
repression in strains of E. coli that are lacI.sup.-, and the
T7-lac promoter. The T7-lac promoter combines the T7 RNA polymerase
promoter sequences with the lacI repressor binding site from the E.
coli lactose operon. This reduces expression of the cloned gene in
the absence of the inducer isopropyl .beta.-D thiogalactopyranoside
(IPTG). Downstream of the T7 promoter is a multiple cloning site
built into a region of sequence which codes for a polyhistidine tag
sequence. Translation initiates at the methionine codon at position
332-330 of the vector sequence and proceeds counter-clockwise to
yield the following peptide: MGSSHHHHEHSSGLVPRGSH. The six
histidine residues allow for purification of the fusion protein by
metal chelation chromatography, whilst the GLPVR motif constitutes
a thrombin cleavage site for removal of the peptide from the fusion
protein after purification. Three restriction enzyme sites are
provided for the insertion of cloned DNA in-frame with the
polyhistidine leader. These are NdeI (CATATG), XhoI (CTCGAG) and
BamHI (GGATCC). Use of the NdeI site to overlap the methionine
initiation codon of the cloned gene removes the possibility of
unwanted amino acids at the N-terminus of the fusion protein. At
the 3' end of the multiple cloning site is the T7 transcriptional
terminator.
[0369] Colorometric Determination of Protein Concentration
[0370] Protein concentration determination utilized a colorometric
method utilizing Coomassie Plus Protein Assay Reagent (Pierce
Chemical Company) according to protein of Example 6.
[0371] Identification of Proteins by Western Blot
[0372] For certain procedures, it is necessary to characterize the
expression of recombinant proteins by an immunological method
termed a Western blot. In this method, proteins to be analyzed are
separated by SDS-PAGE, transferred to a protein binding membrane
such as polyvinylidene difluoride (PVDF), and then probed with an
antibody that is specific for the target protein. Typically, the
binding of the first antibody is detected by the addition of an
enzyme-labelled secondary antibody and an appropriate solution
which contains a chromogenic substrate. One procedure for the
transfer of proteins to a protein-binding membrane was as follows.
After SDS-PAGE, the proteins on the gel were transferred by
electrotransfer to a protein-binding surface such PVDF. In this
procedure, two sheets of filter paper (3M, Whatman) soaked in 0.3M
Tris, 10% (v/v) methanol, pH10.4, were placed on the anode of an
electroblotter (Semi-dry blotter, Biorad). These filter papers were
then overlayed by a further two sheets of filter paper soaked in 25
mM Tris, 10% (v/v) methanol, pH10.4. On top of this stack of filter
papers was placed a sheet of PVDF membrane which had been
pre-wetted in methanol and then soaked in a buffer that comprises
25 mM Tris, 10% (v/v) methanol, pH10.4. The SDS-PAGE gel was then
placed on the top of the PVDF membrane, and overlayed with two
sheets of filter paper soaked in 25 mM Tris, 192 mM
6-amino-n-caproic acid, 10% (v/v) methanol. The cathode of the
electroblotter was then placed on top of the stack of filter
papers, gel and membrane, and the proteins transferred by passing a
current between the electrodes at 15V for 30 minutes. Subsequent
steps for the detection of the transferred proteins were described
in the Novex WesternBreeze System (Invitrogen). For the detection
of human CD59, a rat anti-CD59 monoclonal antibody YTH53.1 (Davies
et al., J. Exp. Med. 170, 637, 1989) was used together with an
enzyme-labeled anti-rat secondary antibody. For the identification
of His-tagged DAF, an anti his-tag monoclonal antibody was
used.
[0373] Purification of CD59 From Human Urine
[0374] Urine was collected into 10 mM sodium azide/5 mM benzamidine
over approximately 48 hrs. The urine was then passed through a
fluted coarse filter to a Pellicon concentrator fitted with a
membrane cassette with a 10 kDa MW cut-off membrane. Insoluble
material was removed by centrifugation at 10000.times.g for 30
minutes. The supernatant was then applied to a CNBr-activated
Sepharose 4B affinity column prepared with the rat monoclonal
anti-CD59 antibody YTH 53.1 (Davies et al. J. Exp. Med. 170, 637,
1989). The column was washed overnight with 1M NaCl and bound
material eluted with 4M MgCl.sub.2. The protein content of each 1
ml fraction eluted from the column was determined by measuring
absorbance at OD280 nm. The fractions containing the most protein
were then pooled and dialysed through a 10 kDa MW cut off membrane
into a solution containing 0.9% NaCl, and then dialysed by a
similar procedure into PBS. The dialysed protein was then
concentrated using a stirred cell ultrafiltration device (Amicon)
fitted with a 10 kDa MW cut-off membrane. The material may be
further purified by gel filtration in 10 mM Hepes, 140 mM NaCl,
pH7.4, on a Superdex S-75 fast protein liquid chromatography system
(Pharmacia) or Sephadex G-75. This method gave a yield of around 7
mg pure protein from 20L urine.
Expression and Purification of Recombinant Soluble CD59 from CHO
Cells
[0375] Soluble CD59 was expressed in a recombinant form from
Chinese Hamster Ovary cells as follows. Briefly, the polymerase
chain reaction was used to produce a truncated cDNA encoding
soluble CD59 from a full length cDNA (Davies et al. J. Exp. Med.
170, 637, 1989). A mutation was introduced into the cDNA at codon
18 of the mature protein which changed the Asn codon for Ala. The
procedure for this site-directed mutagenesis can be performed by a
number of methods including the Quickchange mutagenesis kit
(Stratagene). To introduce the modified gene into the CHO
expression plasmid pDR2EF1alpha, the polymerase chain reaction was
used with two oligonucleotides; the first oligonucleotide was
complementary to the first seven codons at the N-terminus of the
mature CD59 protein; and the 3' oligonucleotide introduces a
termination codon immediately following the codon for Asn-70 of the
CD59 cDNA. These oligonucleotides were also designed to contain
recognition sequences for restriction endonucleases compatible with
the polylinker site of the CHO expression vector. The DNA fragment
resulting from the PCR amplification was ligated into a CHO
expression that had become stabily transfected were selected from
untransfected cells by growth in medium that contained the
antibiotic hygromycin. Individual transformants were picked and for
each clone the expression of CD59 was analyzed by ELISA. The
highest expressing clone was chosen for large-scale production of
CD59 using a variety of techniques including the use of cell
factories (Nunc).
[0376] To purify the CD59, the culture medium was precleared by
centrifugation at 10000.times.g for 30 minutes. The soluble CD59
was then purified using an immunoaffinity column containing the
monoclonal antibody YTH53.1 (Davies et al. J. Exp. Med. 170, 637,
1989), as described above. The protein was then stored in PBS at
concentrations of up to 5 mg/mL at -70.degree. C.
[0377] Preparation of C56 Euglobulin
[0378] C56 euglobulin was an essential reagent that was used for
the C5b6-initiated reactive lysis of erythrocytes. C56 euglobulin
can be generated in and purified from some acute-phase sera from
post-trauma individuals (such as sports injuries, surgery or
childbirth). Blood was drawn from donors in the acute phase of
inflammation and allowed to clot at room temperature. To each 10
mls of serum, 0.5 mls of yeast suspension was added and the mixture
incubated overnight on a rotator at room temperature. The serum was
centrifuged to remove the yeast and dialysed against 0.02M Na/K
phosphate, pH 5.4. The precipitate (containing the C56 euglobulin)
was collected by centrifugation and redissolved in 0.01M Na/K
phosphate/0.05M NaCl, pH7.0 containing 25% v/v glycerol.
[0379] C5b6-Initiated Reactive Lysis of Erythrocytes
[0380] Guinea pig erythrocytes (TCS Microbiological, UK) were
washed twice in PBS and resuspended to 5% by volume in PBS/0.05%
CHAPS. 50 ml of these cells were placed in the wells of a
round-bottomed microtitre plate. Samples to be tested were diluted
in PBS/0.05% CHAPS and 50 ml of these test solutions added to the
wells containing the guinea pig erythrocytes. The plate was then
incubated at 37.degree. C. for 20 minutes to allow binding of the
samples to the erythrocytes. The microtitre plates were then
centrifuged at 1000 rpm for 5 minutes to pellet the cells using a
benchtop centrifuge. The supernatants were removed and the cell
pellets ml of a C56 euglobulin solution that varied in
concentration in different experiments from between 1:50 to 1:500
dilution in PBS/10 mM EDTA. This solution was mixed with the cells
by placing the microtitre plate on a microtitre plate shaker for 2
minutes. To this solution was then added a 90 ml of a dilution of
normal human serum (from 1:50 to 1:500 in PBS/10 mM EDTA). The
solutions were mixed by placing the microtitre plate on a plate
shaker for a further 2 minutes. The plate was then incubated at
37.degree. C. for 30 minutes. To determine the degree of
haemolysis, the plate was then placed in a benchtop centrifuge and
spun at 1800 rpm for 3 minutes. 100 ml of the supernatant was
transferred to a clear flat bottomed microtitre plate and the
absorbance at 410 nm measured spectroscopically. As controls,
guinea pig erythrocytes were treated in an identical manner to the
test samples with the following exceptions. In the first stage of
the assay, the control samples were incubated with 50 ml of PBS/10
mM EDTA for 20 minutes at 37.degree. C. After centrifugation, a
spontaneous lysis control was prepared by resuspending the cells in
150 ml PBS/10 mM EDTA; by contrast, for the maximum lysis control,
the cells were resuspended in 150 ml water.
[0381] Brief Overview of Examples 25 to 36
[0382] Example 25: Synthesis and characterization of a
membrane-targeted derivative of soluble human urinary CD59
(APT632).
[0383] Example 26: Synthesis and characterization of a
membrane-targeted derivative of human recombinant soluble CD59
(APT637). Example 27: An alternative Method for the production of
urinary (APT2047) and recombinant (APT2059) human CD59
membrane-targeted derivatives using linkage through protein
carbohydrate.
[0384] Example 28: A method for the preparation of recombinant
human CD59 with a C-terminal cysteine, expressed in yeast
(APT633).
[0385] Example 29: A method for the preparation of recombinant
human CD59 with a C-terminal cysteine, expressed in E. coli
(APT635).
[0386] Example 30: A method for the preparation of recombinant
human CD59 with a C-terminal cysteine, expressed in
baculovirus/insect cells (APT2060). with a C-terminal cysteine,
expressed in Chinese hamster ovary cells (APT2061).
[0387] Example 32: A Method for the conjugation of the
membrane-localizing agent APT542 to APT633, APT635, APT2060 or
APT2061.
[0388] Example 33: Synthesis and characterization of APT2057 (Human
DAF short consensus repeats 2-4).
[0389] Example 34: Synthesis and characterization of APT2058 (Human
DAF short consensus repeats 1-4).
[0390] Example 35: Synthesis and characterization of APT2160
(APT2058 derivatized with APT542).
[0391] Example 36: Synthesis and characterization of APT2184
(APT2057 derivatized with APT542).
Example 25
Synthesis and Characterization of Urine-Derived CD59 Linked to
MSWP-1 (APT632)
[0392] APT632 was synthesized in two steps from soluble CD59
isolated from human urine (APT631; SEQ. ID NO: 37) as described in
Methods. APT631 in PBS (200 .mu.L of a 1.9 mg/mL solution) was
mixed with 2-iminothiolane (2 .mu.L of a 100 mM solution) and the
mixture incubated at room temperature for 30 minutes. The solution
was then dialysed into PBS to remove unreacted 2-iminothiolane, and
a solution of tris-2-carboxyethyl phosphine (4 .mu.L of a 10 mM
solution in 10 mM Hepes, pH7.4) added, and the mixture left
overnight at room temperature. To this solution, 10 .mu.L of APT542
(MSWP-1; 21 mM in dimethyl sulphoxide; SEQ. ID NO. 38) was added
and incubated at room temperature for 2 h. The product APT632 was
characterized by the appearance of a protein species that migrated
at approximately 21 kDa as analyzed by SDS-PAGE. A reactive lysis
assay (described in Methods) demonstrated that APT632, at
concentrations greater than 0.5 nM, protected guinea pig
erythrocytes from complement-mediated lysis by a 1:100 dilution of
human serum; by contrast, no significant protection from lysis was
observed with the unmodified form (APT631). in CHO Cells Linked to
MSWP-1 (APT637)
[0393] APT637 was synthesized in two steps from soluble human CD59
that is expressed in a recombinant form from chinese hamster ovary
cells (APT634; SEQ ID NO: 39). APT634 in PBS (200 .mu.L of a 300
.mu.M solution) was mixed with 2-iminothiolane (6 .mu.L of a 10 mM
solution) and the mixture incubated at room temperature for 30
minutes. The solution was then dialysed into PBS to remove
unreacted 2-iminothiolane, and a solution of tris-2-carboxyethyl
phosphine (4 .mu.L of a 10 mM solution in 10 mM Hepes, pH7.4)
added, and the mixture left overnight at room temperature. To this
solution, 10 .mu.L of APT542 (21 mM in dimethyl sulphoxide) was
added and incubated at room temperature for 2 h. The product APT637
was characterized by the appearance of a protein species which
migrated at approximately 10 kDa as analyzed by SDS-PAGE as
described in methods. A reactive lysis assay (described in Methods)
demonstrated that APT637, at concentrations greater than 0.5 nM,
protected guinea pig erythrocytes from complement-mediated lysis by
a 1:100 dilution of human serum; by contrast, no significant
protection from lysis was observed with the unmodified form
(APT634).
Example 27
A Method for the Production of CD59 Derivatives Linked to MSWP-1
Via a Carbohydrate Linkage (APT2047 and APT2059)
[0394] APT2047 is a conjugate of APT634 (SEQ ID NO: 39) and APT542
(SEQ ID NO: 38), and APT2059 is a conjugate of APT631 (SEQ ID NO:
37) and APT542, in which the linkage of each pair of compounds is
through a modified carbohydrate moiety on the CD59 protein. APT2047
and APT2059 are synthesized in three steps from APT634 or APT631.
The first step involves the reaction of the proteins APT634 or
APT631 at a concentration of 1 mg/ml with 10 mM sodium periodate
for 1 h in the dark, in a solution of 0.1M sodium acetate, pH5.5.
To this mixture is added glycerol to a final concentration of 15 mM
and the solution placed on ice for 5 minutes. The mixture is then
dialysed into 0.1M sodium acetate, pH5.5 to remove excess sodium
periodate and glycerol. In the second step, the sodium
periodate-treated proteins are reacted with a solution of
(4-[4-N-concentration of 1 mg/ml for 2 h with stirring. After this
procedure, unreacted MPBH is removed by dialysis into a solution of
0.1M phosphate, pH7.0, 50 mM NaCl. In the third step of the
synthesis, the proteins treated with MPBH are reacted with a
solution comprising a 5-fold molar excess of APT544 to CD59 for 2 h
at room temperature to generate APT2047 and APT2059. The synthesis
of these proteins is confirmed by the appearance of a novel
proteinaceous species that migrates at approximately 10 kDa or 20
kDa by SDS-PAGE under non-reducing conditions, respectively. In
addition, these proteins protect guinea pig erythrocytes from
complement-mediated lysis by human serum at a concentration greater
than 0.5 nM.
Example 28
A Method for the Preparation of Recombinant Human CD59 with a
C-terminal Cysteine, Expressed in Yeast (APT633)
[0395] APT633 is a protein that comprises soluble human CD59 and a
C-terminal cysteine residue following position 81 of the mature
CD59 protein. The protein was expressed in a recombinant form in
Pichia pastoris cells. The polymerase chain reaction was used to
produce a truncated cDNA encoding soluble CD59 from a full length
cDNA (Davies et al. J. Exp. Med. 170, 637, 1989). The 5'
oligonucleotide was complementary to 20 bases of the first 7 codons
at the N-terminus of the mature CD59 protein, and the 3'
oligonucleotide introduced a cysteine codon and a termination codon
immediately following the codon for Ser-81 of the mature CD59
protein. These oligonucleotides were also designed to contain
recognition sequences for restriction endonucleases XhoI and EcoRI
which are compatible with the polylinker site of the vector pUCPIC
(a derivative of pUC19 that contains the alpha-factor leader
sequence and multiple cloning site from pPIC9K (Invitrogen). The
DNA fragment resulting from the PCR amplification was then ligated
into pUCPIC DNA and transformed into the XL1-Blue strain of E. coli
(Stratagene). The transfected cells are selected by growth on a
petri dish containing LB medium (Sigma) supplemented with
ampicillin at a concentration of 100 micrograms/ml (LBAMP). The DNA
from single colonies was isolated and sequenced as described in
Methods. The DNA that encodes the alpha factor and CD59 was then
subcloned into the vector pPIC9K that had been from the resulting
plasmid was linearized with the restriction endonuclease PmeI for
transformation into P. pastoris strain GS 115 (Invitrogen) by
spheroplasting according to the manufacturer's instructions. After
preliminary selection for clones that are capable of growth on a
minimal RD medium(1M sorbitol, 2% w/v dextrose, 1.34% yeast
nitrogen base, 4.times.10.sup.-5% biotin, 0.005% amino acids)
lacking histidine. Clones having undergone multiple integration
events were then selected by resistance to the antibiotic geneticin
sulphate (G418). Clones that were capable of growth in medium
containing G418 at a concentration of 2 mg/mL were screened for
expression of CD59. Individual colonies were inoculated in 10 mL
BMG medium (100 mM potassium phosphate, pH6.0, 13.4 mg/mL yeast
nitrogen base, 0.4 mg/L biotin, 1% (w/v) glycerol) and grown at
30.degree. C. with shaking until clones reached an optical density
of 6 as measured spectroscopically at a wavelength of 600 nm. The
cultures were then transferred to BMM medium (100 mM potassium
phosphate, pH6.0, 13.4 g/L yeast nitrogen base, 0.4 mg/L biotin,
0.5% methanol) and grown for 48 h at 30.degree. C. with shaking.
Culture supernatants were then analyzed by SDS-PAGE and Western
blot for the presence of APT633 which was observed as a novel
proteinaceous species which migrated at approximately 8000 Da.
Example 29
A Method for the Preparation of Recombinant Human CD59 with a
C-terminal Cysteine, Expressed in E. coli (APT635; SEQ ID NO:
41)
[0396] APT635 is a protein that comprises soluble human CD59 and a
C-terminal cysteine residue following codon 81 of the mature CD59
protein (SEQ ID NO: 41). The protein is expressed in a recombinant
form in E. coli cells. The polymerase chain reaction was used to
produce a truncated cDNA encoding soluble CD59 from a full length
cDNA (Davies et al. J. Exp. Med. 170, 637, 1989). The 5'
oligonucleotide was complementary to 20 bases of the first 7 codons
at the N-terminus of the mature CD59 protein, and the 3'
oligonucleotide introduced a cysteine codon and a termination codon
immediately following the codon for Ser-81 of the mature CD59
protein. These oligonucleotides were also designed to contain
recognition sequences for restriction endonucleases compatible with
the polylinker site of pBROC413 (described in WO 94/00571).
pBROC413 DNA and transformed into the UT5600(DE3) strain of E. coli
(described in Methods). The transfected cells are selected by
growth on a petri dish containing LB medium (Sigma) supplemented
with ampicillin at a concentration of 100 micrograms/ml (LBAMP).
The DNA from single colonies was isolated and sequenced as
described in Methods. A single colony representing UT5600(DE3)
cells transfected by DNA encoding APT635 was then grown with
shaking overnight at 37.degree. C. in LBAMP. This overnight culture
was then diluted 1:100 in LBAMP medium and grown with shaking at
37.degree. C. until the culture reached an optical density of 1.0
as determined by absorbance at a wavelength of 600 nm. To this
culture was added a solution of isopropyl
beta-D-thiogalactopyranoside to a final concentration of 1 mM. The
culture was then grown for a further 3 hours with shaking at
37.degree. C. The cells are harvested by centrifugation and
inclusion bodies isolated as described in WO 94/00571. The
expression of APT635 was determined by SDS-PAGE and confirmed by
the appearance of a novel protein species that migrated at
approximately 8000 Da.
Example 30
A Method for the Preparation of Recombinant Human CD59 with a
C-terminal Cysteine, Expressed in Baculovirus/Insect Cells
(APT2060)
[0397] APT2060 is a protein that comprises soluble human CD59 and a
C-terminal cysteine residue following codon 81 of the mature CD59
protein (SEQ ID NO: 40) The protein was expressed in a recombinant
form in a baculovirus expression system. The polymerase chain
reaction was used to produce a truncated cDNA encoding soluble CD59
from a full length cDNA (Davies et al. J. Exp. Med. 170, 637,
1989). The 5' oligonucleotide was complementary to 20 bases of the
first 7 codons at the N-terminus of the mature CD59 protein, and
the 3' oligonucleotide introduced a cysteine codon and a
termination codon immediately following the codon for Ser-81 of the
mature CD59 protein. These oligonucleotides were also designed to
contain recognition sequences for restriction endonucleases
compatible with the polylinker site of pBacPAK 8 baculovirus
transfer vector (Clontech). The DNA fragment resulting from the PCR
amplification was then ligated into pBacPAK 8 DNA. This plasmid was
then transfected into Sf9 cells with Bacfectin (Clontech) and
BacPAK6 viral DNA which had been cut with the confluent monolayer
of Sf9 cells and left at 28.degree. C. for 3 days. The supernatant
was removed and a plaque assay performed on serial dilutions of the
transfection supernatant as described in Baculovirus Expression
Protocols, Methods in Molecular Biology series, ed. C. Richardson).
Individual plaques were then picked into 0.5 mL IPL-41 medium
(Gibco BRL) containing 1% foetal calf serum. The mixture was left
at room temperature for 15 minutes and 100 ml of this solution used
to inoculate a 50% confluent monolayer of Sf9 cells. The cells were
then left to become infected for 4-5 days at 28.degree. C. After
this time, the supernatant was removed and assayed for CD59
expression by Western blot as described in methods. For scale-up of
the recombinant virus, the supernatant was used as an inoculum to
infect more Sf9 cell monolayers as described above; alternatively,
the supernatant can be used to infect Sf9 cells grown in suspension
cultures. In this method, 100 mL Sf9 cells at a concentration of
5.times.10.sup.6 cells/ml in IPL-41 medium containing 1% FCS were
inoculated with 50 ml of viral supernatant. The culture was shaken
for 5-7 days at 27.degree. C. and cells removed by centrifugation.
The recombinant virus may be stored at 4.degree. C. until use.
APT2060 may be detected by Western blot as described in Methods and
purified using an affinity column as described.
Example 31
A Method for the Preparation of Recombinant Human CD59 with a
C-terminal Cysteine, Expressed in Chinese Hamster Ovary Cells
(APT2061; SEQ ID. NO: 42)
[0398] APT2061 is a protein that comprises soluble human CD59 and a
C-terminal cysteine residue at position 71 of the mature protein.
The protein may be expressed in a recombinant form in chinese
hamster ovary cells as described in Methods. Briefly, the
polymerase chain reaction is used to produce a truncated cDNA
encoding soluble CD59 from a full length cDNA (Davies et al. J.
Exp. Med. 170, 637, 1989). The 5' oligonucleotide is complementary
to the first codons at the N-terminus of the mature CD59 protein,
and the 3' oligonucleotide introduces a cysteine codon and a
termination codon immediately following the codon for Asn-70 of the
CD59 cDNA. These oligonucleotides can also designed the polylinker
site of a CHO expression vector, as described.
Example 32
A Method for the Conjugation of APT542 to APT633, APT635, APT2060
or APT2061 to Generate Compounds APT2062 (See SEQ ID NO: 43),
APT2063 (SEQ ID NO: 44), APT2064 (see SEQ ID NO: 43) and APT2065
(SEQ ID NO: 45)
[0399] Compounds APT2062, APT2063, APT2064 and APT2065 are
generated by treating their parent compounds APT633, APT635,
APT2060 and APT2061 with a single molar equivalent of
tris-2-carboxyethyl phosphine (TCEP; in 10 mM Hepes, pH7.4)
overnight at room temperature. To this mixture is added a solution
containing 5 molar equivalents of APT542 (MSWP-1) for 2 hours at
room temperature.
Example 33
A Method for the Synthesis and Characterization of APT2057 (SEQ ID
NO: 46)
[0400] APT2057 is a protein that comprises the short consensus
repeats 2,3 and 4 of human CD55 (decay accelerating factor, DAF),
with a carboxyl terminal cysteine residue and an amino terminal
histidine tag motif expressed in a recombinant form in E. coli
cells. cDNA to human DAF mRNA was generated from total brain RNA
(OriGene Technologies, USA). Reverse transcription was primed with
40 .rho.mol of primer DAF-R (5'GGAATTCTAAGTCAGCAAGCCCATGGTTACT 3'),
3 .mu.g human brain total RNA and other reagents as recommended by
the the RT system manufacturers (Promega, Southampton, UK). Half of
the RT reaction (10 .mu.l) was used as template for PCR. Reaction
volume was increased to 50 .mu.l by the addition of water, buffer,
MgCl.sub.2 (to 2 mM), DMSO (to 5%) and 20 .rho.mol oligonucleotide
DAF-F (5'GCATATGACCGTCGCGCGGCCGAGC 3'). One unit of Taq polymerase
(MBI Fermentas, Vilnius, Lithuania) was added, and the reaction
subjected to 35 cycles of PCR (94.degree. C., 30 sec; 64.degree.
C., 30 sec; 72.degree. C., 60 sec). A PCR product of 1156 by was
identified by agarose gel electrophoresis, purified from the gel
and ligated using standard procedures into the T-cloning vector
pUC57/T (MBI-Fermentas, Vilnius, Lithuania). Positive clones were
full sequence analysis. A plasmid to encode APT2057 was generated
by PCR using the pUC-DAF plasmid as template. Primers were designed
to amplify the region of the DAF gene encoding amino acids 97-285
(SCR2-4). The 5' primer incorporated an NdeI restriction enzyme
site, and a codon specifying glutamine, thereby introducing an
amino terminal methionine-glutamine amino acid pair. The 3' primer
added a carboxyl terminal cysteine residue and incorporated an
EcoRI restriction enzyme site. The PCR product was cloned into the
pUC57/T T-vector as described, sequenced, the insert excised with
NdeI and EcoRI, and ligated into pET15b (Novagen, Madison, USA, see
Methods section). The product of this ligation is the plasmid
pET100-02, which expresses DAF(SCR2-4) as an in-frame fusion of a
20 amino acid leader sequence (MGSSHHHAHHSSGLVPRGSH) to the 191
amino acid DAF SCRs2-4. pET100-02 DNA was introduced into E. coli
HAMS113 and transformed cells selected by virtue of their ability
to grow on LB+agar plates in the presence of 50 .mu.g/ml ampicillin
(LBAMP). A single colony representing HAMS113 containing DNA with
the coding capacity for APT2057 was grown overnight at 37.degree.
C. with shaking (200 rpm) in LBAMP medium, then diluted 1:100 into
1 litre fresh LBAMP and growth at 37.degree. C. with shaking.
Growth was monitored by measurement of culture turbidity at 600 nm,
and upon reaching an optical density of 0.6, isopropyl .beta.-D
thiogalactopyranoside (IPTG) was added to a final concentration of
1 mM, followed by a further 3 hours of growth under the same
conditions as described above. The expression of APT2057 was
analyzed by SDS-PAGE (described in methods). APT2057 appeared as a
unique protein product of approximately 24000 Da as estimated by
comparative mobility with molecular weight standards. Cells
containing APT2057 are harvested by centrifugation and inclusion
bodies isolated as follows. Briefly, the cells are resuspended in
lysis buffer (50 mM Tris, 1 mM ethylene diamine tetra-acetic acid
(ETDA), 50 mM NaCl, pH 8.0) at 50 ml per litre of initial culture.
The suspension is lysed by two passages through an Emulsiflex
homogenizer (Glen-Creston, Middlesex UK), followed by
centrifugation at 15000.times.g to purify inclusion bodies.
Inclusion bodies are initially resuspended to approximately 1
mg.ml.sup.-1 (as estimated from SDS-PAGE) in 20 mM Tris, 1 mM EDTA,
50 mM 2-urea by the addition of 10 M urea 20 mM Tris, 1 mM EDTA, 50
mM 2-mercaptoethanol, pH8.5. This suspension is stirred at
4.degree. C. for 16 hours, and insoluble material removed by
centrifugation at 15000.times.g for 30 minutes. The APT2057 is
refolded by 1 in 50 dilution into 20 mM ethanolamine, 1 mM EDTA, pH
11 buffer and static incubation at 4.degree. C. for 24 hours.
Insoluble material is removed by centrifugation (10000.times.g, 10
minutes), and soluble material buffer exchanged into Dulbecco's A
PBS, pH 7.4 using an XK50.times.23 cm Sephadex G25 column. Refolded
APT2058 is analyzed by SDS-PAGE, Western blot and the effectiveness
of the protein in a haemolytic assay (described in methods).
Example 34
A Method for the Synthesis and Characterization of APT2058 (SEQ ID
NO: 47)
[0401] APT2058 is a protein that comprises the short consensus
repeats 1,2,3 and 4 of human CD55 (decay accelerating factor, DAF),
with a carboxyl terminal cysteine residue and an amino terminal
histidine tag motif expressed in a recombinant form in E. coli
cells. cDNA to human DAF mRNA was generated from total brain RNA as
described in Example 9. A plasmid to encode APT2058 was generated
by PCR using the pUC-DAF plasmid as template. Primers were designed
to amplify the region of the DAF gene encoding amino acids 35-285
(SCR1-4). The 5' primer incorporated an NdeI restriction enzyme
site, and a codon specifying glutamine, thereby introducing an
amino terminal methionine-glutamine amino acid pair. The 3' primer
added a carboxyl terminal cysteine residue and incorporated an
EcoRI restriction enzyme site. The PCR product was cloned into the
pUC57/T T-vector as described, sequenced, the insert excised with
NdeI and EcoRI, and ligated into pET15b (Novagen, Madison, USA).
The product of this ligation is the plasmid pET99-02, which
expresses DAF (SCR1-4) as an in-frame fusion of a 20 amino acid
leader sequence (MGSSBHHHHHSSGLVPRGSH) to the 251 amino acid DAF
SCRs1-4 (APT2058). pET99-02 DNA was introduced into E. coli HAMS113
(see methods) and expression of the recombinant protein induced as
described in Example 1. The expression of APT2058 was analyzed by
SDS-PAGE (described in methods). APT2058 appeared as a unique
protein molecular weight standards. Cells containing APT2058 were
harvested by centrifugation and inclusion bodies isolated as
follows. Briefly, the cells were resuspended in lysis buffer (50 mM
Tris, 1 mM ethylene diamine tetra-acetic acid (ETDA), 50 mM NaCl,
pH 8.0) at 50 ml per litre of initial culture. The suspension was
lysed by two passages through an Emulsiflex homogenizer
(Glen-Creston, Middlesex UK), followed by centrifugation at
15000.times.g to purify inclusion bodies. Inclusion bodies were
initially resuspended to approximately 1 mg.ml.sup.-1 (as estimated
from SDS-PAGE) in 20 mM Tris, 1 mM EDTA, 50 mM 2-mercaptoethanol,
pH8.5, and subsequently diluted to a final concentration of 8M urea
by the addition of 10 M urea 20 mM Tris, 1 mM EDTA, 50 mM
2-mercaptoethanol, 018.5. This suspension was stirred at 4.degree.
C. for 16 hours, and insoluble material removed,by centrifugation
at 15000.times.g for 30 minutes. The APT2057 was refolded by 1 in
50 dilution into 20 mM ethanolamine, 1 mM EDTA, pH 11 buffer and
static incubation at 4.degree. C. for 24 hours. Insoluble material
was removed by centrifugation (10000.times.g, 10 minutes), and
soluble material buffer exchanged into Dulbecco's A PBS, pH 7.4
using an XK50.times.23 cm Sephadex G25 column. Refolded APT2058 was
analyzed by SDS-PAGE, Western blot and the effectiveness of the
protein in a haemolytic assay (described in methods). Using this
assay (at 1:400 dilution of human serum), the concentration of
APT2058 required to bring about 50% inhibition of lysis (IH.sub.50)
was approximately 3 nM.
Example 35
A Method for the Synthesis and Characterization of APT2160 (SEQ ID
NO: 48)
[0402] Compound APT2160 was generated by treating the parent
compound APT2058 (at approximately 100 .mu.M) with a three-fold
molar excess of 10 mM tris-2-carboxyethyl phosphine (TCEP: in 50 mM
Hepes, pH 4.5) overnight at room temperature. To this mixture was
added a solution containing five molar equivalents of MSWP-1
(Example 2) in 100% DMSO for 2 hours at room temperature. APT2160
was characterized by observation of a mobility shift on
non-reducing SDS-PAGE of approximately 2000 Da, consistent with the
addition haemolytic assay (at 1:400 dilution of human serum) and an
IH.sub.50 value 0.03 nM was found
Example 36
A Method for the Synthesis and Characterization of APT2184 (SEQ ID
NO: 49)
[0403] Compound APT2184 is generated by treating the parent
compound APT2057 with a three-fold molar excess of 10 mM
tris-2-carboxyethyl phosphine (TCEP: in 50 mM Hepes, pH 4.5)
overnight at room temperature. To this mixture is added a solution
containing five molar equivalents of MSWP-1 in 100% DMSO for 2
hours at room temperature.
[0404] It is to be understood that the description, specific
examples and data, while indicating exemplary embodiments, are
given by way of illustration and are not intended to limit the
present invention. Various changes and modifications within the
present invention will become apparent to the skilled artisan from
the discussion, disclosure and data contained herein, and thus are
considered part of the invention.
[0405] This application claims priority to GB 9614871.3, filed Jul.
15, 1996, there entirety of which is hereby incorporated by
reference.
TABLE-US-00024 TABLE SEQUENCE LISTING (<- next to a peptide
sequence in { } signifies sequence runs C to N terminus) SEQ ID NO:
1: GCACCGCAGTGCATCATCCCGAACAAATGCTAATAAA SEQ ID NO: 2:
AGCTTTTATTAGCATTTGTTCGGGATGATGCACTGCG SEQ ID NO: 3:
GCACCGCAGTGCATCATCCCGAACAAAGACGGTCCGAAAAAGAAGAAAAAGAAATCTCCGTCCAAATCTTCC
GGTTGCTAATAAA SEQ ID NO: 4:
AGCTTTTATTAGCAACCGGAAGATTTGGACGGAGATTTCTTTTTCTTCTTTTTCGGACCGTCTTTGTTCGGG
ATGATGCACTGCG SEQ ID NO: 5:
Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-Cys-NH.sub-
.2 SEQ ID NO: 6: Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala
Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu
Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys
Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser
Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly
Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu
Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile Trp
Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr
Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His Tyr Gly
Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys Val Phe
Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val
Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys Cys SEQ
ID NO: 7: Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro
Thr Asn Leu Thr Asp Glu Phe Glu The Pro Ile Gly Thr Tyr Leu Asn Tyr
Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro The Ser Ile Ile Cys Leu Lys
Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg
Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile Gln
Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu Ile Gly
Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile Trp Asp Asn
Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr Ile Thr
Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His Tyr Gly Ser Val
Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys Val Phe Glu Leu
Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile
Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys Asp Gly Pro Lys
Lys Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser Gly Cys SEQ ID NO: 8:
linear, 2 polypeptide chains disulphide linked Met Gln Cys Asn Ala
Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu
Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly
Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys
Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly
Met Val His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser
Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile
Ser Gly Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile
Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn
Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly
Ser Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys
Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys
##STR00014## SEQ ID NO: 9: linear, 2 polypeptide chains disulphide
linked Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr
Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu
Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn
Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn
Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile Gln Phe
Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser
Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile Trp Asp Asn Glu
Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn
Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His Tyr Gly Ser Val Val
Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys Val Phe Glu Leu Val
Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp
Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys Asp Gly Pro Lys Lys
Lys Lys Lys Lys Ser Pro ##STR00015## SEQ ID NO 10 linear, 2
polypeptide chains disulphide linked 1 [QCNAPEWLPF ARPTNLTDEF
EFPIGTYLNY ECRPGYSGRP 41 FSIICLKNSV WTGAXDRCRR KSCRNPPDPV
NGMVHVIKGI 81 QFGSQIKYSC TKGYRLIGSS SATCIISGDT VIWDNETPIC 121
DRIPCGLPPT ITNGDFISTN RENFHYGSVV TYRCNPGSGG 161 RKVFELVGEP
SIYCTSNDDQ VGIWSGPAPQ CIIPNKCTPP 201 NVENGILVSD NRSLFSLNEV
VEFRCQPGFV MKGPRRVKCQ 241 ALNKWEPELP SCSRVCQPPP DVLHAERTQR
DKDNFSPGQE 281 VFYSCEPGYD LRGAASMRCT PQGDWSPAAP TCEVKSCDDF 321
MGQLLNGRVL FPVNLQLGAK VDFVCDEGFQ LKGSSASYCV 361 LAGMESLWNS
SVPVCEQIFC PSPPVIPNGR HTGKPLEVFP 401 FGKAVNYTCD PHPDRGTSFD
LIGESTIRCT SDPQGNGVWS 441 SPAPRCGILG HCQAPDHFLF AKLKTQTNAS
DFPIGTSLKY 481 ECRPEYYGRP FSITCLDNLV WSSPKDVCKR KSCKTPPDPV 521
NGMVHVITDI QVGSRINYSC TTGHRLIGHS SAECILSGNA 561 AHWSTKPPIC
QRIPCGLPPT IANGDFISTN RENFHYGSVV 601 TYRCNPGSGG RKVFELVGEP
SIYCTSNDDQ VGIWSGPAPQ 641 CIIPNXCTPP NVENGILVSD NRSLFSLNEV
VEFRCQPGFV 681 MKGPRRVKCQ ALNKWEPELP SCSRVCQPPP DVLHAERTQR 721
DKDNFSPGQE VFYSCEPGYD LRGAASMRCT PQGDWSPAAP 761 TCEVKSCDDF
MGQLLNGRVL FPVNLQLGAK VDFVCDEGFQ 801 LXGSSASYCV LAGMESLWNS
SVPVCEQIFC PSPPVIPNGR 841 HTGKPLEVFP FGKAVNYTCD PHPDRGTSFD
LIGESTIRCT 881 SDPQGNGVWS SPAPRCGILG HCQAPDHFLF AKLKTQTNAS 921
DFPIGTSLKY ECRPEYYGRP FSITCLDNLV WSSPKDVCKR 961 KSCKTPPDPV
NGMVHVITDI QVGSRINYSC TTGHRLIGHS 1001 SAECILSGNT AHWSTKPPIC
QRIPCGLPPT IANGDFISTN 1041 RENFHYGSVV TYRCNLGSRG RKVFELVGEP
SIYCTSNDDQ 1081 VGIWSGPAPQ CIIPNKCTPP NVENGILVSD NRSLFSLNEV 1121
VEFRCQPGFV MKGPRRVKCQ ALNKWEPELP SCSRVCQPPP 1161 EILHGEHTPS
HQDNFSPGQE VFYSCEPGYD LRGAASLHCT 1201 PQGDWSPEAP RCAVKSCDDF
LGQLPHGRVL FPLNLQLGAK 1241 VSFVCDEGFR LKGSSVSHCV LVGMRSLWNN
SVPVCEHIFC 1281 PNPPAILNGR HTGTPSGDIP YGKEISYTCD PHPDRGMTFN 1321
LIGESTIRCT SDPHGNGVWS SPAPRCELSV RAGHCKTPEQ 1361 FPFASPTIPI
NDFEFPVGTS LNYECRPGYF GKMFSISCLE 1401 NLVWSSVEDN CRRKSCGPPP
EPFNGMVHIN TCTQFGSTVN 1441 YSCNEGFRLI GSPSTTCLVS GNNVTWDKKA
PICEIISCEP 1481 PPTISNGDFY SNNRTSFHNG TVVTYQCHTG PDGEQLFELV 1521
GERSIYCTSK DDQVGVWSSP PPRCISTNKC TAPEVENAIR 1561 VPGNRSFFSL
TEIIRFRCQP GFVMVGSHTV QCQTNGRWGP 1601 KLPHCSRVCQ PPPEILHGEH
TLSHQDNFSP GQEVFYSCEP 1641 SYDLRGAASL HCTPQGDWSP EAPRCTVKSC
DDFLGQLPHG 1681 RVLLPLNLQL GAKVSFVCDE GFRLKGRSAS HCVLAGMKAL 1721
WNSSVPVCEQ IFCPNPPAIL NGRHTGTPFG DIPYGKEISY 1761 ACDTHPDRGM
TFNLIGESSI RCTSDPQGNG VWSSPAPRCE 1801 LSVPAACPHP PKIQNGHYIG
GHVSLYLPGM TISYTCDPGY 1841 LLVGKGFIFC TDQGIWSQLD HYCKEVNCSF
PLFMNGISKE 1881 LEMKXVYHYG DYVTLKCEDG YTLEGSPWSQ CQADDRWDPP 1921
##STR00016## In SEQ ID NO 10, peptide sequences are given in
brackets in single letter amino acid code. SEQ ID No. 11 linear, 2
polypeptide chains disulphide linked Met Gln Cys Asn Ala Pro Glu
Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro
Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro
Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg
Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val
His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr
Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly
Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys
Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu
Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly
Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser
Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys
##STR00017## SEQ ID No. 12 linear, 2 polypeptide chains disulphide
linked Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr
Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu
Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn
Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn
Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly Ile Gln Phe
Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser
Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile Trp Asp Asn Glu
Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn
Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His Tyr Gly Ser Val Val
Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys Val Phe Glu Leu Val
Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp
Ser Gly Pro Ala Pro Gln Cys ##STR00018## SEQ ID No. 13 linear, 2
polypeptide chains disulphide linked Met Gln Cys Asn Ala Pro Glu
Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro
Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro
Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg
Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val
His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr
Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly
Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys
Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu
Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly
Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser
Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys
##STR00019## SEQ ID NO:14 Met Gln Cys Asn Ala Pro Glu Trp Leu Pro
Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr
Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile
Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg
Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile
Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr
Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val
Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro
Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His
Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys
Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp
Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Thr Asn
Ala Asn Lys Ser Leu Ser Ser Ile Ser Cys Gln Thr SEQ ID No 15
CTGGAGCGGGCCCGCACCGCAGTGCATCATCCCGAACAAATGCTAATAAAAGC SEQ ID No 16
GCTTTTATTAGCATTTGTTCGGGATGATGCACTGCGGTGCGGGCCCGCTCCAG SEQ ID No 17
linear, 2 polypeptide chains disulphide linked Met Gln Cys Asn Ala
Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu
Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly
Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys
Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly
Met Val His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser
Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile
Ser Gly Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile
Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn
Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly
Ser Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys
Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys
##STR00020## SEQ ID No 18
Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Pro-Ser-Lys-Ser-Ser-Lys-NH.sub-
.2 SEQ ID No 19
Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-Cys SEQ ID
No 20
Cys-Ser-Ala-Ala-Pro-Ser-Ser-Gly-Phe-Arg-Ile-Leu-Leu-Leu-Lys-Val SEQ
ID No 21 linear, 2 polypeptide chains disulphide linked
##STR00021## SEQ ID No 22 Single chain form of the 527 amino acid
residue intact t-PA molecule. Residue 478 (serine) has been
modified as shown below
[SYQVICRDEKTQMIYQQHQSWLRPVLRSNRVEYCWCNSGRAQCHSVPVKSCSEPRCFN
GGTCQQALYFSDFVCQCPEGFAGKCCEIDTRATCYEDQGISYRGTWSTAESGAECTNW
NSSALAQKPYSGRRPDAIRLGLGNHNYCRNPDRDSKPWCYVFKAGKYSSEVCSTPACS
EGNSDCYFGNGSAYRGTHSLTESGASCLPWNSMILIGKVYTAQNPSAQALGLGKHNYC
RNPDGDAKPWCHVLKNRRLTWEYCDVPSCSTCGLRQYSQPQFRIKGGLFADIASHPWQA
AIFAKHRRSPGERFLCGGILISSCWILSAAHCFQERFPPHHLTVILGRTYRVVPGEE
EQKFEVEKYIVHKEFDDDTYDNDIALLQLKSDSSRCAQESSVVRTVCLPPADLQLPDW
TECELSGYGKHEALSPFYSERLKEAHVRLYPSSRCTSQHLLNRTVTDNMLCAGDTRSG
GPQANLHDACQGDSGGPLVCLNDGRMTLVGIISWGLGCGQKDVPGVYTKVTNYLDWIRDNMR]
##STR00022## SEQ ID No 23 Met Gln Cys Asn Ala Pro Glu Trp Leu Pro
Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr
Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile
Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg
Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile
Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr
Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val
Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro
Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His
Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys
Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp
Gin Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys
Asp Gly Pro Ser Glu Ile Leu Arg Gly Asp Phe Ser Ser Cys SEQ ID No
24 linear, 2 polypeptide chains disulphide linked Met Gln Cys Asn
Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe
Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser
Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr Gly Ala
Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn
Gly Met Val His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr
Ser Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile
Ile Ser Gly Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg
Ile Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr
Asn Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro
Gly Ser Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr
Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln
Cys Ile Ile Pro Asn Lys Asp-Gly-Pro-Ser-Glu-Ile-Leu-Arg-Gly
Asp-Phe- <- Ser-Ser-Cys-S--S-(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys
Lys-Lys-Ser-Pro-Ser- Lys-Ser-Ser-Gly)-NH-(Myristoyl) SEQ ID No 25
CGCACCGCAGTGCATCATCCCGAACAAAGATGGCCCGAGCGAAATTCTGCGTGGCGATTTTAGCAGCTGCTA
SEQ ID No 26
ACGTTAGCAGCTGCTAAAATCGCCACGCAGAATTTCGCTCGGGCCATCTTTGTTCGGGATGATGCACTGCGG
TGCGGGCC SEQ ID No 27
N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-
Gly-Asp-(S-2-thiopyridyl)Cys-NH.sub.2 SEQ ID No 28
N-acetyl-(S-2-thiopyridyl)Cys
Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser- Pro-Ser
Lys-Ser-Ser-(.epsilon.N-(Myristoyl))Lys-NH.sub.2 SEQ ID No 29
N-(Myristoyl)-Ser-Lys-Asp-Gly-Lys-Lys-Lys-Lys-Lys-Lys-Ser-Lys-Thr-Lys-
(S-2-Thiopyridyl)Cys-NH.sub.2 SEQ ID No 30
N-acetyl-(S-2-thiopyridyl)Cys-Ser-Ala-Ala-Pro-Ser-Ser-Gly-Phe-Arg-Ile-
Leu-Leu-Leu-Lys-Val-NH(CH.sub.2).sub.9CH.sub.3 SEQ ID No 31 Met Gln
Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn Leu Thr Asp
Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu Cys Arg Pro Gly
Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys Asn Ser Val Trp Thr
Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys Arg Asn Pro Pro Asp Pro
Val Asn Gly Met Val His Val Ile Lys Gly Ile Gln Phe Gly Ser Gln Ile
Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu Ile Gly Ser Ser Ser Ala Thr
Cys Ile Ile Ser Gly Asp Thr Val Ile Trp Asp Asn Glu Thr Pro Ile Cys
Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile
ser Thr Asn Arg Glu Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys
Asn Pro Gly Ser Gly Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser
Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala
Pro Gln Cys Ile Ile Pro Asn Lys Asp Cly Pro Lys Lys Lys Lys Lys Lys
Ser Pro Ser Lys Ser Ser
Gly-Cys-S--S--(CH.sub.2).sub.2--CONH--(CH.sub.2).sub.12CH.sub.3 SEQ
ID No 32 linear, 2 polypeptide chains disulphide linked
##STR00023## SEQ ID No 33 Met Gln Cys Asn Ala Pro Glu Trp Leu Pro
Phe Ala Arg Pro Thr Asn Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr
Tyr Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile
Ile Cys Leu Lys Asn ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg
Lys Ser Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile
Lys Gly Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr
Arg Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val
Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro
Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His
Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys
Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp
Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys
Ala Ala Pro Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Gly Cys
SEQ ID No 34
CGCACCGCAGTGCATCATCCCGAACAAAGCGGCGCCCAGCGTGATTGGCTTCCGTATTCTGCTGCTGAAAGT
GGCGGGCTGCTA SEQ ID No 35
AGCTTAGCAGCCCGCCACTTTCAGCAGCAGAATACGGAAGCCAATCACGCTGGGCGCCGCTTTGTTCGGGAT
GATGCACTGCGGTGCGGGCC SEQ ID NO 36 linear, 2 polypeptide chains
disulphide linked Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala
Arg Pro Thr Asn Leu Thr Asp Glu The Glu Phe Pro Ile Gly Thr Tyr Leu
Asn Tyr Glu Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys
Leu Lys Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser
Cys Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys Gly
Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg Leu
Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val Ile Trp
Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro Pro Thr
Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe His Tyr Gly
Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly Gly Arg Lys Val The
Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr Ser Asn Asp Asp Gln Val
Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys Ile Ile Pro Asn Lys Ala Ala
Pro Ser Val Ile Gly Phe Arg Ile Leu <- Leu Leu Lys Val Gly Cys
S--S-(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys Lys-Lys-
Ser-Pro-Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
[0406] (i) SEQUENCE CHARACTERISTICS: [0407] (A) LENGTH: 77 amino
acids [0408] (B) TYPE: amino acid [0409] (D) TOPOLOGY: linear
[0410] (ii) MOLECULE TYPE: peptide
[0411] (v) FRAGMENT TYPE:
[0412] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
TABLE-US-00025 Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-Asp-
Cys-Lys-Thr-Ala-Val-Asn-Cys-Ser-Ser-Asp-Phe-Asp-
Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-Tyr-
Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-Asn-
Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-
Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Phe-Asn-
Glu-Gln-Leu-Glu-Asn
[0413] INFORMATION FOR SEQ ID NO:38: [0414] (i) SEQUENCE
CHARACTERISTICS: [0415] (A) LENGTH: 17 amino acids [0416] (B) TYPE:
amino acid [0417] (D) TOPOLOGY: linear [0418] (ii) MOLECULE TYPE:
peptide [0419] (v) FRAGMENT TYPE: [0420] (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:38:
TABLE-US-00026 [0420]
N-(myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-
Lys-Lys-Lys-Lys-Pro-Gly-Asp-Cys-(2-thiopyridyl)- NH2
[0421] INFORMATION FOR SEQ ID NO:39: [0422] (i) SEQUENCE
CHARACTERISTICS: [0423] (A) LENGTH: 70 amino acids [0424] (B) TYPE:
amino acid [0425] (D) TOPOLOGY: linear [0426] (ii) MOLECULE TYPE:
peptide [0427] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
TABLE-US-00027 [0427]
Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-Asp-
Cys-Lys-Thr-Ala-Val-Ala-Cys-Ser-Ser-Asp-Phe-Asp-
Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-Tyr-
Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-Asn-
Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-
Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn
[0428] INFORMATION FOR SEQ ID NO:40: [0429] (i) SEQUENCE
CHARACTERISTICS: [0430] (A) LENGTH: 82 amino acids [0431] (B) TYPE:
amino acid [0432] (D) TOPOLOGY: linear [0433] (ii) MOLECULE TYPE:
peptide [0434] (v) FRAGMENT TYPE: [0435] (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:40:
TABLE-US-00028 [0435]
Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-Asp-
Cys-Lys-Thr-Ala-Val-Asn-Cys-Ser-Ser-Asp-Phe-Asp-
Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-Tyr-
Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-Asn-
Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-
Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Phe-Asn-
Glu-Gln-Leu-Glu-Asn-Gly-Gly-Thr-Ser-Cys
[0436] INFORMATION FOR SEQ ID NO:41: [0437] (i) SEQUENCE
CHARACTERISTICS: [0438] (A) LENGTH: 83 amino acids [0439] (B) TYPE:
amino acid [0440] (D) TOPOLOGY: linear [0441] (ii) MOLECULE TYPE:
peptide [0442] (v) FRAGMENT TYPE: [0443] (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:41:
TABLE-US-00029 [0443]
Met-Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-
Asp-Cys-Lys-Thr-Ala-Val-Asn-Cys-Ser-Ser-Asp-Phe-
Asp-Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Thr-
Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-Tyr-Tyr-Cys-Cys-
Lys-Lys-Asp-Leu-Cys-Asn-Phe-Asn-Glu-Gln-Leu-Glu-
Asn-Gly-Gly-Thr-Ser-Cys
[0444] INFORMATION FOR SEQ ID NO:42: [0445] (i) SEQUENCE
CHARACTERISTICS: [0446] (A) LENGTH: 71 amino acids [0447] (B) TYPE:
amino acid [0448] (D) TOPOLOGY: linear [0449] (ii) MOLECULE TYPE:
peptide [0450] (v) FRAGMENT TYPE: [0451] (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:42:
TABLE-US-00030 [0451]
Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-Asp-
Cys-Lys-Thr-Ala-Val-Ala-Cys-Ser-Ser-Asp-Phe-Asp-
Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-Tyr-
Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-Asn-
Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-
Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Cys
[0452] INFORMATION FOR SEQ ID NO:43: [0453] (i) SEQUENCE
CHARACTERISTICS: [0454] (A) LENGTH: 99 amino acids [0455] (B) TYPE:
amino acid [0456] (D) TOPOLOGY: linear, two polypeptide chains
disulphide linked [0457] (ii) MOLECULE TYPE: peptide [0458] (v)
FRAGMENT TYPE: [0459] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
TABLE-US-00031 [0459]
Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-Asp-
Cys-Lys-Thr-Ala-Val-Asn-Cys-Ser-Ser-Asp-Phe-Asp-
Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-Tyr-
Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-Asn-
Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-Thr-
Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Phe-Asn-
Glu-Gln-Leu-Glu-Asn-Gly-Gly-Thr-Ser-Cys-(S--S)-
.sup..rarw.(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-
Pro-Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
[0460] INFORMATION FOR SEQ ID NO:44: [0461] (i) SEQUENCE
CHARACTERISTICS: [0462] (A) LENGTH: 100 amino acids [0463] (B)
TYPE: amino acid [0464] (D) TOPOLOGY: linear, 2 polypeptide chains
disulphide linked [0465] (ii) MOLECULE TYPE: peptide [0466] (v)
FRAGMENT TYPE: [0467] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
TABLE-US-00032 [0467]
Met-Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-
Asp-Cys-Lys-Thr-Ala-Val-Asn-Cys-Ser-Ser-Asp-Phe-
Asp-Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-
Tyr-Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-
Asn-Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-
Thr-Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Phe-
Asn-Glu-Gln-Leu-Glu-Asn-Gly-Gly-Thr-Ser-Cys-(S--S)-
.sup..rarw.(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-Ser-
Pro-Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
[0468] INFORMATION FOR SEQ ID NO:45: [0469] (i) SEQUENCE
CHARACTERISTICS: [0470] (A) LENGTH: 88 amino acids [0471] (B) TYPE:
amino acid [0472] (D) TOPOLOGY: linear, 2 polypeptides, disulphide
linked [0473] (ii) MOLECULE TYPE: peptide [0474] (v) FRAGMENT TYPE:
[0475] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
TABLE-US-00033 [0475] Leu-Gln-Cys-Tyr-Asn-Cys-Pro-Asn-Pro-Thr-Ala-
Asp-Cys-Lys-Thr-Ala-Val-Ala-Cys-Ser-Ser-Asp-Phe-
Asp-Ala-Cys-Leu-Ile-Thr-Lys-Ala-Gly-Leu-Gln-Val-
Tyr-Asn-Lys-Cys-Trp-Lys-Phe-Glu-His-Cys-Asn-Phe-
Asn-Asp-Val-Thr-Thr-Arg-Leu-Arg-Glu-Asn-Glu-Leu-
Thr-Tyr-Tyr-Cys-Cys-Lys-Lys-Asp-Leu-Cys-Asn-Cys-
(S--S)-.sup..rarw.(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-
Ser-Pro-Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
[0476] (i) SEQUENCE CHARACTERISTICS: [0477] (A) LENGTH: 211 amino
acids [0478] (B) TYPE: amino acid [0479] (D) TOPOLOGY: linear
[0480] (ii) MOLECULE TYPE: peptide
[0481] (v) FRAGMENT TYPE:
[0482] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46
TABLE-US-00034
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-
Ser-His-Met-Ser-Cys-Glu-Val-Pro-Thr-Arg-Leu-Asn-Ser-Ala-Ser-Leu-Lys-Gln-
Pro-Tyr-Ile-Thr-Gln-Asn-Tyr-Phe-Pro-Val-Gly-Thr-Val-Val-Glu-Tyr-Glu-Cys-
Arg-Pro-Gly-Tyr-Arg-Arg-Glu-Pro-Ser-Leu-Ser-Pro-Lys-Leu-Thr-Cys-Leu-Gln-
Asn-Leu-Lys-Trp-Ser-Thr-Ala-Val-Glu-Phe-Cys-Lys-Lys-Lys-Ser-Cys-Pro-Asn-
Pro-Gly-Glu-Ile-Arg-Asn-Gly-Gln-Ile-Asp-Val-Pro-Gly-Gly-Ile-Leu-Phe-Gly-
Ala-Thr-Ile-Ser-Phe-Ser-Cys-Asn-Thr-Gly-Tyr-Lys-Leu-Phe-Gly-Ser-Thr-Ser-
Ser-Phe-Cys-Leu-Ile-Ser-Gly-Ser-Ser-Val-Gln-Trp-Ser-Asp-Pro-Leu-Pro-Glu-
Cys-Arg-Glu-Ile-Tyr-Cys-Pro-Ala-Pro-Pro-Gln-Ile-Asp-Asn-Gly-Ile-Ile-Gln-Gl-
y-
Glu-Arg-Asp-His-Tyr-Gly-Tyr-Arg-Gln-Ser-Val-Thr-Tyr-Ala-Cys-Asn-Lys-Gly-
Phe-Thr-Met-Ile-Gly-Glu-His-Ser-Ile-Tyr-Cys-Thr-Val-Asn-Asn-Asp-Glu-Gly-
Glu-Trp-Ser-Gly-Pro-Pro-Pro-Glu-Cys-Arg-Gly-Cys
[0483] INFORMATION FOR SEQ ID NO:47: [0484] (i) SEQUENCE
CHARACTERISTICS: [0485] (A) LENGTH: 274 amino acids [0486] (B)
TYPE: amino acid [0487] (D) TOPOLOGY: linear [0488] (ii) MOLECULE
TYPE: peptide [0489] (v) FRAGMENT TYPE: [0490] (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:47
TABLE-US-00035 [0490]
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Ar-
g-Gly-
Ser-His-Met-Gln-Asp-Cys-Gly-Leu-Pro-Pro-Asp-Val-Pro-Asn-Ala-Gln-Pro-Ala-
Leu-Glu-Gly-Arg-Thr-Ser-Phe-Pro-Glu-Asp-Thr-Val-Ile-Thr-Tyr-Lys-Cys-Glu-
Ser-Gln-Trp-Ser-Asp-Ile-Glu-Glu-Phe-Cys-Asn-Arg-Ser-Cys-Glu-Val-Pro-Thr-
Arg-Leu-Asn-Ser-Ala-Ser-Leu-Lys-Gln-Pro-Tyr-Ile-Thr-Gln-Asn-Tyr-Phe-Pro-
Val-Gly-Thr-Val-Val-Glu-Tyr-Glu-Cys-Arg-Pro-Gly-Tyr-Arg-Arg-Glu-Pro-Ser-
Leu-Ser-Pro-Lys-Leu-Thr-Cys-Leu-Gln-Asn-Leu-Lys-Trp-Ser-Thr-Ala-Val-Glu-
Phe-Cys-Lys-Lys-Lys-Ser-Cys-Pro-Asn-Pro-Gly-Glu-Ile-Arg-Asn-Gly-Gln-Ile-
Asp-Val-Pro-Gly-Gly-Ile-Leu-Phe-Gly-Ala-Thr-Ile-Ser-Phe-Ser-Cys-Asn-Thr-
Gly-Tyr-Lys-Leu-Phe-Gly-Ser-Thr-Ser-Ser-Phe-Cys-Leu-Ile-Ser-Gly-Ser-Ser-
Val-Gln-Trp-Ser-Asp-Pro-Leu-Pro-Glu-Cys-Arg-Glu-Ile-Tyr-Cys-Pro-Ala-Pro-
Pro-Gln-Ile-Asp-Asn-Gly-Ile-Ile-Gln-Gly-Glu-Arg-Asp-His-Tyr-Gly-Tyr-Arg-
Gln-Ser-Val-Thr-Tyr-Ala-Cys-Asn-Lys-Gly-Phe-Thr-Met-Ile-Gly-Glu-His-Ser-
Ile-Tyr-Cys-Thr-Val-Asn-Asn-Asp-Glu-Gly-Glu-Trp-Ser-Gly-Pro-Pro-Pro-Glu-
Cys-Arg-Gly-Cys
[0491] INFORMATION FOR SEQ ID NO:48: [0492] (i) SEQUENCE
CHARACTERISTICS: [0493] (A) LENGTH: 291 amino acids [0494] (B)
TYPE: amino acid [0495] (D) TOPOLOGY: linear, 2 polypeptide chains,
disulphide linked [0496] (ii) MOLECULE TYPE: peptide [0497] (v)
FRAGMENT TYPE: [0498] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48
TABLE-US-00036 [0498]
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Ar-
g-Gly-
Ser-His-Met-Gln-Asp-Cys-Gly-Leu-Pro-Pro-Asp-Val-Pro-Asn-Ala-Gln-Pro-Ala-
Leu-Glu-Gly-Arg-Thr-Ser-Phe-Pro-Glu-Asp-Thr-Val-Ile-Thr-Tyr-Lys-Cys-Glu-
Glu-Ser-Phe-Val-Lys-Ile-Pro-Gly-Glu-Lys-Asp-Ser-Val-Ile-Cys-Leu-Lys-Gly-
Ser-Gln-Trp-Ser-Asp-Ile-Glu-Glu-Phe-Cys-Asn-Arg-Ser-Cys-Glu-Val-Pro-Thr-
Arg-Leu-Asn-Ser-Ala-Ser-Leu-Lys-Gln-Pro-Tyr-Ile-Thr-Gln-Asn-Tyr-Phe-Pro-
Val-Gly-Thr-Val-Val-Glu-Tyr-Glu-Cys-Arg-Pro-Gly-Tyr-Arg-Arg-Glu-Pro-Ser-
Leu-Ser-Pro-Lys-Leu-Thr-Cys-Leu-Gln-Asn-Leu-Lys-Trp-Ser-Thr-Ala-Val-Glu-
Phe-Cys-Lys-Lys-Lys-Ser-Cys-Pro-Asn-Pro-Gly-Glu-Ile-Arg-Asn-Gly-Gln-Ile-
Asp-Val-Pro-Gly-Gly-Ile-Leu-Phe-Gly-Ala-Thr-Ile-Ser-Phe-Ser-Cys-Asn-Thr-
Val-Gln-Trp-Ser-Asp-Pro-Leu-Pro-Glu-Cys-Arg-Glu-Ile-Tyr-Cys-Pro-Ala-Pro-
Pro-Gln-Ile-Asp-Asn-Gly-Ile-Ile-Gln-Gly-Glu-Arg-Asp-His-Tyr-Gly-Tyr-Arg-
Gln-Ser-Val-Thr-Tyr-Ala-Cys-Asn-Lys-Gly-Phe-Thr-Met-Ile-Gly-Glu-His-Ser-
Ile-Tyr-Cys-Thr-Val-Asn-Asn-Asp-Glu-Gly-Glu-Trp-Ser-Gly-Pro-Pro-Pro-Glu-
Cys-Arg-Gly-Cys-(S--S)-.sup..rarw.(Cys-Asp-Gly-Pro-Lys-Lys-Lys-Lys-Lys-Lys-
-Ser-Pro- Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
[0499] INFORMATION FOR SEQ ID NO:49: [0500] (i) SEQUENCE
CHARACTERISTICS: [0501] (A) LENGTH: 228 amino acids [0502] (B)
TYPE: amino acid [0503] (D) TOPOLOGY: linear, 2 polypeptides,
disulphide linked [0504] (ii) MOLECULE TYPE: peptide [0505] (v)
FRAGMENT TYPE: [0506] (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49
TABLE-US-00037 [0506]
Met-Gly-Ser-Ser-His-His-His-His-His-His-Ser-Ser-Gly-Leu-Val-Pro-Ar-
g-Gly-
Ser-His-Met-Ser-Cys-Glu-Val-Pro-Thr-Arg-Leu-Asn-Ser-Ala-Ser-Leu-Lys-Gln-
Pro-Tyr-Ile-Thr-Gln-Asn-Tyr-Phe-Pro-Val-Gly-Thr-Val-Val-Glu-Tyr-Glu-Cys-
Arg-Pro-Gly-Tyr-Arg-Arg-Glu-Pro-Ser-Leu-Ser-Pro-Lys-Leu-Thr-Cys-Leu-Gln-
Asn-Leu-Lys-Trp-Ser-Thr-Ala-Val-Glu-Phe-Cys-Lys-Lys-Lys-Ser-Cys-Pro-Asn-
Pro-Gly-Glu-Ile-Arg-Asn-Gly-Gln-Ile-Asp-Val-Pro-Gly-Gly-Ile-Leu-Phe-Gly-
Ala-Thr-Ile-Ser-Phe-Ser-Cys-Asn-Thr-Gly-Tyr-Lys-Leu-Phe-Gly-Ser-Thr-Ser-
Ser-Phe-Cys-Leu-Ile-Ser-Gly-Ser-Ser-Val-Gln-Trp-Ser-Asp-Pro-Leu-Pro-Glu-
Cys-Arg-Glu-Ile-Tyr-Cys-Pro-Ala-Pro-Pro-Gln-Ile-Asp-Asn-Gly-Ile-Ile-Gln-Gl-
y-
Glu-Arg-Asp-His-Tyr-Gly-Tyr-Arg-Gln-Ser-Val-Thr-Tyr-Ala-Cys-Asn-Lys-Gly-
Phe-Thr-Met-Ile-Gly-Glu-His-Ser-Ile-Tyr-Cys-Thr-Val-Asn-Asn-Asp-Glu-Gly-
Glu-Trp-Ser-Gly-Pro-Pro-Pro-Glu-Cys-Arg-Gly-Cys-.sup..rarw.(Cys-Asp-Gly-Pr-
o-Lys-
Lys-Lys-Lys-Lys-Lys-Ser-Pro-Ser-Lys-Ser-Ser-Gly)-NH-(Myristoyl)
Sequence CWU 1
1
53137DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gcaccgcagt gcatcatccc gaacaaatgc taataaa
37237DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 2agcttttatt agcatttgtt cgggatgatg cactgcg
37385DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 3gcaccgcagt gcatcatccc gaacaaagac
ggtccgaaaa agaagaaaaa gaaatctccg 60tccaaatctt ccggttgcta ataaa
85485DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4agcttttatt agcaaccgga agatttggac
ggagatttct ttttcttctt tttcggaccg 60tctttgttcg ggatgatgca ctgcg
85517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide used to synthesize MSWP-1 5Gly Ser Ser Lys Ser
Pro Ser Lys Lys Lys Lys Lys Lys Pro Gly Asp1 5 10
15Cys6198PRTArtificial SequenceDescription of Artificial Sequence
[SCR1-3]-Cys protein 6Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe
Ala Arg Pro Thr Asn1 5 10 15Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly
Thr Tyr Leu Asn Tyr Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly Arg Pro
Phe Ser Ile Ile Cys Leu Lys 35 40 45Asn Ser Val Trp Thr Gly Ala Lys
Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp Pro Val
Asn Gly Met Val His Val Ile Lys Gly65 70 75 80Ile Gln Phe Gly Ser
Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95Leu Ile Gly Ser
Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105 110Ile Trp
Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120
125Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn
130 135 140Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly
Ser Gly145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro
Ser Ile Tyr Cys Thr 165 170 175Ser Asn Asp Asp Gln Val Gly Ile Trp
Ser Gly Pro Ala Pro Gln Cys 180 185 190Ile Ile Pro Asn Lys Cys
1957214PRTArtificial SequenceDescription of Artificial Sequence
[SCR1-3]/switch fusion protein 7Met Gln Cys Asn Ala Pro Glu Trp Leu
Pro Phe Ala Arg Pro Thr Asn1 5 10 15Leu Thr Asp Glu Phe Glu Phe Pro
Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly
Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45Asn Ser Val Trp Thr Gly
Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp
Pro Val Asn Gly Met Val His Val Ile Lys Gly65 70 75 80Ile Gln Phe
Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95Leu Ile
Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105
110Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu
115 120 125Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg
Glu Asn 130 135 140Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn
Pro Gly Ser Gly145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly
Glu Pro Ser Ile Tyr Cys Thr 165 170 175Ser Asn Asp Asp Gln Val Gly
Ile Trp Ser Gly Pro Ala Pro Gln Cys 180 185 190Ile Ile Pro Asn Lys
Asp Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro 195 200 205Ser Lys Ser
Ser Gly Cys 210816PRTArtificial SequenceDescription of Artificial
Sequence Illustrative amino acid sequence 8Asp Gly Pro Lys Lys Lys
Lys Lys Lys Ser Pro Ser Lys Ser Ser Gly1 5 10 15916PRTArtificial
SequenceDescription of Artificial Sequence Illustrative amino acid
sequence 9Gly Ser Ser Lys Ser Pro Ser Lys Lys Lys Lys Lys Lys Pro
Gly Asp1 5 10 151020PRTArtificial SequenceDescription of Artificial
Sequence Illustrative amino acid sequence 10Ser Pro Ser Asn Glu Thr
Pro Lys Lys Lys Lys Lys Arg Phe Ser Phe1 5 10 15Lys Lys Ser Gly
201116PRTArtificial SequenceDescription of Artificial Sequence
Illustrative amino acid sequence 11Asp Gly Pro Lys Lys Lys Lys Lys
Lys Ser Pro Ser Lys Ser Ser Lys1 5 10 151214PRTArtificial
SequenceDescription of Artificial Sequence Illustrative amino acid
sequence 12Ser Lys Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys1
5 10136PRTArtificial SequenceDescription of Artificial Sequence
Illustrative amino acid sequence 13Gly Arg Gly Asp Ser Pro1
514209PRTArtificial SequenceDescription of Artificial Sequence
SCR1-3 with the c-terminal amino acids N195 and K196 replaced by a
14 amino acid peptide 14Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe
Ala Arg Pro Thr Asn1 5 10 15Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly
Thr Tyr Leu Asn Tyr Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly Arg Pro
Phe Ser Ile Ile Cys Leu Lys 35 40 45Asn Ser Val Trp Thr Gly Ala Lys
Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp Pro Val
Asn Gly Met Val His Val Ile Lys Gly65 70 75 80Ile Gln Phe Gly Ser
Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95Leu Ile Gly Ser
Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105 110Ile Trp
Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120
125Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn
130 135 140Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly
Ser Gly145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro
Ser Ile Tyr Cys Thr 165 170 175Ser Asn Asp Asp Gln Val Gly Ile Trp
Ser Gly Pro Ala Pro Gln Cys 180 185 190Ile Ile Pro Thr Asn Ala Asn
Lys Ser Leu Ser Ser Ile Ser Cys Gln 195 200 205Thr
1553DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 15ctggagcggg cccgcaccgc agtgcatcat
cccgaacaaa tgctaataaa agc 531653DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 16gcttttatta
gcatttgttc gggatgatgc actgcggtgc gggcccgctc cag 531713PRTArtificial
SequenceDescription of Artificial Sequence Illustrative amino acid
sequence 17Asp Gly Pro Ser Glu Ile Leu Arg Gly Asp Phe Ser Ser1 5
101817PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide used to generate MSWP-2 18Cys Asp Gly Pro Lys Lys
Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser1 5 10
15Lys1915PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide used to generate MSWP-3 19Ser Lys Asp Gly Lys Lys
Lys Lys Lys Lys Ser Lys Thr Lys Cys1 5 10 152016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide used
to generate TCTP-1 20Cys Ser Ala Ala Pro Ser Ser Gly Phe Arg Ile
Leu Leu Leu Lys Val1 5 10 152117PRTArtificial SequenceDescription
of Artificial Sequence Illustrative amino acid sequence 21Gly Asn
Glu Gln Ser Phe Arg Val Asp Leu Arg Thr Leu Leu Arg Tyr1 5 10
15Ala229PRTArtificial SequenceDescription of Artificial Sequence
Illustrative amino acid sequence 22Gly Phe Arg Ile Leu Leu Leu Lys
Val1 523211PRTArtificial SequenceDescription of Artificial Sequence
SCR1-3 with an additional 14 amino acid residues at the c-terminus
23Met Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn1
5 10 15Leu Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr
Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys
Leu Lys 35 40 45Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg
Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His
Val Ile Lys Gly65 70 75 80Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser
Cys Thr Lys Gly Tyr Arg 85 90 95Leu Ile Gly Ser Ser Ser Ala Thr Cys
Ile Ile Ser Gly Asp Thr Val 100 105 110Ile Trp Asp Asn Glu Thr Pro
Ile Cys Asp Arg Ile Pro Cys Gly Leu 115 120 125Pro Pro Thr Ile Thr
Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn 130 135 140Phe His Tyr
Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly145 150 155
160Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr
165 170 175Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro
Gln Cys 180 185 190Ile Ile Pro Asn Lys Asp Gly Pro Ser Glu Ile Leu
Arg Gly Asp Phe 195 200 205Ser Ser Cys 2102415PRTArtificial
SequenceDescription of Artificial Sequence Illustrative amino acid
sequence 24Ser Ala Ala Pro Ser Ser Gly Phe Arg Ile Leu Leu Leu Lys
Val1 5 10 152572DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 25cgcaccgcag tgcatcatcc
cgaacaaaga tggcccgagc gaaattctgc gtggcgattt 60tagcagctgc ta
722680DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26acgttagcag ctgctaaaat cgccacgcag
aatttcgctc gggccatctt tgttcgggat 60gatgcactgc ggtgcgggcc
802717PRTArtificial SequenceDescription of Artificial Sequence
Myristoyl/Electrostatic Swith Peptide Reagent 1 (MSWP-1) 27Gly Ser
Ser Lys Ser Pro Ser Lys Lys Lys Lys Lys Lys Pro Gly Asp1 5 10
15Cys2817PRTArtificial SequenceDescription of Artificial Sequence
Myristoyl/Electrostatic Switch Peptide Reagent 2 (MSWP-2) 28Cys Asp
Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser1 5 10
15Lys2915PRTArtificial SequenceDescription of Artificial Sequence
Myristoyl/Electrostatic Switch Peptide Reagent 3 (MSWP-3) 29Ser Lys
Asp Gly Lys Lys Lys Lys Lys Lys Ser Lys Thr Lys Cys1 5 10
153016PRTArtificial SequenceDescription of Artificial Sequence
T-cell targeting peptide reagent 1 (TCTP-1) 30Cys Ser Ala Ala Pro
Ser Ser Gly Phe Arg Ile Leu Leu Leu Lys Val1 5 10
1531214PRTArtificial SequenceDescription of Artificial Sequence
[SCR1-3/switch fusion]-[MAET] 31Met Gln Cys Asn Ala Pro Glu Trp Leu
Pro Phe Ala Arg Pro Thr Asn1 5 10 15Leu Thr Asp Glu Phe Glu Phe Pro
Ile Gly Thr Tyr Leu Asn Tyr Glu 20 25 30Cys Arg Pro Gly Tyr Ser Gly
Arg Pro Phe Ser Ile Ile Cys Leu Lys 35 40 45Asn Ser Val Trp Thr Gly
Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys 50 55 60Arg Asn Pro Pro Asp
Pro Val Asn Gly Met Val His Val Ile Lys Gly65 70 75 80Ile Gln Phe
Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys Gly Tyr Arg 85 90 95Leu Ile
Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser Gly Asp Thr Val 100 105
110Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu
115 120 125Pro Pro Thr Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg
Glu Asn 130 135 140Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn
Pro Gly Ser Gly145 150 155 160Gly Arg Lys Val Phe Glu Leu Val Gly
Glu Pro Ser Ile Tyr Cys Thr 165 170 175Ser Asn Asp Asp Gln Val Gly
Ile Trp Ser Gly Pro Ala Pro Gln Cys 180 185 190Ile Ile Pro Asn Lys
Asp Gly Pro Lys Lys Lys Lys Lys Lys Ser Pro 195 200 205Ser Lys Ser
Ser Gly Cys 2103217PRTArtificial SequenceDescription of Artificial
Sequence Illustrative amino acid sequence 32Ala Ala Pro Ser Val Ile
Gly Phe Arg Ile Leu Leu Leu Lys Val Ala1 5 10
15Gly33215PRTArtificial SequenceDescription of Artificial Sequence
SCR1-3 with an additional c-terminal 18 amino acids 33Met Gln Cys
Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro Thr Asn1 5 10 15Leu Thr
Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu Asn Tyr Glu20 25 30Cys
Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile Ile Cys Leu Lys35 40 45
Asn Ser Val Trp Thr Gly Ala Lys Asp Arg Cys Arg Arg Lys Ser Cys50
55 60 Arg Asn Pro Pro Asp Pro Val Asn Gly Met Val His Val Ile Lys
Gly65 70 75 80 Ile Gln Phe Gly Ser Gln Ile Lys Tyr Ser Cys Thr Lys
Gly Tyr Arg 85 90 95Leu Ile Gly Ser Ser Ser Ala Thr Cys Ile Ile Ser
Gly Asp Thr Val 100 105 110Ile Trp Asp Asn Glu Thr Pro Ile Cys Asp
Arg Ile Pro Cys Gly Leu 115 120 125Pro Pro Thr Ile Thr Asn Gly Asp
Phe Ile Ser Thr Asn Arg Glu Asn 130 135 140Phe His Tyr Gly Ser Val
Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly145 150 155 160 Gly Arg Lys
Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys Thr 165 170 175 Ser
Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro Gln Cys 180 185
190 Ile Ile Pro Asn Lys Ala Ala Pro Ser Val Ile Gly Phe Arg Ile Leu
195 200 205 Leu Leu Lys Val Ala Gly Cys 210 2153484DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34cgcaccgcag tgcatcatcc cgaacaaagc ggcgcccagc
gtgattggct tccgtattct 60gctgctgaaa gtggcgggct gcta
843592DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 35agcttagcag cccgccactt tcagcagcag
aatacggaag ccaatcacgc tgggcgccgc 60tttgttcggg atgatgcact gcggtgcggg
cc 923617PRTArtificial SequenceDescription of Artificial Sequence
Illustrative amino acid sequence 36Asp Gly Pro Lys Lys Lys Lys Lys
Lys Ser Pro Ser Lys Ser Ser Gly1 5 10 15Cys3777PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein APT631
37Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala1
5 10 15Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala
Gly 20 25 30Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn
Phe Asn 35 40 45Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr
Tyr Cys Cys 50 55 60Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu
Asn65 70 753817PRTArtificial SequenceDescription of Artificial
Sequence Synthetic protein APT542 38Gly Ser Ser Lys Ser Pro Ser Lys
Lys Lys Lys Lys Lys Pro Gly Asp1 5 10 15Cys3970PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein APT634
39Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala1
5 10 15Val Ala Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala
Gly 20 25 30Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn
Phe Asn 35 40 45Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr
Tyr Cys Cys 50 55 60Lys Lys Asp Leu Cys Asn65 704082PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
APT2060 40Leu Gln Cys Tyr Asn
Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala1 5 10 15Val Asn Cys Ser
Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30Leu Gln Val
Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45Asp Val
Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60Lys
Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Gly Gly Thr65 70 75
80Ser Cys4183PRTArtificial SequenceDescription of Artificial
Sequence Synthetic protein APT635 41Met Leu Gln Cys Tyr Asn Cys Pro
Asn Pro Thr Ala Asp Cys Lys Thr1 5 10 15Ala Val Asn Cys Ser Ser Asp
Phe Asp Ala Cys Leu Ile Thr Lys Ala 20 25 30Gly Leu Gln Val Tyr Asn
Lys Cys Trp Lys Phe Glu His Cys Asn Phe 35 40 45Asn Asp Val Thr Thr
Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys 50 55 60Cys Lys Lys Asp
Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Gly Gly65 70 75 80Thr Ser
Cys4271PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein APT2061 42Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr
Ala Asp Cys Lys Thr Ala1 5 10 15Val Ala Cys Ser Ser Asp Phe Asp Ala
Cys Leu Ile Thr Lys Ala Gly 20 25 30Leu Gln Val Tyr Asn Lys Cys Trp
Lys Phe Glu His Cys Asn Phe Asn 35 40 45Asp Val Thr Thr Arg Leu Arg
Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60Lys Lys Asp Leu Cys Asn
Cys65 704318PRTArtificial SequenceDescription of Artificial
Sequence Illustrative amino acid sequence 43Ala Ala Pro Ser Val Ile
Gly Phe Arg Ile Leu Leu Leu Lys Val Ala1 5 10 15Gly
Cys4414PRTArtificial SequenceDescription of Artificial Sequence
Illustrative amino acid sequence 44Asp Gly Pro Ser Glu Ile Leu Arg
Gly Asp Phe Ser Ser Cys1 5 104536DNAArtificial SequenceDescription
of Artificial Sequence Illustrative oligonucleotide 45cctctggcca
aatgtacctc tcgtgcacat tgctga 3646211PRTArtificial
SequenceDescription of Artificial Sequence Synthetic protein
APT2057 46Met Gly Ser Ser His His His His His His Ser Ser Gly Leu
Val Pro1 5 10 15Arg Gly Ser His Met Ser Cys Glu Val Pro Thr Arg Leu
Asn Ser Ala 20 25 30Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe
Pro Val Gly Thr 35 40 45 Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr
Arg Arg Glu Pro Ser Leu 50 55 60 Ser Pro Lys Leu Thr Cys Leu Gln
Asn Leu Lys Trp Ser Thr Ala Val65 70 75 80 Glu Phe Cys Lys Lys Lys
Ser Cys Pro Asn Pro Gly Glu Ile Arg Asn 85 90 95Gly Gln Ile Asp Val
Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile Ser 100 105 110Phe Ser Cys
Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser Phe 115 120 125Cys
Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro Glu 130 135
140Cys Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly
Ile145 150 155 160Ile Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln
Ser Val Thr Tyr 165 170 175Ala Cys Asn Lys Gly Phe Thr Met Ile Gly
Glu His Ser Ile Tyr Cys 180 185 190Thr Val Asn Asn Asp Glu Gly Glu
Trp Ser Gly Pro Pro Pro Glu Cys 195 200 205Arg Gly Cys
21047274PRTArtificial SequenceDescription of Artificial Sequence
Synthetic protein APT2058 47Met Gly Ser Ser His His His His His His
Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Gln Asp Cys Gly
Leu Pro Pro Asp Val Pro Asn 20 25 30Ala Gln Pro Ala Leu Glu Gly Arg
Thr Ser Phe Pro Glu Asp Thr Val 35 40 45Ile Thr Tyr Lys Cys Glu Glu
Ser Phe Val Lys Ile Pro Gly Glu Lys 50 55 60Asp Ser Val Ile Cys Leu
Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu65 70 75 80Phe Cys Asn Arg
Ser Cys Glu Val Pro Thr Arg Leu Asn Ser Ala Ser 85 90 95Leu Lys Gln
Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly Thr Val 100 105 110Val
Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser 115 120
125Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala Val Glu
130 135 140Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg
Asn Gly145 150 155 160Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly
Ala Thr Ile Ser Phe 165 170 175Ser Cys Asn Thr Gly Tyr Lys Leu Phe
Gly Ser Thr Ser Ser Phe Cys 180 185 190Leu Ile Ser Gly Ser Ser Val
Gln Trp Ser Asp Pro Leu Pro Glu Cys 195 200 205Arg Glu Ile Tyr Cys
Pro Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile 210 215 220Gln Gly Glu
Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala225 230 235
240Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr Cys Thr
245 250 255Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu
Cys Arg 260 265 270Gly Cys 4820PRTArtificial SequenceDescription of
Artificial Sequence Synthetic leader sequence 48Met Gly Ser Ser His
His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His
204931DNAArtificial SequenceDescription of Artificial Sequence
Primer DAF-R 49ggaattctaa gtcagcaagc ccatggttac t
315025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide DAF-F 50gcatatgacc gtcgcgcggc cgagc
2551527PRTHomo sapienstissue plasminogen activator 51Ser Tyr Gln
Val Ile Cys Arg Asp Glu Lys Thr Gln Met Ile Tyr Gln1 5 10 15Gln His
Gln Ser Trp Leu Arg Pro Val Leu Arg Ser Asn Arg Val Glu 20 25 30Tyr
Cys Trp Cys Asn Ser Gly Arg Ala Gln Cys His Ser Val Pro Val 35 40
45Lys Ser Cys Ser Glu Pro Arg Cys Phe Asn Gly Gly Thr Cys Gln Gln
50 55 60Ala Leu Tyr Phe Ser Asp Phe Val Cys Gln Cys Pro Glu Gly Phe
Ala65 70 75 80Gly Lys Cys Cys Glu Ile Asp Thr Arg Ala Thr Cys Tyr
Glu Asp Gln 85 90 95Gly Ile Ser Tyr Arg Gly Thr Trp Ser Thr Ala Glu
Ser Gly Ala Glu 100 105 110Cys Thr Asn Trp Asn Ser Ser Ala Leu Ala
Gln Lys Pro Tyr Ser Gly 115 120 125Arg Arg Pro Asp Ala Ile Arg Leu
Gly Leu Gly Asn His Asn Tyr Cys 130 135 140Arg Asn Pro Asp Arg Asp
Ser Lys Pro Trp Cys Tyr Val Phe Lys Ala145 150 155 160Gly Lys Tyr
Ser Ser Glu Phe Cys Ser Thr Pro Ala Cys Ser Glu Gly 165 170 175Asn
Ser Asp Cys Tyr Phe Gly Asn Gly Ser Ala Tyr Arg Gly Thr His 180 185
190Ser Leu Thr Glu Ser Gly Ala Ser Cys Leu Pro Trp Asn Ser Met Ile
195 200 205Leu Ile Gly Lys Val Tyr Thr Ala Gln Asn Pro Ser Ala Gln
Ala Leu 210 215 220Gly Leu Gly Lys His Asn Tyr Cys Arg Asn Pro Asp
Gly Asp Ala Lys225 230 235 240Pro Trp Cys His Val Leu Lys Asn Arg
Arg Leu Thr Trp Glu Tyr Cys 245 250 255Asp Val Pro Ser Cys Ser Thr
Cys Gly Leu Arg Gln Tyr Ser Gln Pro 260 265 270Gln Phe Arg Ile Lys
Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro 275 280 285Trp Gln Ala
Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg 290 295 300Phe
Leu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser Ala305 310
315 320Ala His Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr Val
Ile 325 330 335Leu Gly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu
Gln Lys Phe 340 345 350Glu Val Glu Lys Tyr Ile Val His Lys Glu Phe
Asp Asp Asp Thr Tyr 355 360 365Asp Asn Asp Ile Ala Leu Leu Gln Leu
Lys Ser Asp Ser Ser Arg Cys 370 375 380Ala Gln Glu Ser Ser Val Val
Arg Thr Val Cys Leu Pro Pro Ala Asp385 390 395 400Leu Gln Leu Pro
Asp Trp Thr Glu Cys Glu Leu Ser Gly Tyr Gly Lys 405 410 415His Glu
Ala Leu Ser Pro Phe Tyr Ser Glu Arg Leu Lys Glu Ala His 420 425
430Val Arg Leu Tyr Pro Ser Ser Arg Cys Thr Ser Gln His Leu Leu Asn
435 440 445Arg Thr Val Thr Asp Asn Met Leu Cys Ala Gly Asp Thr Arg
Ser Gly 450 455 460Gly Pro Gln Ala Asn Leu His Asp Ala Cys Gln Gly
Asp Ser Gly Gly465 470 475 480Pro Leu Val Cys Leu Asn Asp Gly Arg
Met Thr Leu Val Gly Ile Ile 485 490 495Ser Trp Gly Leu Gly Cys Gly
Gln Lys Asp Val Pro Gly Val Tyr Thr 500 505 510Lys Val Thr Asn Tyr
Leu Asp Trp Ile Arg Asp Asn Met Arg Pro515 520 525521947PRTHomo
sapiensCR1 52Gln Cys Asn Ala Pro Glu Trp Leu Pro Phe Ala Arg Pro
Thr Asn Leu1 5 10 15Thr Asp Glu Phe Glu Phe Pro Ile Gly Thr Tyr Leu
Asn Tyr Glu Cys 20 25 30Arg Pro Gly Tyr Ser Gly Arg Pro Phe Ser Ile
Ile Cys Leu Lys Asn 35 40 45Ser Val Trp Thr Gly Ala Lys Asp Arg Cys
Arg Arg Lys Ser Cys Arg 50 55 60Asn Pro Pro Asp Pro Val Asn Gly Met
Val His Val Ile Lys Gly Ile65 70 75 80Gln Phe Gly Ser Gln Ile Lys
Tyr Ser Cys Thr Lys Gly Tyr Arg Leu 85 90 95Ile Gly Ser Ser Ser Ala
Thr Cys Ile Ile Ser Gly Asp Thr Val Ile 100 105 110Trp Asp Asn Glu
Thr Pro Ile Cys Asp Arg Ile Pro Cys Gly Leu Pro 115 120 125Pro Thr
Ile Thr Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu Asn Phe 130 135
140His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser Gly
Gly145 150 155 160Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile
Tyr Cys Thr Ser 165 170 175Asn Asp Asp Gln Val Gly Ile Trp Ser Gly
Pro Ala Pro Gln Cys Ile 180 185 190Ile Pro Asn Lys Cys Thr Pro Pro
Asn Val Glu Asn Gly Ile Leu Val 195 200 205Ser Asp Asn Arg Ser Leu
Phe Ser Leu Asn Glu Val Val Glu Phe Arg 210 215 220Cys Gln Pro Gly
Phe Val Met Lys Gly Pro Arg Arg Val Lys Cys Gln225 230 235 240Ala
Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg Val Cys 245 250
255Gln Pro Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg Asp Lys
260 265 270Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu
Pro Gly 275 280 285Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr
Pro Gln Gly Asp 290 295 300Trp Ser Pro Ala Ala Pro Thr Cys Glu Val
Lys Ser Cys Asp Asp Phe305 310 315 320Met Gly Gln Leu Leu Asn Gly
Arg Val Leu Phe Pro Val Asn Leu Gln 325 330 335Leu Gly Ala Lys Val
Asp Phe Val Cys Asp Glu Gly Phe Gln Leu Lys 340 345 350Gly Ser Ser
Ala Ser Tyr Cys Val Leu Ala Gly Met Glu Ser Leu Trp 355 360 365Asn
Ser Ser Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser Pro Pro 370 375
380Val Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val Phe
Pro385 390 395 400Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His
Pro Asp Arg Gly 405 410 415Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr
Ile Arg Cys Thr Ser Asp 420 425 430Pro Gln Gly Asn Gly Val Trp Ser
Ser Pro Ala Pro Arg Cys Gly Ile 435 440 445Leu Gly His Cys Gln Ala
Pro Asp His Phe Leu Phe Ala Lys Leu Lys 450 455 460Thr Gln Thr Asn
Ala Ser Asp Phe Pro Ile Gly Thr Ser Leu Lys Tyr465 470 475 480Glu
Cys Arg Pro Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr Cys Leu 485 490
495Asp Asn Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg Lys Ser
500 505 510Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val
Ile Thr 515 520 525Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys
Thr Thr Gly His 530 535 540Arg Leu Ile Gly His Ser Ser Ala Glu Cys
Ile Leu Ser Gly Asn Ala545 550 555 560Ala His Trp Ser Thr Lys Pro
Pro Ile Cys Gln Arg Ile Pro Cys Gly 565 570 575Leu Pro Pro Thr Ile
Ala Asn Gly Asp Phe Ile Ser Thr Asn Arg Glu 580 585 590Asn Phe His
Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Pro Gly Ser 595 600 605Gly
Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile Tyr Cys 610 615
620Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser Gly Pro Ala Pro
Gln625 630 635 640Cys Ile Ile Pro Asn Lys Cys Thr Pro Pro Asn Val
Glu Asn Gly Ile 645 650 655Leu Val Ser Asp Asn Arg Ser Leu Phe Ser
Leu Asn Glu Val Val Glu 660 665 670Phe Arg Cys Gln Pro Gly Phe Val
Met Lys Gly Pro Arg Arg Val Lys 675 680 685Cys Gln Ala Leu Asn Lys
Trp Glu Pro Glu Leu Pro Ser Cys Ser Arg 690 695 700Val Cys Gln Pro
Pro Pro Asp Val Leu His Ala Glu Arg Thr Gln Arg705 710 715 720Asp
Lys Asp Asn Phe Ser Pro Gly Gln Glu Val Phe Tyr Ser Cys Glu 725 730
735Pro Gly Tyr Asp Leu Arg Gly Ala Ala Ser Met Arg Cys Thr Pro Gln
740 745 750Gly Asp Trp Ser Pro Ala Ala Pro Thr Cys Glu Val Lys Ser
Cys Asp 755 760 765Asp Phe Met Gly Gln Leu Leu Asn Gly Arg Val Leu
Phe Pro Val Asn 770 775 780Leu Gln Leu Gly Ala Lys Val Asp Phe Val
Cys Asp Glu Gly Phe Gln785 790 795 800Leu Lys Gly Ser Ser Ala Ser
Tyr Cys Val Leu Ala Gly Met Glu Ser 805 810 815Leu Trp Asn Ser Ser
Val Pro Val Cys Glu Gln Ile Phe Cys Pro Ser 820 825 830Pro Pro Val
Ile Pro Asn Gly Arg His Thr Gly Lys Pro Leu Glu Val 835 840 845Phe
Pro Phe Gly Lys Ala Val Asn Tyr Thr Cys Asp Pro His Pro Asp 850 855
860Arg Gly Thr Ser Phe Asp Leu Ile Gly Glu Ser Thr Ile Arg Cys
Thr865 870 875 880Ser Asp Pro Gln Gly Asn Gly Val Trp Ser Ser Pro
Ala Pro Arg Cys 885 890 895Gly Ile Leu Gly His Cys Gln Ala Pro Asp
His Phe Leu Phe Ala Lys 900 905 910Leu Lys Thr Gln Thr Asn Ala Ser
Asp Phe Pro Ile Gly Thr Ser Leu 915 920 925Lys Tyr Glu Cys Arg Pro
Glu Tyr Tyr Gly Arg Pro Phe Ser Ile Thr 930 935 940Cys Leu Asp Asn
Leu Val Trp Ser Ser Pro Lys Asp Val Cys Lys Arg945 950 955 960Lys
Ser Cys Lys Thr Pro Pro Asp Pro Val Asn Gly Met Val His Val 965 970
975Ile Thr Asp Ile Gln Val Gly Ser Arg Ile Asn Tyr Ser Cys Thr Thr
980 985 990Gly His Arg Leu Ile Gly His Ser Ser Ala Glu Cys Ile Leu
Ser Gly 995 1000 1005Asn Thr Ala His Trp Ser Thr Lys Pro
Pro Ile Cys Gln Arg Ile Pro 1010 1015 1020Cys Gly Leu Pro Pro Thr
Ile Ala Asn Gly Asp Phe Ile Ser Thr Asn1025 1030 1035 1040Arg Glu
Asn Phe His Tyr Gly Ser Val Val Thr Tyr Arg Cys Asn Leu 1045 1050
1055Gly Ser Arg Gly Arg Lys Val Phe Glu Leu Val Gly Glu Pro Ser Ile
1060 1065 1070Tyr Cys Thr Ser Asn Asp Asp Gln Val Gly Ile Trp Ser
Gly Pro Ala 1075 1080 1085Pro Gln Cys Ile Ile Pro Asn Lys Cys Thr
Pro Pro Asn Val Glu Asn 1090 1095 1100Gly Ile Leu Val Ser Asp Asn
Arg Ser Leu Phe Ser Leu Asn Glu Val1105 1110 1115 1120Val Glu Phe
Arg Cys Gln Pro Gly Phe Val Met Lys Gly Pro Arg Arg 1125 1130
1135Val Lys Cys Gln Ala Leu Asn Lys Trp Glu Pro Glu Leu Pro Ser Cys
1140 1145 1150Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu His Gly
Glu His Thr 1155 1160 1165Pro Ser His Gln Asp Asn Phe Ser Pro Gly
Gln Glu Val Phe Tyr Ser 1170 1175 1180Cys Glu Pro Gly Tyr Asp Leu
Arg Gly Ala Ala Ser Leu His Cys Thr1185 1190 1195 1200Pro Gln Gly
Asp Trp Ser Pro Glu Ala Pro Arg Cys Ala Val Lys Ser 1205 1210
1215Cys Asp Asp Phe Leu Gly Gln Leu Pro His Gly Arg Val Leu Phe Pro
1220 1225 1230Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe Val Cys
Asp Glu Gly 1235 1240 1245Phe Arg Leu Lys Gly Ser Ser Val Ser His
Cys Val Leu Val Gly Met 1250 1255 1260Arg Ser Leu Trp Asn Asn Ser
Val Pro Val Cys Glu His Ile Phe Cys1265 1270 1275 1280Pro Asn Pro
Pro Ala Ile Leu Asn Gly Arg His Thr Gly Thr Pro Ser 1285 1290
1295Gly Asp Ile Pro Tyr Gly Lys Glu Ile Ser Tyr Thr Cys Asp Pro His
1300 1305 1310Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly Glu Ser
Thr Ile Arg 1315 1320 1325Cys Thr Ser Asp Pro His Gly Asn Gly Val
Trp Ser Ser Pro Ala Pro 1330 1335 1340Arg Cys Glu Leu Ser Val Arg
Ala Gly His Cys Lys Thr Pro Glu Gln1345 1350 1355 1360Phe Pro Phe
Ala Ser Pro Thr Ile Pro Ile Asn Asp Phe Glu Phe Pro 1365 1370
1375Val Gly Thr Ser Leu Asn Tyr Glu Cys Arg Pro Gly Tyr Phe Gly Lys
1380 1385 1390Met Phe Ser Ile Ser Cys Leu Glu Asn Leu Val Trp Ser
Ser Val Glu 1395 1400 1405Asp Asn Cys Arg Arg Lys Ser Cys Gly Pro
Pro Pro Glu Pro Phe Asn 1410 1415 1420Gly Met Val His Ile Asn Thr
Asp Thr Gln Phe Gly Ser Thr Val Asn1425 1430 1435 1440Tyr Ser Cys
Asn Glu Gly Phe Arg Leu Ile Gly Ser Pro Ser Thr Thr 1445 1450
1455Cys Leu Val Ser Gly Asn Asn Val Thr Trp Asp Lys Lys Ala Pro Ile
1460 1465 1470Cys Glu Ile Ile Ser Cys Glu Pro Pro Pro Thr Ile Ser
Asn Gly Asp 1475 1480 1485Phe Tyr Ser Asn Asn Arg Thr Ser Phe His
Asn Gly Thr Val Val Thr 1490 1495 1500Tyr Gln Cys His Thr Gly Pro
Asp Gly Glu Gln Leu Phe Glu Leu Val1505 1510 1515 1520Gly Glu Arg
Ser Ile Tyr Cys Thr Ser Lys Asp Asp Gln Val Gly Val 1525 1530
1535Trp Ser Ser Pro Pro Pro Arg Cys Ile Ser Thr Asn Lys Cys Thr Ala
1540 1545 1550Pro Glu Val Glu Asn Ala Ile Arg Val Pro Gly Asn Arg
Ser Phe Phe 1555 1560 1565Ser Leu Thr Glu Ile Ile Arg Phe Arg Cys
Gln Pro Gly Phe Val Met 1570 1575 1580Val Gly Ser His Thr Val Gln
Cys Gln Thr Asn Gly Arg Trp Gly Pro1585 1590 1595 1600Lys Leu Pro
His Cys Ser Arg Val Cys Gln Pro Pro Pro Glu Ile Leu 1605 1610
1615His Gly Glu His Thr Leu Ser His Gln Asp Asn Phe Ser Pro Gly Gln
1620 1625 1630Glu Val Phe Tyr Ser Cys Glu Pro Ser Tyr Asp Leu Arg
Gly Ala Ala 1635 1640 1645Ser Leu His Cys Thr Pro Gln Gly Asp Trp
Ser Pro Glu Ala Pro Arg 1650 1655 1660Cys Thr Val Lys Ser Cys Asp
Asp Phe Leu Gly Gln Leu Pro His Gly1665 1670 1675 1680Arg Val Leu
Leu Pro Leu Asn Leu Gln Leu Gly Ala Lys Val Ser Phe 1685 1690
1695Val Cys Asp Glu Gly Phe Arg Leu Lys Gly Arg Ser Ala Ser His Cys
1700 1705 1710Val Leu Ala Gly Met Lys Ala Leu Trp Asn Ser Ser Val
Pro Val Cys 1715 1720 1725Glu Gln Ile Phe Cys Pro Asn Pro Pro Ala
Ile Leu Asn Gly Arg His 1730 1735 1740Thr Gly Thr Pro Phe Gly Asp
Ile Pro Tyr Gly Lys Glu Ile Ser Tyr1745 1750 1755 1760Ala Cys Asp
Thr His Pro Asp Arg Gly Met Thr Phe Asn Leu Ile Gly 1765 1770
1775Glu Ser Ser Ile Arg Cys Thr Ser Asp Pro Gln Gly Asn Gly Val Trp
1780 1785 1790Ser Ser Pro Ala Pro Arg Cys Glu Leu Ser Val Pro Ala
Ala Cys Pro 1795 1800 1805His Pro Pro Lys Ile Gln Asn Gly His Tyr
Ile Gly Gly His Val Ser 1810 1815 1820Leu Tyr Leu Pro Gly Met Thr
Ile Ser Tyr Thr Cys Asp Pro Gly Tyr1825 1830 1835 1840Leu Leu Val
Gly Lys Gly Phe Ile Phe Cys Thr Asp Gln Gly Ile Trp 1845 1850
1855Ser Gln Leu Asp His Tyr Cys Lys Glu Val Asn Cys Ser Phe Pro Leu
1860 1865 1870Phe Met Asn Gly Ile Ser Lys Glu Leu Glu Met Lys Lys
Val Tyr His 1875 1880 1885Tyr Gly Asp Tyr Val Thr Leu Lys Cys Glu
Asp Gly Tyr Thr Leu Glu 1890 1895 1900Gly Ser Pro Trp Ser Gln Cys
Gln Ala Asp Asp Arg Trp Asp Pro Pro1905 1910 1915 1920Leu Ala Lys
Cys Thr Ser Arg Ala His Cys Cys Asp Gly Pro Lys Lys 1925 1930
1935Lys Lys Lys Lys Ser Pro Ser Lys Ser Ser Gly 1940
19455310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5
10
* * * * *