U.S. patent application number 11/982085 was filed with the patent office on 2008-10-16 for hybrid immunoglobulins with moving parts.
Invention is credited to Daniel J. Capon.
Application Number | 20080254512 11/982085 |
Document ID | / |
Family ID | 39854063 |
Filed Date | 2008-10-16 |
United States Patent
Application |
20080254512 |
Kind Code |
A1 |
Capon; Daniel J. |
October 16, 2008 |
Hybrid immunoglobulins with moving parts
Abstract
Hybrid immunoglobulins containing moving parts are provided as
well as related compositions and methods of use and methods of
production. In addition, analogous genetic devices are provided as
well as related compositions and methods of use and methods of
production.
Inventors: |
Capon; Daniel J.;
(Hillsborough, CA) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Family ID: |
39854063 |
Appl. No.: |
11/982085 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60856864 |
Nov 2, 2006 |
|
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Current U.S.
Class: |
435/69.6 ;
530/387.3; 530/402 |
Current CPC
Class: |
A61P 17/06 20180101;
A61P 29/00 20180101; A61K 47/6875 20170801; A61K 51/1084 20130101;
C07K 2317/52 20130101; C07K 2319/30 20130101; A61P 35/04 20180101;
C12N 15/625 20130101; A61P 11/06 20180101; C12N 15/62 20130101;
A61P 19/02 20180101; A61P 35/02 20180101; A61K 47/6835 20170801;
A61P 35/00 20180101 |
Class at
Publication: |
435/69.6 ;
530/387.3; 530/402 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C07K 16/18 20060101 C07K016/18; C07K 1/00 20060101
C07K001/00 |
Claims
1. A compound comprising a first stretch of consecutive amino
acids, each of which is joined to the preceding amino acid by a
peptide bond and the sequence of which comprises a binding site for
a target; and a second stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which is identical to the sequence of the first
stretch of consecutive amino acids and which comprises an identical
binding site for the target; wherein each of the first stretch of
amino acids and the second stretch of amino acids has at a
predefined end thereof a cysteine residue or a selenocysteine
residue and such cysteine residues or such selenocysteine residues
are joined by a bond having the structure: ##STR00012## wherein
each X is the same and represents a sulfur (S) or a selenium (Se)
and each C represents a beta-carbon of one of such cysteine or
selenocysteine residues.
2-7. (canceled)
8. A compound comprising a first stretch of consecutive amino
acids, each of which is joined to the preceding amino acid by a
peptide bond and the sequence of which comprises a binding site for
a target; and a second stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which is different from the sequence of the first
stretch of consecutive amino acids and which comprises a binding
site for a different moiety; wherein each of the first stretch of
amino acids and the second stretch of amino acids has at a
predefined end thereof a cysteine residue or a selenocysteine
residue and such residues are joined by a bond having the
structure: ##STR00013## wherein each X may be the same or different
and represents a sulfur (S) or a selenium (Se) and each C
represents a beta-carbon of one of such cysteine or selenocysteine
residues.
9-17. (canceled)
18. A multimer comprising two or more identical compounds according
to any one of claims 1 or 8 joined together by at least one
bond.
19-50. (canceled)
51. A method of affecting the activity of a target comprising
contacting the target with a composition comprising the compound of
claim 1 under conditions such that the compound binds to and
affects the activity of the target.
52-62. (canceled)
63. A process of making the compound of claim 1, comprising: (a)
transfecting a cell with a recombinant nucleic acid which comprises
(i) a first portion, the sequence of which is a N-terminal signal
sequence, contiguous with (ii) a second portion, the sequence of
which encodes a stretch of consecutive amino acids contiguous with
(iii) a third portion, the sequence of which encodes a C-terminal
intein-containing binding domain, under conditions permitting
synthesis of a chimeric polypeptide comprising the stretch of
consecutive amino acids contiguous with the C-terminal
intein-containing binding domain; (b) isolating the chimeric
polypeptide produced in step (a); (c) treating the chimeric
polypeptide so as to cause thio-mediated cleavage of the C-terminal
intein-containing binding domain from the stretch of consecutive
amino acids and its replacement with a C-terminal thioester; (d)
treating the product of step (c) to permit the attachment of a
cysteine residue to the product so as to form product with a
C-terminal cysteine; and (e) oxidizing the product of step (e) in
the presence of another product of step (e) under conditions
permitting formation of the compound.
64-69. (canceled)
70. A compound comprising an independently folding protein domain
fused to a second independently folding protein domain by
non-peptide bond.
71-74. (canceled)
75. A method of making a stretch of consecutive amino acids
comprising an N-terminal cysteine comprising: (a) transfecting a
cell with a recombinant nucleic acid which comprises (i) a first
portion, the sequence of which encodes a N-terminal signal sequence
contiguous with (ii) a second portion, the sequence of which
encodes a stretch of consecutive amino acids comprising a
N-terminal cysteine residue, under conditions permitting (i)
synthesis of a chimeric polypeptide which comprises the N-terminal
signal sequence joined by a peptide bond at its C-terminus to the
N-terminal cysteine of the stretch of consecutive amino acids and
(ii) cleavage of the N-terminal signal sequence from the chimeric
polypeptide within the cell so as to produce a stretch of
consecutive amino acids comprising an N-terminal cysteine; (b)
recovering the stretch of consecutive amino acids produced in step
(a).
76-154. (canceled)
155. A process for making a compound comprising contacting a
stretch of consecutive amino acids, each of which is joined to the
preceding amino acid by a peptide bond and the sequence of which
comprises a binding site for a target with a second stretch of
consecutive amino acids, each of which is joined to the preceding
amino acid by a peptide bond and the sequence of which is identical
to the sequence of the first stretch of consecutive amino acids and
which comprises an identical binding site for the target, wherein
each of the first stretch of amino acids and the second stretch of
amino acids has at a predefined end thereof a cysteine residue or a
selenocysteine residue, under reducing conditions so as to make the
compound.
156. A process for making a compound comprising contacting a
stretch of consecutive amino acids, each of which is joined to the
preceding amino acid by a peptide bond and the sequence of which
comprises a binding site for a target with a second stretch of
consecutive amino acids, each of which is joined to the preceding
amino acid by a peptide bond and the sequence of which is different
to the sequence of the first stretch of consecutive amino acids and
which comprises an identical binding site for the target, wherein
each of the first stretch of amino acids and the second stretch of
amino acids has at a predefined end thereof a cysteine residue or a
selenocysteine residue, under reducing conditions so as to make the
compound.
157-164. (canceled)
165. A compound comprising a first stretch of consecutive amino
acids, each of which is joined to the preceding amino acid by a
peptide bond and the sequence of which comprises a binding site for
a target; and a second stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which is identical to the sequence of the first
stretch of consecutive amino acids and which comprises an identical
binding site for the target; wherein each of the first stretch of
amino acids and the second stretch of amino acids has at a
predefined end thereof, independently, a natural amino acid or
non-natural amino having a linear aliphatic side-chain acid
comprising a sulfur (S) or a selenium (Se) and wherein such sulfur
(S) or a selenium (Se) are joined by a bond having the structure:
##STR00014## wherein each X is a sulfur (S) or a selenium (Se) and
each (C) represents a carbon of the linear aliphatic side-chain of
one of such natural or non-natural amino acid and wherein n and m
are, independently, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
166-170. (canceled)
171. A compound comprising a first stretch of consecutive amino
acids, each of which is joined to the preceding amino acid by a
peptide bond and the sequence of which comprises a binding site for
a target; and a second stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which is different from the sequence of the first
stretch of consecutive amino acids and which comprises a binding
site for a different moiety; wherein each of the first stretch of
amino acids and the second stretch of amino acids has at a
predefined end thereof, independently, a natural amino acid or
non-natural amino having a linear aliphatic side-chain acid
comprising a sulfur (S) or a selenium (Se) and wherein such sulfur
(S) or a selenium (Se) are joined by a bond having the structure:
##STR00015## wherein each X may be the same or different and
represents a sulfur (S) or a selenium (Se) and each (C) represents
a carbon of the linear aliphatic side-chain of one of such natural
or non-natural amino acid and wherein n and m are, independently,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
172-176. (canceled)
177. A method of producing a protein which comprises a first
polypeptide contiguous with an intein, which intein is contiguous
with a second polypeptide comprising a binding domain, the method
comprising transfecting an animal cell with a nucleic acid, which
nucleic acid comprises (i) a first portion which encodes the
polypeptide contiguous with (ii) a second portion which encodes the
intein, contiguous with a and the third portion of which encodes
the binding domain, under conditions such that the animal cell
expresses and secretes the protein.
178-181. (canceled)
182. A compound comprising: a first stretch of consecutive amino
acids each of which is joined to the preceding amino acid by a
peptide bond and which first stretch of consecutive amino acids
comprises an amino acid residue having a chalcogen functional
group-containing side chain; and a second stretch of consecutive
amino acids, comprising at least 100 amino acids, each of which is
joined to the preceding amino acid by a peptide bond, wherein at
least 90 consecutive amino acids thereof of the second stretch of
consecutive amino acids have a sequence identical to portion of a
human immunoglobulin constant region polypeptide, and wherein the
second stretch of consecutive amino acids comprises an amino acid
residue having a chalcogen functional group-containing side chain
at a predefined terminus thereof, wherein said amino acid residue
having a chalcogen functional group-containing side chain of the
first stretch of consecutive amino acids and said amino acid
residue having a chalcogen functional group-containing side chain
of the second stretch of consecutive amino acids are joined by a
bond having the structure: ##STR00016## wherein each X represents,
independently, a chalcogen, and wherein C.sub.1 represents a side
chain carbon of the amino acid residue having a chalcogen
functional group-containing side chain of the first stretch of
consecutive amino acids and C.sub.2 represents a side chain carbon
of the second stretch of consecutive amino acids.
183-194. (canceled)
195. The compound of claim 182, wherein the human immunoglobulin
constant region polypeptide is a human IgG1, human IgG2, human
IgG3, or human IgG4.
196. The compound of claim 195, wherein the side chain of at least
one of amino acid residues having a chalcogen functional
group-containing side chain comprises a C1-C10 alkylene.
197. A composition comprising two of the compounds of claim 182
bonded together via at least one disulfide bond between the second
stretch of consecutive amino acids of each of the compounds.
198. A polypeptide consisting of consecutive amino acids having the
sequence set forth in one of SEQ ID NOS:35 through 46, or having
the sequence set forth in one of SEQ ID NOS:53 through 67, or
having the sequence set forth in one of SEQ ID NOS:74 through 82,
or having the sequence set forth in one of SEQ ID NOS:89 through
97.
199-201. (canceled)
202. A polypeptide consisting of consecutive amino acids having a
sequence identical to a portion of the sequence set forth in SEQ ID
NO:44, SEQ ID NO:64, SEQ ID NO:81 or SEQ ID NO:96, wherein at least
one of the terminal residues of the polypeptide has a chalcogen
functional group-containing side chain.
203. The polypeptide of claim 202, wherein the terminal residue
having a chalcogen functional group-containing side chain is a
cysteine or analog thereof
Description
[0001] This application claims benefit of U.S. Provisional
Application No. 60/856,864, filed Nov. 2, 2006, the contents of
which are hereby incorporated by reference into this
application.
[0002] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art as
known to those skilled therein as of the date of the invention
described and claimed herein.
BACKGROUND OF THE INVENTION
[0003] All machines and devices have moving parts. The function of
the moving parts is to perform work, by transforming a source of
energy, in order to carry out a useful task. Moving parts cover a
spectrum of sizes and shapes. At one end of the spectrum is a
visible world evident in machines that perform mechanical tasks. At
the other end of the spectrum is an invisible world of charge
carriers utilized by devices that carry out electrical work.
[0004] This spectrum is so vast that certain of its regions have
only begun to be technologically exploited. Among these are devices
with moving parts of several nano-meters to several hundred
nano-meters. This size range holds considerable interest to many
scientists and engineers because it is comparable to the very size
of molecules, the fundamental units of chemical matter.
Nano-machines have the potential to exploit the unique properties
of molecules, such as intermolecular binding or catalysis.
[0005] The ability to make molecules of any imaginable size and
shape is one activity crucial in building nano-machines. As such it
has been widely anticipated in medicine, electronics, optics, and
many other fields. Tremendous commercial activity has been focused
on the synthesis very large numbers of chemically distinct
molecules. However, molecular configuration (differences in
bonding) is just one practical means of generating diversity.
Molecular conformation (differences in bond rotation) offers
another important avenue for generating a universe of continuous
size and shape.
[0006] Molecular conformation has certain unique advantages in
strategies for creating molecules with moving parts. While atoms
and chemical bonds have precise linear and angular dimensions,
conformational change can provide limitless variation in the size
and shape of molecules. Covalent and non-covalent chemical bonds
both afford rotational degrees of freedom. Dihedral rotation around
each of a series of bonds connecting distinct parts (domains) of a
molecule is capable of providing the essential dynamic ingredient
of nano-machines.
[0007] In general, any two given atoms interconnected by a single
bond (i.e., a single electron pair) can rotate fully 360 degrees
with respect to each other and with respect to the other atoms that
each is bonded to. A series of consecutive single bonds is like a
series of interconnected ball joints. Although limited to rotary
motions, a series of consecutive single bonds, like a series of
consecutive ball joints, can recapitulate the movement of other
types of interconnected moving parts (e.g., a series of consecutive
hinges).
[0008] One challenging aspect of creating useful nano-machines is
striking a balance in the number of moving parts and the number of
interconnections. Above a certain threshold, increasing the number
of parts or connections in any machine is counterproductive. Thus
automobile engines employ an optimal number of pistons, valves,
camshafts, pulleys, and so forth.
[0009] The analogous challenge in the chemical field is illustrated
by two related, but very different types of molecules, namely
organic and biological polymers. A good comparison is provided by
polyethylene and proteins. Polyethylenes are stretches of
consecutive ethylenes, (CH.sub.2).sub.n, interconnected by
consecutive single bonds (--C--).sub.n, while proteins are
stretches of consecutive amino acids, (NHCHRCO).sub.n,
interconnected by consecutive peptide bonds
(.dbd.N--C--C.dbd.).sub.n. Unbranched polyethylenes are repeating
chains of single bonds, while proteins are repeating chains of one
double bond followed by two single bonds. The most important
difference between these two types of chains is that polyethylene
can adopt almost any conformation and thus has no definite size or
shape (only a statistically averaged one), while proteins are
extremely rigid and thus have very definite (and unchanging) size
and shape.
[0010] A simple but reasonable comparison to a mechanical device
would represent polyethylene as a machine with a high ratio of
moving parts to connections, and a protein as a machine with an low
ratio of moving parts to connections. Neither molecule is very
suited to a machine-like task unless one takes advantage of higher
order structures that each can form. For example, polyethylene is
useful when its ability to form intermolecular fibers is exploited.
Interesting, the ability of polyethylene to display such tertiary
structure depends upon its inherent flexibility. Although some
proteins can form fibers of commercial value (e.g., silk, wool and
collagen), most proteins are globular and do not.
[0011] Globular proteins are, for nearly all practical purposes,
machines with few if any moving parts, like a crowbar that must
exert its leverage in combination with other objects, such as the
human that wields it and the objects against which it is wedged.
Notwithstanding, there are many instances in which there would be
great value to protein-like molecules having distinct regions
(e.g., binding domains) that are joined together in some manner
permitting relative, yet coordinated movement. One example would be
protein-like molecules capable of cooperatively binding a disease
target having two or more identical binding sites. This would take
full advantage of the unique properties of globular protein binding
domains, namely their great specificity for targets, particularly
other proteins associated with disease.
[0012] The potential commercial value of protein-like molecules
that are able to cooperatively bind a disease target may be
estimated quantitatively. A starting assumption is that most
therapeutics in current use, whether small molecules or
biopharmaceuticals, typically bind their targets non-cooperatively
with affinity constants on the order of nano-molar (10.sup.-9 M).
Remarkably, a cooperative therapeutic could conceivably bind the
same target, with an affinity of nano-molar.times.nano-molar
(10.sup.-9 M.times.10.sup.-9 M) [i.e., atto-molar (10.sup.-18
M)].
[0013] Because therapeutics are typically required in great molar
excess over their targets (about one million-fold), a cooperative
therapeutic would thus be equivalent to a non-cooperative
therapeutic at a 10.sup.-6 smaller dose. For many current
biopharmaceuticals (e.g. antibodies and immunoadhesins) this
difference amounts to 1 microgram per single dose instead of 1 gram
per single dose. With patient costs exceeding $1,000 per gram, this
factor has great significance in new drug discovery and development
as well as for existing biopharmaceuticals.
[0014] One irony associated with antibodies and immunoadhesins is
that while they are symmetric proteins having two identical binding
domains, they do not generally bind symmetrically to symmetric
targets. The inflexible connections between the two binding domains
do not provide the machine-like motion that would permit
cooperative binding. Numerous attempts to engineer antibodies and
immunoadhesins that bind symmetrically have failed because of the
difficulty in achieving the precise geometry needed for
complementary symmetries between the binding sites and target
sites. Unlike materials used to make conventional machines, such as
wood, metals, plastics, ceramics, and the like, molecules cannot
simply be cut, wrought, cast, machined or joined to an exact size
and shape.
[0015] While cooperative binding is thus not readily achieved with
any single fixed size and shape, conformational flexibility between
binding domains does provides a potential solution. A "one size
fits all" strategy is based upon the proposition that a
protein-like molecule with binding domains that move symmetrically
will also be capable of binding symmetrically (i.e., cooperativity)
The binding domains are driven thermodynamically into a
conformation most compatible with simultaneous binding of both
target sites because it represents the energetically favored
conformational minima.
SUMMARY OF THE INVENTION
[0016] In an embodiment this invention provides a compound
comprising a first stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which comprises a binding site for a target; and a
second stretch of consecutive amino acids, each of which is joined
to the preceding amino acid by a peptide bond and the sequence of
which is identical to the sequence of the first stretch of
consecutive amino acids and which comprises an identical binding
site for the target; wherein each of the first stretch of amino
acids and the second stretch of amino acids has at a predefined end
thereof a cysteine residue or a selenocysteine residue and such
cysteine residues or such selenocysteine residues are joined by a
bond having the structure:
##STR00001##
wherein each X is the same and represents a sulfur (S) or a
selenium (Se) and each C represents a beta-carbon of one of such
cysteine or selenocysteine residues.
[0017] In an embodiment this invention also provides a compound
comprising a first stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which comprises a binding site for a target; and a
second stretch of consecutive amino acids, each of which is joined
to the preceding amino acid by a peptide bond and the sequence of
which is different from the sequence of the first stretch of
consecutive amino acids and which comprises a binding site for a
different moiety; wherein each of the first stretch of amino acids
and the second stretch of amino acids has at a predefined end
thereof a cysteine residue or a selenocysteine residue and such
residues are joined by a bond having the structure:
##STR00002##
wherein each X may be the same or different and represents a sulfur
(S) or a selenium (Se) and each C represents a beta-carbon of one
of such cysteine or selenocysteine residues.
[0018] Genetic devices disclosed herein comprise two or more
stretches of consecutive amino acids that are connected at a
predefined terminus by a non-peptide bond. Such genetic devices are
both symmetric and symmetrically binding with respect to one or
more important targets (i.e., cooperative). The genetic devices
herein are protein-like molecules may be described by a number of
related terms that include symmetroadhesins,
immuno-symmetroadhesins, hemi-symmetroadhesins, and
bi-symmetroadhesins [meaning "stick to proportionately," from the
Gk. symmetros "having a common measure, even, proportionate," and
the L. adhaerentem, prp. of adhaerere "stick to"].
[0019] Disclosed herein is a compound comprising two or more
independently-folding protein domains linked to one another through
one or more non-peptide bonds, around which bond(s) dihedral
rotation may occur.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1: Stretches of consecutive amino acids, with one
X-terminus, depicting positions of N-, C-, S-, and Se-termini.
N-terminal and C-terminal amino acid residues, drawn as a
Newman-style projection, are shown above and below the projection
plane, respectively: (i) A stretch of consecutive amino acids
(generalized structure), having, an N-terminal amino acid residue
(sidechain=R1) having a free .alpha.-amino (NH.sub.2) group, and a
C-terminal amino acid residue (sidechain=Rn) having a free
.alpha.-carboxyl (COOH) group. (ii) A stretch of consecutive amino
acids, with N-terminal S-terminus, having, an N-terminal cysteine
having free .alpha.-amino (NH.sub.2) and .beta.-sulfhydryl (SH)
groups, and a C-terminal amino acid residue having a free
.alpha.-carboxyl (COOH) group. (iii) A stretch of consecutive amino
acids, with C-terminal S-terminus, having, an N-terminal amino acid
residue having a free .alpha.-amino (NH.sub.2) group, and a
C-terminal cysteine having free .alpha.-carboxyl (COOH) and
.beta.-sulfhydryl (SH) groups. (iv) A stretch of consecutive amino
acids, with N-terminal Se-terminus, having, an N-terminal
selenocysteine having free .alpha.-amino (NH.sub.2) and
.beta.-selenohydryl (SeH) groups, and a C-terminal amino acid
residue having a free .alpha.-carboxyl (COOH) group. (v) A stretch
of consecutive amino acids, with C-terminal Se-terminus, having, a
N-terminal amino acid residue having a free .alpha.-amino
(NH.sub.2) group, and a C-terminal selenocysteine having free
.alpha.-carboxyl (COOH) and .beta.-selenohydryl (SeH) groups.
[0021] FIG. 2: Stretches of consecutive amino acids, with two
X-termini, depicting positions of N-, C-, S-, and Se-termini.
N-terminal and C-terminal amino acid residues, drawn as a
Newman-style projection, are shown above and below the projection
plane, respectively. (i) A stretch of consecutive amino acids, with
two X-termini (generalized structure), having, an N-terminal amino
acid residue (sidechain=X1) having a free .alpha.-amino (NH.sub.2)
group, and a C-terminal amino acid residue (sidechain=Xn) having a
free .alpha.-carboxyl (COOH) group. (ii) A stretch of consecutive
amino acids, with N-terminal S-terminus and C-terminal S-terminus,
having, an N-terminal cysteine having free .alpha.-amino (NH.sub.2)
and .beta.-sulfhydryl (SH) groups, and a C-terminal cysteine having
free .alpha.-carboxyl (COOH) and .beta.-sulfhydryl (SH) groups.
(iii) A stretch of consecutive amino acids, with N-terminal
S-terminus and C-terminal Se-terminus, having, an N-terminal
cysteine having free .alpha.-amino (NH.sub.2) and .beta.-sulfhydryl
(SH) groups, and a C-terminal selenocysteine having free
.alpha.-carboxyl (COOH) and .beta.-selenohydryl (SeH) groups. (iv)
A stretch of consecutive amino acids, with N-terminal Se-terminus
and C-terminal S-terminus, having, an N-terminal selenocysteine
having free .alpha.-amino (NH.sub.2) and .beta.-selenohydryl (SeH)
groups, and a C-terminal cysteine having free .alpha.-carboxyl
(COOH) and .beta.-sulfhydryl (SH) groups. (v) A stretch of
consecutive amino acids, with N-terminal Se-terminus and C-terminal
Se-terminus, having, an N-terminal selenocysteine having free
.alpha.-amino (NH.sub.2) and .beta.-selenohydryl (SeH) groups, and
a C-terminal selenocysteine having free .alpha.-carboxyl (COOH) and
.beta.-selenohydryl (SeH) groups.
[0022] FIG. 3: General structure of a chimeric polypeptide
consisting of a first stretch of consecutive amino acids joined at
its C-terminus by a peptide bond to the N-terminus of a second
stretch of consecutive amino acids. Chimeric polypeptides, like
proteins found in nature, are continuous stretches of consecutive
amino acids, each of which is joined to the preceding amino acid by
a peptide bond. Like other proteins, chimeric polypeptides have
limited conformational flexibility because the peptide bond by
itself provides no more than two consecutive single bonds capable
of dihedral rotation along the polypeptide chain. Amino acid
residues in the figure are numbered as follows: The first stretch
of consecutive amino acids has length=n residues, and numbering=1,
2, 3, . . . , (n-2), (n-1), n. The second stretch of consecutive
amino acids has length=p residues, and numbering=1', 2', 3', . . .
, (p-a2), (p-a1), p. The chimeric polypeptide has length=(n+p)
residues, and numbering=1, 2, 3, . . . , (n-2), (n-1), n, (n+1),
(n+2), (n+3), (n+p-2), (n+p-1), (n+p). The major and minor
tautomeric forms and the resonance structure are shown on the left,
center and right, respectively.
[0023] FIG. 4A: General structure of a "symmetroadhesin" with a
head-to-tail configuration consisting of a first stretch of
consecutive amino acids joined at its C-terminal-X-terminus by an
-X-X- bond to the N-terminal-X-terminus of a second stretch of
consecutive amino acids. The -X-X- bond is not a peptide bond.
Non-limiting examples of the bonds envisaged here include any
combination wherein each X is a S or a Se atom. The overall
polarity of head-to-tail symmetroadhesins is N- to C-terminal.
Symmetroadhesins, like proteins, are stretches of consecutive amino
acids each of which is joined to the preceding amino acid, but
differ from proteins by substituting one or more -X-X- bonds for
peptide bonds. Symmetroadhesins have greater conformational
flexibility than polypeptides because each -X-X- bond provides
seven adjacent single bonds capable of dihedral rotation. Amino
acid residues are numbered as follows: The first stretch of
consecutive amino acids has length=n residues, and numbering=1, 2,
3, . . . , (n-2), (n-1), n. The second stretch of consecutive amino
acids has length=p residues, and numbering=1', 2', 3', (p-a2),
(p-a1), p. The head-to-tail symmetroadhesin has length=(n+p)
residues, and numbering=1, 2, 3, . . . , (n-2), (n-1), n, (n+1),
(n+2), (n+3), . . . , (n+p-2), (n+p-1), (n+p). The major and minor
tautomeric forms and the resonance structure are shown on the left,
center and right, respectively.
[0024] FIG. 4B: The symmetroadhesin of FIG. 4A (left) compared with
a symmetroadhesin (right) consisting of a first stretch of
consecutive amino acids joined at the X-terminus of its penultimate
C-terminal residue by an -X-X- bond to the X-terminus of the
penultimate N-terminal residue of a second stretch of consecutive
amino acids. Resonance structures are shown for each.
[0025] FIG. 4C: The symmetroadhesin of FIG. 4A (left) compared with
a symmetroadhesin (right) consisting of a first stretch of
consecutive amino acids joined at the X-terminus of its
antepenultimate C-terminal residue by an -X-X- bond to the
X-terminus of the antepenultimate N-terminal residue of a second
stretch of consecutive amino acids. Resonance structures are shown
for each.
[0026] FIG. 4D: The symmetroadhesin of FIG. 4A (left) compared with
a symmetroadhesin (right) consisting of a first stretch of
consecutive amino acids joined at the X-terminus of its
preantepenultimate C-terminal residue by an -X-X- bond to the
X-terminus of the preantepenultimate N-terminal residue of a second
stretch of consecutive amino acids. Resonance structures are shown
for each.
[0027] FIG. 5: General structure of a symmetroadhesin with a
head-to-head configuration consisting of a first stretch of
consecutive amino acids, joined at its N-terminal-X-terminus by an
-X-X- bond to the N-terminal-X-terminus of a second stretch of
consecutive amino acids. The overall polarity of head-to-head
symmetroadhesins changes at the position of the -X-X- bond, going
from C- to N-terminal to N- to C-terminal. Amino acid residues are
numbered as follows: The first stretch of consecutive amino acids
has length=n residues, and numbering=1, 2, 3, . . . , (n-2), (n-1),
n. The second stretch of consecutive amino acids has length=p
residues, and numbering=1', 2', 3', (p-a2), (p-a1), p. The
head-to-head symmetroadhesin has length=(n+p) residues, and
numbering=n, (n-1), (n-2), . . . , 3, 2, 1, (inversion), 1', 2',
3', . . . , (p-a2), (p-a1), p. The major and minor tautomeric forms
and the resonance structure are shown on the left, center and
right, respectively.
[0028] FIG. 6: General structure of a symmetroadhesin with a
tail-to-tail configuration consisting of a first stretch of
consecutive amino acids joined at its C-terminal-X-terminus by an
-X-X- bond to the C-terminal-X-terminus of a second stretch of
consecutive amino acids. The overall polarity of tail-to-tail
symmetroadhesins changes at the position of the -X-X- bond, going
from N- to C-terminal to C- to N-terminal. Amino acid residues are
numbered as follows: The first stretch of consecutive amino acids
has length=n residues, and numbering=1, 2, 3, . . . , (n-2), (n-1),
n. The second stretch of consecutive amino acids has length=p
residues, and numbering=1', 2', 3', (p-a2), (p-a1), p. The
tail-to-tail symmetroadhesin has length=n+p residues, and
numbering=1, 2, 3, . . . , (n-2), (n-1), n, (inversion), p, (p-a1),
(p-a2), . . . , 3', 2', 1'. The major and minor tautomeric forms
and the resonance structure are shown on the left, center and
right, respectively.
[0029] FIG. 7: A schematic representation of a head-to-tail
hemi-symmetroadhesin showing the all-trans conformation. All of the
seven consecutive single bonds joining the C-terminal-X-terminus
with the N-terminal-X-terminus are trans (N--C--C--X--X--C--C--C).
The two binding domains are pointed away from one another in this
conformation; a rotation of 180 degrees around any one of the seven
consecutive single bonds will point the two binding domains towards
one another. Hemi-symmetroadhesins with the head-to-tail
configuration are asymmetric molecules regardless of conformation
(compare FIGS. 7 and 8); however, two or more head-to-tail
hemi-symmetroadhesins can together form a symmetric molecule.
[0030] FIG. 8: A schematic representation of a head-to-tail
hemi-symmetroadhesin showing the X-cis-X conformation. All but one
of seven consecutive single bonds joining the C-terminal-X-terminus
with the N-terminal-X-terminus are trans
(N--C--C--X-cis-X--C--C--C). The two binding domains are pointed
towards one another in this conformation; further rotations of 180
degrees around any one of the seven consecutive single bonds will
point the two binding domains away from one another.
Hemi-symmetroadhesins with the head-to-tail configuration are
asymmetric molecules regardless of conformation (compare FIGS. 7
and 8); however, two or more head-to-tail hemi-symmetroadhesins can
together form a symmetric molecule.
[0031] FIG. 9: A schematic representation of a head-to-head
hemi-symmetroadhesin showing the all-trans conformation. All of the
seven consecutive single bonds joining the 1.sup.st
N-terminal-X-terminus and 2.sup.nd N-terminal-X-terminus are trans
(C--C--C--X--X--C--C--C). The two binding domains are pointed away
from one another in this conformation; a rotation of 180 degrees
around any one of the seven consecutive single bonds will point the
two binding domains towards one another. Hemi-symmetroadhesins with
the head-to-head configuration are symmetric molecules in only two
of their possible conformations: the all-trans and the X-cis-X
(compare FIGS. 9 and 10); however, two or more head-to-head
hemi-symmetroadhesins subunits can form a molecule that has an
unlimited number of symmetric conformations.
[0032] FIG. 10: A schematic representation of a head-to-head
hemi-symmetroadhesin showing the X-cis-X conformation. All but one
of the seven consecutive single bonds joining the 1.sup.st
N-terminal-X-terminus and 2.sup.nd N-terminal-X-terminus are trans
(C--C--C--X-cis-X--C--C--C). The two binding domains are pointed
towards one another in this conformation; further rotations of 180
degrees around any one of the seven consecutive single bonds will
point the two binding domains away from one another.
Hemi-symmetroadhesins with the head-to-head configuration are
symmetric molecules in only two of their possible conformations:
the all-trans and the X-cis-X (compare FIGS. 9 and 10); however,
two or more head-to-head hemi-symmetroadhesins subunits can form a
molecule that has an unlimited number of symmetric
conformations.
[0033] FIG. 11: A schematic representation of a tail-to-tail
hemi-symmetroadhesin showing the all-trans conformation. All of the
seven consecutive single bonds joining the 1.sup.st
C-terminal-X-terminus and 2.sup.nd C-terminal-X-terminus are trans
(N--C--C--X--X--C--C--N). The two binding domains are pointed away
from one another in this conformation; a rotation of 180 degrees
around any one of the seven consecutive single bonds will point the
two binding domains towards one another. Hemi-symmetroadhesins with
the tail-to-tail configuration are symmetric molecules in only two
of their possible conformations: the all-trans and the X-cis-X
(compare FIGS. 11 and 12); however, two or more tail-to-tail
hemi-symmetroadhesins subunits can form a molecule that has an
unlimited number of symmetric conformations.
[0034] FIG. 12: A schematic representation of a tail-to-tail
hemi-symmetroadhesin showing the X-cis-X conformation. All but one
of the seven consecutive single bonds joining the 1.sup.st
C-terminal-X-terminus and 2.sup.nd C-terminal-X-terminus are trans
(N--C--C--X-cis-X--C--C--N). The two binding domains are pointed
towards one another in this conformation; further rotations of 180
degrees around any one of the seven consecutive single bonds will
point the two binding domains away from one another.
Hemi-symmetroadhesins with the tail-to-tail configuration are
symmetric molecules in only two of their possible conformations:
the all-trans and the X-cis-X (compare FIGS. 9 and 10); however,
two or more tail-to-tail hemi-symmetroadhesins subunits can form a
molecule that has an unlimited number of symmetric
conformations.
[0035] FIG. 13: A schematic representation of a tail-to-tail
hemi-symmetroadhesin consisting of two immunoglobulin Fab binding
domains. The all-trans conformation is shown here. All of the seven
consecutive single bonds joining the 1.sup.st C-terminal-X-terminus
and 2.sup.nd C-terminal-X-terminus are trans
(N--C--C--X--X--C--C--N). The two Fab binding domains are pointed
away from one another in this conformation; a rotation of 180
degrees around any one of the seven consecutive single bonds will
point the Fab binding domains towards one another (compare FIGS. 13
and 14). The heavy chain regions are joined together by the X-X
bond; the light chain regions are joined to the heavy chain regions
by internal disulfide bonds. Abbreviations: VL, light chain
variable region; CL, light chain constant region; VH, heavy chain
variable region; CH1, heavy chain constant region 1.
[0036] FIG. 14: A schematic representation of a tail-to-tail
hemi-symmetroadhesin consisting of two immunoglobulin Fab binding
domains. The X-cis-X conformation is shown here. All but one of
seven consecutive single bonds joining the 1.sup.st
C-terminal-X-terminus and 2.sup.nd C-terminal-X-terminus are trans
(N--C--C--X-cis-X--C--C--N). The two Fab binding domains are
pointed towards one another in this conformation; further rotation
of 180 degrees around any one of the seven consecutive single bonds
will point the Fab binding domains away from one another (compare
FIGS. 13 and 14). The heavy chain regions are joined together by
the X-X bond; the light chain regions are joined to the heavy chain
regions by internal disulfide bonds. Abbreviations: VL, light chain
variable region; CL, light chain constant region; VH, heavy chain
variable region; CH1, heavy chain constant region 1.
[0037] FIG. 15: Schematic representation of the immunoadhesin
molecule (Capon et al. (1989) Nature 337, 525-530). Immunoadhesins
are chimeric polypeptides that form disulfide-linked dimers. Each
chimeric polypeptide consists of a binding domain joined at its
C-terminus by a peptide bond to the N-terminus of an immunoglobulin
Fc domain. Although immunoadhesins are structurally symmetric, they
do not generally bind cooperatively to dimeric or multimeric target
molecules. Abbreviations: CH2, heavy chain constant region 2; CH3,
heavy chain constant region 3.
[0038] FIG. 16: Schematic representation of the immunoglobulin
(antibody) molecule. Immunoglobulins are heterotetramers consisting
of two heavy chains and two light chains. Although immunoglobulins
are structurally symmetric, they do not generally bind
cooperatively to dimeric or multimeric target molecules.
Abbreviations: VL, light chain variable region; CL, light chain
constant region; VH, heavy chain variable region; CH1, heavy chain
constant region 1; CH2, heavy chain constant region 2; CH3, heavy
chain constant region 3.
[0039] FIG. 17: A schematic representation of a head-to-tail
immunosymmetroadhesin showing the all-trans conformation.
Head-to-tail immunosymmetroadhesins are head-to-tail
hemi-symmetroadhesins that form disulfide-linked dimers. Each
hemi-symmetroadhesin consists of an immunoglobulin Fab binding
domain having a C-terminal-X-terminus joined by an -X-X- bond to a
immunoglobulin Fc subunit having an N-terminal-X-terminus. The
dimer contains two functional Fab binding domains and one
functional Fc binding domain. The seven consecutive single bonds
which join each Fab domain to an Fc subunit are all trans
(N--C--C--X--X--C--C--C). Symmetric rotations of 180 degrees around
the first (N--C), third (C--X), fifth (X--C), or seventh (C--C)
pairs of consecutive single bonds will move the two Fab domains in
a first general direction (compare FIGS. 17 and 18). Symmetric
rotations of 180 degrees around the second (C--C), fourth (X--X),
or sixth (C--C) single bond pairs will move the two Fab domains in
a second general direction (compare FIGS. 17 and 19).
[0040] FIG. 18: A schematic representation of a head-to-tail
immunosymmetroadhesin showing the X-cis-C conformation. The X-cis-C
conformation is obtained from the all-trans conformation by the
symmetric rotation of the fifth pair of seven consecutive single
bonds (N--C--C--X--X-cis-C--C--C) (compare FIGS. 17 and 18). Other
conformations that are similar to the X-cis-C conformation shown
here are obtained from the all-trans conformation following the
symmetric rotation of the first (N-cis-C--C--X--X--C--C--C), the
third (N--C--C-cis-X--X--C--C--C), or the seventh pairs
(N--C--C--X--X--C--C-cis-C) of consecutive single bonds.
[0041] FIG. 19: A schematic representation of a head-to-tail
immunosymmetroadhesin showing the X-cis-X conformation. The X-cis-X
conformation is obtained from the all-trans conformation by the
symmetric rotation of the fourth pair of seven consecutive single
bonds (N--C--C--X-cis-X--C--C--C) (compare FIGS. 17 and 19). Other
conformations that are similar to the X-cis-X conformation shown
here are obtained from the all-trans conformation following the
symmetric rotation of the second (N--C-cis-C--X--X--C-C--C), or the
sixth pairs (N--C-C--X--X--C-cis-C--C) of consecutive single
bonds.
[0042] FIG. 20: A schematic representation of a tail-to-tail
immunosymmetroadhesin showing the all-trans conformation.
Tail-to-tail immunosymmetroadhesins are tail-to-tail
hemi-symmetroadhesins that form disulfide-linked dimers. Each
hemi-symmetroadhesin consists of an immunoglobulin Fab binding
domain having a C-terminal-X-terminus joined by an X-X bond to an
immunoglobulin Fc subunit having a C-terminal-X-terminus. The dimer
contains two functional Fab binding domains and one functional Fc
binding domain. The seven consecutive single bonds which join each
Fab domain to an Fc subunit are all trans (N--C--C--X--X--C--C--N).
Symmetric rotations of 180 degrees around the first (N--C), third
(C--X), fifth (X--C), or seventh (C--N) pairs of consecutive single
bonds will move the two Fab domains in a first general direction
(compare FIGS. 20 and 21). Symmetric rotations of 180 degrees
around the second (C--C), fourth (X--X), or sixth (C--C) single
bond pairs will move the two Fab domains in a second general
direction (compare FIGS. 20 and 22).
[0043] FIG. 21: A schematic representation of a tail-to-tail
immunosymmetroadhesin showing the X-cis-C conformation. The X-cis-C
conformation is obtained from the all-trans conformation by the
symmetric rotation of the fifth pair of seven consecutive single
bonds (N--C--C--X--X-cis-C--C--N) (compare FIGS. 20 and 21). Other
conformations that are similar to the X-cis-C conformation shown
here are obtained from the all-trans conformation following the
symmetric rotation of the first (N-cis-C--C--X--X--C--C--N), the
third (N--C--C-cis-X--X--C--C--N), or the seventh pairs
(N--C--C--X--X--C--C-cis-N) of consecutive single bonds.
[0044] FIG. 22: A schematic representation of a tail-to-tail
immunosymmetroadhesin showing the X-cis-X conformation. The X-cis-X
conformation is obtained from the all-trans conformation by the
symmetric rotation of the fourth pair of seven consecutive single
bonds (N--C--C--X-cis-X--C--C--N) (compare FIGS. 20 and 22). Other
conformations that are similar to the X-cis-X conformation shown
here are obtained from the all-trans conformation following the
symmetric rotation of the second (N--C-cis-C--X--X--C--C--N), or
the sixth pairs (N--C--C--X--X--C-cis-C--N) of consecutive single
bonds.
[0045] FIG. 23: Schematic representation of bi-symmetroadhesin with
four Fab binding domains, showing the all-trans conformation. The
molecule is a dimer of two hemi-symmetroadhesins each consisting of
three stretches of consecutive amino acids. The bi-symmetroadhesin
shown here is a head-to-tail, tail-to-tail hemi-symmetroadhesin
that forms disulfide-linked dimers. Each hemi-symmetroadhesin
consists of two immunoglobulin Fab domains having a
C-terminal-X-terminus that is joined by an -X-X- bond to a
immunoglobulin Fc subunit; the first Fab domain is joined to the Fc
N-terminal-X-terminus, and the second Fab is joined to the Fc
C-terminal-X-terminus. The dimer has four functional Fab binding
domains, and one functional Fc binding domain. The seven
consecutive single bonds which join all four Fab domains to an Fc
subunit are all trans (N--C--C--X--X--C--C--C/N). Symmetric
rotations of 180 degrees around the first (N--C), third (C--X),
fifth (X--C), or seventh (C--C/N) pairs of consecutive single bonds
will move the four Fab domains in a first general direction
(compare FIGS. 23 and 24). Symmetric rotations of 180 degrees
around the second (C--C), fourth (X--X), or sixth (C--C) single
bond pairs will move the four Fab domains in a second general
direction (compare FIGS. 23 and 25).
[0046] FIG. 24: Schematic representation of the X-cis-C
conformation of a bi-immunosymmetroadhesin consisting of four Fab
binding domains and one Fc domain. The molecule is a dimer of two
hemi-symmetroadhesins each consisting of three stretches of
consecutive amino acids.
[0047] FIG. 25: Schematic representation of the X-cis-X
conformation of a bi-immunosymmetroadhesin consisting of four Fab
binding domains and one Fc domain. The molecule is a dimer of two
hemi-symmetroadhesins each consisting of three stretches of
consecutive amino acids.
[0048] FIG. 26: Schematic representation of the all-trans
conformation of a bi-immunosymmetroadhesin consisting of two Fab
binding domains, one Fc domain, and two non-Fab binding domains.
The molecule is a dimer of two hemi-symmetroadhesins each
consisting of three stretches of consecutive amino acids. The
bi-symmetroadhesin shown here is a head-to-tail, tail-to-tail
hemi-symmetroadhesin that forms disulfide-linked dimers. Each
hemi-symmetroadhesin consists of one immunoglobulin Fab domain
having a C-terminal-X-terminus that is joined by an -X-X- bond to
the N-terminal-X-terminus of an immunoglobulin Fc subunit, and one
non-immunoglobulin binding domain having a C-terminal-X-terminus
that is joined by an -X-X- bond to the C-terminal-X-terminus of an
immunoglobulin Fc subunit. The dimer has two functional Fab binding
domains, two functional non-immunoglobulin binding domains, and one
functional Fc binding domain. The seven consecutive single bonds
which join all four binding domain to an Fc subunit are all trans
(N--C--C--X--X--C--C--C/N). Symmetric rotations of 180 degrees
around the first (N--C), third (C--X), fifth (X--C), or seventh
(C--C/N) pairs of consecutive single bonds will move the four
binding domains in a first general direction (compare FIGS. 26 and
27). Symmetric rotations of 180 degrees around the second (C--C),
fourth (X--X), or sixth (C--C) single bond pairs will move the four
binding domains in a second general direction (compare FIGS. 26 and
28).
[0049] FIG. 27: Schematic representation of the X-cis-C
conformation of a bi-immunosymmetroadhesin consisting of two Fab
binding domains, one Fc domain, and two non-Fab binding domains.
The molecule is a dimer of two hemi-symmetroadhesins each
consisting of three stretches of consecutive amino acids.
[0050] FIG. 28: Schematic representation of the X-cis-X
conformation of a bi-immunosymmetroadhesin consisting of two Fab
binding domains, one Fc domain, and two non-Fab binding domains.
The molecule is a dimer of two hemi-symmetroadhesins each
consisting of three stretches of consecutive amino acids.
[0051] FIG. 29: Schematic representation of an immunoglobulin
binding to a first symmetric target. The interaction is symmetric
and cooperative. Both targets are bound by both
immunoglobulins.
[0052] FIG. 30: Schematic representation of a symmetroadhesin
binding to a first symmetric target. The interaction is symmetric
and cooperative. Both targets are bound by both symmetroadhesins in
a first conformation (all-trans).
[0053] FIG. 31: Schematic representation of an immunoglobulin
binding to a second symmetric target. The interaction is neither
symmetric and nor cooperative. Only one target is bound by each
immunoglobulin.
[0054] FIG. 32: Schematic representation of a symmetroadhesin
binding to a second symmetric target. The interaction is symmetric
and cooperative. Both targets are bound by both symmetroadhesins in
a second conformation (X-cis-C).
[0055] FIG. 33: Schematic representation of an immunoglobulin
binding to a third symmetric target. The interaction is neither
symmetric and nor cooperative. Only one target is bound by each
immunoglobulin.
[0056] FIG. 34: Schematic representation of a symmetroadhesin
binding to a third symmetric target. The interaction is symmetric
and cooperative. Both targets are bound by both symmetroadhesins in
a third conformation (X-cis-X).
[0057] FIG. 35A: Amino acid sequences of various polypeptide
synthetic intermediates of a human IgG1 Fc symmetroadhesin
precursor subunit with an N-terminal-S-terminus. Part (i) shows
three distinct pre-Fc polypeptides comprising, alternatively, the
human sonic hedgehog (SHH), human interferon alpha-2 (IFN), or
human cholesterol ester transferase (CETP) signal sequences
(residues -23 to -1, -23 to -1, or -17 to -1, respectively), and
the human IGHG1 Fc domain (residues 1 to 228) beginning at the
fifth amino acid encoded by the hinge exon, CDKTHTCPPCP (Ellison et
al. (1982) Nuc. Acids Res. 10, 4071-4079). The three distinct
pre-Fc polypeptides have lengths of 251, 251 and 245 residues,
respectively. Part (ii) shows the mature Fc domain (length=228)
with an N-terminal-S-terminus. The N-terminal cysteine residue is
underlined. (IGHG1, UniProtKB/Swiss-Prot entry P01857, Ig gamma-1
chain C region, Homo sapiens).
[0058] FIG. 35B: Amino acid sequences of various polypeptide
synthetic intermediates of a human IgG2 Fc symmetroadhesin
precursor subunit with an N-terminal-S-terminus. Part (i) shows
three distinct pre-Fc polypeptides comprising, alternatively, the
human sonic hedgehog (SHH), human interferon alpha-2 (IFN), or
human cholesterol ester transferase (CETP) signal sequences
(residues -23 to -1, -23 to -1, or -17 to -1, respectively), and
the human IGHG2 Fc domain (residues 1 to 225) beginning at the
fourth amino acid encoded by the hinge exon, CCVECPPCP (Ellison et
al. (1982) Nuc. Acids Res. 10, 4071-4079). The three distinct
pre-Fc polypeptides have lengths of 248, 248 and 242 residues,
respectively. Part (ii) shows the mature Fc domain (length=225)
with an N-terminal-S-terminus. The N-terminal cysteine residue is
underlined. (IGHG2, UniProtKB/Swiss-Prot entry P01859, Ig gamma-2
chain C region, Homo sapiens).
[0059] FIG. 35C: Amino acid sequences of various polypeptide
synthetic intermediates of a human IgG3 Fc symmetroadhesin
precursor subunit with an N-terminal-S-terminus. Part (i) shows
three distinct pre-Fc polypeptides comprising, alternatively, the
human sonic hedgehog (SHH), human interferon alpha-2 (IFN), or
human cholesterol ester transferase (CETP) signal sequences
(residues -23 to -1, -23 to -1, or -17 to -1, respectively), and
the human IGHG3 Fc domain (residues 1 to 267) beginning at the
thirteenth amino acid encoded by the first hinge exon, CPRCP
(Strausberg et al. (2002) Proc. Natl. Acad. Sci. 99, 16899-1690).
The three distinct pre-Fc polypeptides have lengths of 290, 290 and
284 residues, respectively. Part (ii) shows the mature Fc domain
(length=267) with an N-terminal-S-terminus. The N-terminal cysteine
residue is underlined. (IGHG3, UniProtKB/Swiss-Prot entry Q8N4Y9,
Ig gamma-3 chain C region, Homo sapiens).
[0060] FIG. 35D: Amino acid sequences of various polypeptide
synthetic intermediates of a human IgG4 Fc symmetroadhesin
precursor subunit with an N-terminal-S-terminus. Part (i) shows
three distinct pre-Fc polypeptides comprising, alternatively, the
human sonic hedgehog (SHH), human interferon alpha-2 (IFN), or
human cholesterol ester transferase (CETP) signal sequences
(residues -23 to -1, -23 to -1, or -17 to -1, respectively), and
the human IGHG4 Fc domain (residues 1 to 222) beginning at the
eighth amino acid encoded by the hinge exon, CPSCP (Strausberg et
al. (2002) Proc. Natl. Acad. Sci. 99, 16899-1690). The three
distinct pre-Fc polypeptides have lengths of 245, 245 and 239
residues, respectively. Part (ii) shows the mature Fc domain
(length=222) with an N-terminal-S-terminus. The N-terminal cysteine
residue is underlined. (IGHG4, UniProtKB/Swiss-Prot entry Q8TC63,
Ig gamma-4 chain C region, Homo sapiens).
[0061] FIG. 36A: Amino acid sequences of various polypeptide
synthetic intermediates of a human Fc symmetroadhesin precursor
subunit with an N-terminal-X-terminus. Part (i) shows three
distinct pre-Fc polypeptides comprising, alternatively, the human
sonic hedgehog (SHH), human interferon alpha-2 (IFN), or human
cholesterol ester transferase (CETP) signal sequences (residues -23
to -1, -23 to -1, or -17 to -1, respectively), and the human IGHG1
Fc domain (residues 1 to 222) beginning at the eleventh amino acid
encoded by the hinge exon, CPPCP (Ellison et al. (1982) Nuc. Acids
Res. 10, 4071-4079). The three distinct pre-Fc polypeptides have
lengths of 245, 245 and 239 residues, respectively. Part (ii) shows
the mature Fc domain (length=222) having an N-terminal-S-terminus.
The N-terminal cysteine residue is underlined. Part (iii) shows the
mature Fc domain extended by native chemical ligation (length=226)
to have an N-terminal-X-terminus. The N-terminal X amino acid
(e.g., cysteine, selenocysteine) is underlined; it is followed by
the sixth amino acid encoded by the hinge exon, XDKTHTCPPCP.
(IGHG1, UniProtKB/Swiss-Prot entry P01857, Ig gamma-1 chain C
region, Homo sapiens).
[0062] FIG. 36B: Amino acid sequences of various polypeptide
synthetic intermediates of a human Fc symmetroadhesin precursor
subunit with an N-terminal-X-terminus. Part (i) shows three
distinct pre-Fc polypeptides comprising, alternatively, the human
sonic hedgehog (SHH), human interferon alpha-2 (IFN), or human
cholesterol ester transferase (CETP) signal sequences (residues -23
to -1, -23 to -1, or -17 to -1, respectively), and the human IGHG1
Fc domain (residues 1 to 219) beginning at the fourteenth amino
acid encoded by the hinge exon, CP (Ellison et al. (1982) Nuc.
Acids Res. 10, 4071-4079). The three distinct pre-Fc polypeptides
have lengths of 242, 242 and 236 residues, respectively. Part (ii)
shows the mature Fc domain (length=219) having an
N-terminal-S-terminus. The N-terminal cysteine residue is
underlined. Part (iii) shows the mature Fc domain extended by
native chemical ligation (length=222) to have an
N-terminal-X-terminus. The N-terminal X amino acid (e.g., cysteine,
selenocysteine) is underlined; it is followed by the twelfth amino
acid encoded by the hinge exon, XPPCP. (IGHG1, UniProtKB/Swiss-Prot
entry P01857, Ig gamma-1 chain C region, Homo sapiens).
[0063] FIG. 37A: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human Fc
symmetroadhesin precursor subunit with a C-terminal-X-terminus.
Part (i) shows two distinct pre-Fc-intein polypeptides comprising,
alternatively, the human CD2 or CD4 signal sequences (residues -24
to -1, or -25 to -1, respectively), the human IGHG1 Fc domain
(residues 1 to 224) beginning at the seventh amino acid encoded by
the hinge exon (KTHTCPPCP), the human IGHG3 M1 domain (residues 225
to 241), and an Mth RIR1 intein-chitin binding domain (residues 242
to 441). The two distinct pre-Fc-intein chimeric polypeptides have
lengths of 465 and 466 residues, respectively. Part (ii) shows the
mature Fc-intein chimeric polypeptide (length=441) comprising the
human Fc/M1 domain and the Mth RIR1 intein-chitin binding domain.
The intein autocleavage site is underlined. Part (iii) shows the
thioester-terminated human Fc/M1 domain (length=242). The
C-terminal thio-glycine residue (Z) is underlined. Part (iv) shows
the human Fc/M1 domain (length=243) with a C-terminal-X-terminus.
The C-terminal X amino acid residue (e.g., cysteine,
selenocysteine) is underlined. (IGHG1, UniProtKB/Swiss-Prot entry
P01857, Ig gamma-1 chain C region, Homo sapiens; IGHG3,
NCBI/GenBank accession BAA11363, membrane-bound-type Ig
gamma-chain, Homo sapiens).
[0064] FIG. 37B: Amino acid sequences of various polypeptide
synthetic intermediates of a human Fc symmetroadhesin precursor
subunit with an C-terminal-S-terminus. Part (i) shows two distinct
pre-Fc polypeptides comprising, alternatively, the human CD2 or CD4
signal sequences (residues -24 to -1, or -25 to -1, respectively),
the human IGHG1 Fc domain (residues 1 to 224) beginning at the
seventh amino acid encoded by the hinge exon (KTHTCPPCP), and a
portion of the human IGHG3 M1 domain (residues 225 to 232). The two
distinct pre-Fc polypeptides have lengths of 256 and 257 residues,
respectively. Part (ii) shows the mature Fc domain (length=232)
with an N-terminal-S-terminus. The N-terminal cysteine residue is
underlined. (IGHG1, UniProtKB/Swiss-Prot entry P01857, Ig gamma-1
chain C region, Homo sapiens).
[0065] FIGS. 38A-38B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human Fc
symmetroadhesin precursor subunit, with an N-terminal-S-terminus
and a C-terminal-X-terminus. (A) Part (i) shows three distinct
pre-Fc-intein polypeptides comprising, alternatively, the human
sonic hedgehog (SHH), human interferon alpha-2 (IFN), or human
cholesterol ester transferase (CETP) signal sequences (residues -23
to -1, -23 to -1, or -17 to -1, respectively), the human IGHG1 Fc
domain (residues 1 to 226) beginning at the fifth amino acid
encoded by the hinge exon (CDKTHTCPPCP), the human IGHG3 M1 domain
(residues 227 to 243), and an Mth RIR1 intein-chitin binding domain
(residues 244 to 443). The three distinct pre-Fc-intein chimeric
polypeptides have lengths of 466, 466 and 460 residues,
respectively. Part (ii) shows the mature Fc-intein chimeric
polypeptide (length=443) comprising the human Fc/M1 domain and the
Mth RIR1 intein-chitin binding domain with an
N-terminal-S-terminus. The N-terminal cysteine residue and intein
autocleavage site are underlined. (B) Part (iii) shows the
thioester-terminated human Fc/M1 domain (length=244). The
N-terminal cysteine residue and C-terminal thio-glycine residue (Z)
are underlined. Part (iv) shows the Fc/M1 domain (length=245) with
an N-terminal-S-terminus and a C-terminal-X-terminus. The
N-terminal cysteine residue and C-terminal X amino acid residue
(e.g., cysteine, selenocysteine) are underlined. (IGHG1,
UniProtKB/Swiss-Prot entry P01857, Ig gamma-1 chain C region, Homo
sapiens; IGHG3, NCBI/GenBank accession BAA11363,
membrane-bound-type Ig gamma-chain, Homo sapiens).
[0066] FIGS. 39A-39B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human Fc
symmetroadhesin precursor subunit, with an N-terminal-X-terminus
and a C-terminal-X-terminus. (A) Part (i) shows three distinct
pre-Fc-intein polypeptides comprising, alternatively, the human
sonic hedgehog (SHH), human interferon alpha-2 (IFN), or human
cholesterol ester transferase (CETP) signal sequences (residues -23
to -1, -23 to -1, or -17 to -1, respectively), the human IGHG1 Fc
domain (residues 1 to 220) beginning at the eleventh amino acid
encoded by the hinge exon (CPPCP), the human IGHG3 M1 domain
(residues 221 to 237), and an Mth RIR1 intein-chitin binding domain
(residues 238 to 437). The three distinct pre-Fc-intein chimeric
polypeptides have lengths of 460, 460 and 454 residues,
respectively. Part (ii) shows the mature Fc-intein chimeric
polypeptide (length=437) comprising the human Fc/M1 domain and the
Mth RIR1 intein-chitin binding domain with an
N-terminal-S-terminus. The N-terminal cysteine residue and intein
autocleavage site are underlined. (B) Part (iii) shows the mature
Fc-intein chimeric polypeptide extended by native chemical ligation
(length=443) to have an N-terminal-X-terminus. The N-terminal X
amino acid (e.g., cysteine, selenocysteine) is underlined. Part
(iv) shows the thioester-terminated human Fc/M1 domain
(length=244). The N-terminal X amino acid residue and C-terminal
thio-glycine residue (Z) are underlined. Part (v) shows the Fc/M1
domain (length=245) with an N-terminal-X-terminus and a
C-terminal-X-terminus. The N-terminal X amino acid residue and
C-terminal X amino acid residue are underlined. (IGHG1,
UniProtKB/Swiss-Prot entry P01857, Ig gamma-1 chain C region, Homo
sapiens; IGHG3, NCBI/GenBank accession BAA11363,
membrane-bound-type Ig gamma-chain, Homo sapiens).
[0067] FIGS. 40A-40B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human CD4
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-CD4-intein polypeptide (length=596)
comprising the human CD4 signal sequence (residues -25 to -1) and
extracellular domain (residues 1 to 371), and an Mth RIR1
intein-chitin binding domain (residues 372 to 571). Part (ii) shows
the mature CD4-intein chimeric polypeptide (length=571) comprising
the human CD4 extracellular domain and the Mth RIR1 intein-chitin
binding domain. The position of the intein autocleavage site is
underlined. (B) Part (iii) shows the thioester-terminated human CD4
extracellular domain (length=372). The C-terminal thio-glycine
residue (Z) is underlined. Part (iv) shows the human CD4
extracellular domain (length=373) with a C-terminal-X-terminus. The
C-terminal X amino acid residue (e.g., cysteine, selenocysteine) is
underlined. (CD4, UniProtKB/Swiss-Prot entry P01730, T-cell surface
glycoprotein CD4).
[0068] FIGS. 41A-41B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a Di62-VH
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-Di62-VH-intein polypeptide (length=444)
comprising the mouse Di62-VH signal sequence (residues -19 to -1)
and variable domain (residues 1 to 117), the human CH1 contant
domain (residues 118 to 225), and an Mth RIR1 intein-chitin binding
domain (residues 226 to 425). Part (ii) shows the mature
Di62-VH-intein chimeric polypeptide (length=425) comprising the
mouse Di62-VH variable domain, the human CH1 contant domain, and
the Mth RIR1 intein-chitin binding domain. The position of the
intein autocleavage site is underlined. (B) Part (iii) shows the
thioester-terminated mouse Di62-VH variable domain/human CH1
contant domain (length=226). The C-terminal thio-glycine residue
(Z) is underlined. Part (iv) shows the mouse Di62-VH variable
domain/human CH1 contant domain (length=227) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (Di62-VH, NCBI/GenBank
accession CAA05416, IgG heavy chain, antigen binding of human TNF
alpha subunit, Mus musculus).
[0069] FIG. 42: Amino acid sequences of various polypeptide
synthetic intermediates of a Di62-Vk symmetroadhesin precursor
subunit. Part (i) shows the pre-Di62-Vk polypeptide (length=234)
comprising the mouse Di62-Vk signal sequence (residues -20 to -1)
and variable domain (residues 1 to 107), and the human Ck contant
domain (residues 108 to 214). Part (ii) shows the mature Di62-Vk
chimeric polypeptide (length=214) comprising the mouse Di62-Vk
variable domain, and the human Ck contant domain. (Di62-Vk,
NCBI/GenBank accession CAA05417, IgG light chain, antigen binding
of human TNF alpha subunit, Mus musculus).
[0070] FIG. 43: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human TNR1A
symmetroadhesin precursor subunit with a C-terminal-X-terminus.
Part (i) shows the pre-TNR1A-intein polypeptide (length=411)
comprising the human TNR1A signal sequence (residues -21 to -1) and
extracellular domain (residues 1 to 190), and an Mth RIR1
intein-chitin binding domain (residues 191 to 390). Part (ii) shows
the mature TNR1A-intein chimeric polypeptide (length=390)
comprising the human TNR1A extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. Part (iii) shows the
thioester-terminated human TNR1A extracellular domain (length=191).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human TNR1A extracellular domain (length=192) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (TNR1A,
UniProtKB/Swiss-Prot entry P19438, Tumor necrosis factor receptor
superfamily member 1A).
[0071] FIG. 44A: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human TNR1B
symmetroadhesin precursor subunit with a C-terminal-X-terminus.
Part (i) shows the pre-TNR1B-intein polypeptide (length=457)
comprising the human TNR1B signal sequence (residues -22 to -1) and
extracellular domain (residues 1 to 235), and an Mth RIR1
intein-chitin binding domain (residues 236 to 435). Part (ii) shows
the mature TNR1B-intein chimeric polypeptide (length=435)
comprising the human TNR1B extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. Part (iii) shows the
thioester-terminated human TNR1B extracellular domain (length=236).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human TNR1B extracellular domain (length=237) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (TNR1B,
UniProtKB/Swiss-Prot entry 20333, Tumor necrosis factor receptor
superfamily member 1B).
[0072] FIG. 44B: Amino acid sequences of various polypeptide
synthetic intermediates of a TNR1B immunoadhesin precursor subunit.
Part (i) shows the pre-TNR1B-immunoadhesin polypeptide (length=489)
comprising the TNR1B signal sequence (residues -22 to -1) and
extracellular domain (residues 1 to 235), and the human heavy
contant domain (residues 236 to 467). Part (ii) shows the mature
TNR1B-immunoadhesin (length=467).
[0073] FIGS. 45A-45C: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human VGFR1
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-VGFR1-intein polypeptide (length=958)
comprising the human VGFR1 signal sequence (residues -26 to -1) and
extracellular domain (residues 1 to 732), and an Mth RIR1
intein-chitin binding domain (residues 733 to 932). (B) Part (ii)
shows the mature VGFR1-intein chimeric polypeptide (length=932)
comprising the human VGFR1 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (C) Part (iii) shows the
thioester-terminated human VGFR1 extracellular domain (length=733).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human VGFR1 extracellular domain (length=734) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (VGFR1,
UniProtKB/Swiss-Prot entry P17948, Vascular endothelial growth
factor receptor 1).
[0074] FIGS. 46A-46C: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human VGFR2
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-VGFR2-intein polypeptide (length=964)
comprising the human VGFR2 signal sequence (residues -19 to -1) and
extracellular domain (residues 1 to 745), and an Mth RIR1
intein-chitin binding domain (residues 746 to 945). (B) Part (ii)
shows the mature VGFR2-intein chimeric polypeptide (length=945)
comprising the human VGFR2 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (C) Part (iii) shows the
thioester-terminated human VGFR2 extracellular domain (length=746).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human VGFR2 extracellular domain (length=747) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (VGFR2,
UniProtKB/Swiss-Prot entry P35968, Vascular endothelial growth
factor receptor 2).
[0075] FIGS. 47A-47C: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human VGFR3
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-VGFR3-intein polypeptide (length=975)
comprising the human VGFR3 signal sequence (residues -24 to -1) and
extracellular domain (residues 1 to 751), and an Mth RIR1
intein-chitin binding domain (residues 752 to 951). (B) Part (ii)
shows the mature VGFR3-intein chimeric polypeptide (length=951)
comprising the human VGFR3 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (C) Part (iii) shows the
thioester-terminated human VGFR3 extracellular domain (length=752).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human VGFR3 extracellular domain (length=753) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (VGFR3,
UniProtKB/Swiss-Prot entry P35916, Vascular endothelial growth
factor receptor 3).
[0076] FIGS. 48A-48B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human ERBB1
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-ERBB1-intein polypeptide (length=845)
comprising the human ERBB1 signal sequence (residues -24 to -1) and
extracellular domain (residues 1 to 621), and an Mth RIR1
intein-chitin binding domain (residues 622 to 821). Part (ii) shows
the mature ERBB1-intein chimeric polypeptide (length=821)
comprising the human ERBB1 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (B) Part (iii) shows the
thioester-terminated human ERBB1 extracellular domain (length=622).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human ERBB1 extracellular domain (length=623) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (ERBB1,
UniProtKB/Swiss-Prot entry P00533, Epidermal growth factor
receptor).
[0077] FIGS. 49A-49B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human ERBB2
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-ERBB2-intein polypeptide (length=852)
comprising the human ERBB2 signal sequence (residues -22 to -1) and
extracellular domain (residues 1 to 630), and an Mth RIR1
intein-chitin binding domain (residues 631 to 830). Part (ii) shows
the mature ERBB2-intein chimeric polypeptide (length=830)
comprising the human ERBB2 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (B) Part (iii) shows the
thioester-terminated human ERBB2 extracellular domain (length=631).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human ERBB2 extracellular domain (length=632) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (ERBB2,
UniProtKB/Swiss-Prot entry P04626, Receptor tyrosine-protein kinase
erbB-2).
[0078] FIGS. 50A-50B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human ERBB3
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-ERBB3-intein polypeptide (length=843)
comprising the human ERBB3 signal sequence (residues -19 to -1) and
extracellular domain (residues 1 to 624), and an Mth RIR1
intein-chitin binding domain (residues 625 to 824). Part (ii) shows
the mature ERBB3-intein chimeric polypeptide (length=824)
comprising the human ERBB3 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (B) Part (iii) shows the
thioester-terminated human ERBB3 extracellular domain (length=625).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human ERBB3 extracellular domain (length=626) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (ERBB3,
UniProtKB/Swiss-Prot entry P21860, Receptor tyrosine-protein kinase
erbB-3).
[0079] FIGS. 51A-51B: Amino acid sequences of various polypeptide
intermediates in an intein-based synthesis of a human ERBB4
symmetroadhesin precursor subunit with a C-terminal-X-terminus. (A)
Part (i) shows the pre-ERBB4-intein polypeptide (length=851)
comprising the human ERBB4 signal sequence (residues -25 to -1) and
extracellular domain (residues 1 to 626), and an Mth RIR1
intein-chitin binding domain (residues 627 to 826). Part (ii) shows
the mature ERBB4-intein chimeric polypeptide (length=826)
comprising the human ERBB4 extracellular domain and the Mth RIR1
intein-chitin binding domain. The position of the intein
autocleavage site is underlined. (B) Part (iii) shows the
thioester-terminated human ERBB4 extracellular domain (length=627).
The C-terminal thio-glycine residue (Z) is underlined. Part (iv)
shows the human ERBB4 extracellular domain (length=628) with a
C-terminal-X-terminus. The C-terminal X amino acid residue (e.g.,
cysteine, selenocysteine) is underlined. (ERBB4,
UniProtKB/Swiss-Prot entry Q15303, Receptor tyrosine-protein kinase
erbB-4).
[0080] FIG. 52: Expression in 293 kidney cells of human IgG1 Fc
symmetroadhesin subunits with N-terminal-S-termini. Lanes 1-6 and
lanes 7-12 show the IgG1 Fc polypeptides of FIG. 35A (ii) and FIG.
36A (ii), respectively. Cell supernatants: lanes 1, 3, 5, 7, 9 and
11; cell lysates: lanes 2, 4, 6, 8, 10 and 12. Signal sequences
used: SHH (lanes 1, 2, 7 and 8); IFN.alpha. (lanes 3, 4, 9, 10);
CETP (lanes 5, 6, 11 and 12).
[0081] FIG. 53: Expression in 293 kidney cells of human IgG1 Fc
symmetroadhesin subunits. Lanes 1-2, 3-4 and 5-6 show the IgG1 Fc
polypeptides of FIG. 35A (ii), FIG. 36A (ii) and FIG. 37B (ii),
respectively. Cell supernatants: (lanes 1-6). Signal sequences
used: SHH (lanes 1-6).
[0082] FIG. 54: Protein A purification of human IgG1 Fc
symmetroadhesin subunits expressed in 293 kidney cells. Lane 2 and
8 show the IgG1 Fc polypeptides of FIG. 36A and FIG. 35A,
respectively. Lanes 1-7: proteinA-sepharose column fractions for
the IgG1 Fc polypeptide of FIG. 36A.
[0083] FIG. 55: Thiol-sepharose binding of proteinA-purified human
IgG1 Fc symmetroadhesin subunits shown in FIG. 54. Lanes 1-3 and
lanes 4-6 show the human IgG1 Fc polypeptides of FIG. 35A and FIG.
36A, respectively. Lanes 1 and 4: starting material; lanes 2 and 5:
thiol-sepharose flow-thru fraction; lanes 3 and 6: thiol-sepharose
bound fraction.
[0084] FIG. 56: Expression in human 293 kidney cells of human
CD4-intein fusion proteins. Lanes 1-4 show the CD4-intein fusion
polypeptide of FIG. 40A (ii). Cell supernatants: lanes 1 and 3;
cell lysates: lanes 2 and 4.
[0085] FIG. 57: Expression in human 293 kidney cells of human TNR1B
fusion proteins. Lanes 2 and 5 show the TNR1B-intein fusion protein
of FIG. 44A (ii). Lanes 1 and 3 show the TNR1B-immunoadhesin fusion
protein of FIG. 44B (ii). Lanes 3 and 6 show proteins from
mock-transfected cells. Cell supernatants: lanes 1-3; cell lysates:
lanes 4-7. Lane 7: control TNR1B-immunoadhesin (R&D
Systems).
[0086] FIG. 58: TNR1B symmetroadhesin subunits with
C-terminal-S-termini. Lanes 1-2 show the TNR1B polypeptide of FIG.
44A (iii) following purification by chitin affinity chromatography
and cleavage/elution with MESNA. Lanes 3 shows the native ligation
product between the TNR1B polypeptide of FIG. 44A (iii) with a
fluorescent-labeled peptide (New England Biolabs). Panel (i):
direct fluorescence; panel (ii): western blot with anti-TNR1B
antibody (R&D Systems); panel (iii): SYPRO Ruby staining
(Sigma-Aldrich).
[0087] FIG. 59: TNR1B symmetroadhesin subunits with
C-terminal-S-termini. Lane 5 shows the TNR1B polypeptide of FIG.
44A (iv) following purification by chitin affinity chromatography
and cleavage/elution with cysteine. Lanes 1-4 show
TNR1B-immunoadhesin.
[0088] FIG. 60: TNR1B symmetroadhesin. Lanes 1-4 show the TNR1B
symmetroadhesin of FIG. 44A (iv) before oxidation (lanes 1 and 4)
and after oxidation in the presence of 10 mM CuSO4. Lanes 3 and 6
show a TNR1B-immunoadhesin control. Lanes 1-3: reducing conditions;
lanes 4-6: non-reducing conditions. The TNR1B symmetroadhesin
monomer (42 kd) and dimer (84 kd) are apparent in lanes 2 and 5,
and lane 5, respectively.
[0089] FIG. 61A-61C: TNF-alpha saturation binding analysis with
various TNR1B polypeptides on the Biacore T-100. (A) The TNR1B
symmetroadhesin of FIG. 44A (iv) was covalently coupled to a
Biacore CM-5 chip using standard Biacore amine chemistry. (B) TNR1B
immunoadhesin (R&D Systems) was covalently coupled to a Biacore
CM-5 chip using standard Biacore amine chemistry. (C) The TNR1B
symmetroadhesin of FIG. 44A (iv) was covalently coupled to a
Biacore CM-5 chip using standard Biacore thiol chemistry. Following
coupling, saturation binding analysis was carried out using
TNF-alpha (R&D Systems) at the indicated concentations.
[0090] FIG. 62A-62C: Scatchard analysis of the TNF-alpha saturation
binding analysis shown in FIG. 61A-61C. (A) TNR1B symmetroadhesin
of FIG. 44A (iv) covalently coupled using amine chemistry;
Kd=Kd=4.697.times.10.sup.-9 M. (B) TNR1B-immunoadhesin (R&D
Systems) covalently coupled using amine chemistry;
Kd=4.089.times.10.sup.-9 M. (C) TNR1B symmetroadhesin of FIG. 44A
(iv) covalently coupled using thiol chemistry;
Kd=0.8476.times.10.sup.-9 M.
DETAILED DESCRIPTION
[0091] This invention provides a compound comprising a first
stretch of consecutive amino acids, each of which is joined to the
preceding amino acid by a peptide bond and the sequence of which
comprises a binding site for a target; and a second stretch of
consecutive amino acids, each of which is joined to the preceding
amino acid by a peptide bond and the sequence of which is identical
to the sequence of the first stretch of consecutive amino acids and
which comprises an identical binding site for the target; wherein
each of the first stretch of amino acids and the second stretch of
amino acids has at a predefined end thereof a cysteine residue or a
selenocysteine residue and such cysteine residues or such
selenocysteine residues are joined by a bond having the
structure:
##STR00003##
wherein each X is the same and represents a sulfur (S) or a
selenium (Se) and each C represents a beta-carbon of one of such
cysteine or selenocysteine residues.
[0092] In an embodiment, the bond has the structure:
##STR00004##
[0093] In an embodiment, the residue at the predefined end of each
of the first stretch and the second stretch of consecutive amino
acids is a cysteine residue. In an embodiment the residue at the
predefined end of each of the first stretch and the second stretch
of consecutive amino acids is a selenocysteine residue.
[0094] This invention also provides a compound comprising a first
stretch of consecutive amino acids, each of which is joined to the
preceding amino acid by a peptide bond and the sequence of which
comprises a binding site for a target; and a second stretch of
consecutive amino acids, each of which is joined to the preceding
amino acid by a peptide bond and the sequence of which is different
from the sequence of the first stretch of consecutive amino acids
and which comprises a binding site for a different moiety; wherein
each of the first stretch of amino acids and the second stretch of
amino acids has at a predefined end thereof a cysteine residue or a
selenocysteine residue and such residues are joined by a bond
having the structure:
##STR00005##
wherein each X may be the same or different and represents a sulfur
(S) or a selenium (Se) and each C represents a beta-carbon of one
of such cysteine or selenocysteine residues.
[0095] In an embodiment the bond has the structure:
##STR00006##
[0096] In an embodiment both of the residues at the predefined ends
of each of the first stretch of amino acids and the second stretch
of amino acids are cysteine residues. In an embodiment both of the
residues at the predefined ends of each of the first stretch of
amino acids and the second stretch of amino acids are
selenocysteine residues. In an embodiment the residue at one
predefined end of one of the first stretch or second stretch of
consecutive amino acids is a cysteine residue and the residue at
the other predefined end is a selenocysteine residue.
[0097] This invention provides a multimer comprising two or more
identical instant compounds joined together by at least one bond.
In an embodiment the multimer is a dimer. In an embodiment the
multimer is a trimer. In an embodiment the multimer is a tetramer.
In an embodiment of the multimer, the one or more bonds comprises a
disulfide bond.
[0098] In an embodiment of the compounds the predefined end of both
the first stretch of amino acids and the second stretch of amino
acids is a N-terminal end thereof. In an embodiment the predefined
end of both the first stretch of amino acids and the second stretch
of amino acids is a C-terminal end thereof. In an embodiment the
predefined end of one of the first stretch of amino acids and the
second stretch of amino acids is a C-terminal end and the other
predefined end is a N-terminal end.
[0099] In an embodiment of the compounds, the first stretch of
amino acids comprises L-amino acids. In an embodiment, the first
stretch of amino acids comprises D-amino acids. In an embodiment,
the first stretch of amino acids comprises L-amino acids and
D-amino acids
[0100] In an embodiment, the second stretch of amino acids
comprises L-amino acids. In an embodiment, the second stretch of
amino acids comprises D-amino acids. In an embodiment, the second
stretch of amino acids comprises L-amino acids and D-amino
acids.
[0101] In an embodiment, the first stretch of amino acids comprises
at least 50 consecutive amino acids. In an embodiment the second
stretch of amino acids comprises at least 50 consecutive amino
acids. In an embodiment, the first and/or second stretch of amino
acids is between 1 and 100, 100 and 200 or 200 and 300 amino acids
in length. In an embodiment the first stretch or second stretch of
amino acids comprises at least 20, 25, 30, 35, 40, or 45
consecutive amino acids.
[0102] In an embodiment, the first stretch of amino acids comprises
more than one type of amino acid. In an embodiment, the second
stretch of amino acids comprises more than one type of amino
acid.
[0103] In an embodiment of the instant compounds, the sequence of
the first and/or second stretch of amino acids corresponds to the
sequence of a constant region of an immunoglobulin. In an
embodiment, the immunoglobulin is a human immunoglobulin. In an
embodiment, the constant region of the immunoglobulin is a constant
region of an IgG, an IgA, an IgE, an IgD, or an IgM immunoglobulin.
In an embodiment, the constant region of the immunoglobulin is a
constant region of an IgG-1, IgG-2, IgG-3 or IgG-4 immunoglobulin.
In an embodiment, the constant region of the immunoglobulin which
is a constant region of an IgG-1, IgG-2, IgG-3 or IgG-4
immunoglobulin has one of the sequences set forth herein. In an
embodiment, the constant region of the immunoglobulin is a constant
region of an IgG immunoglobulin and comprises a hinge region, a CH6
region and a CH3 region. In an embodiment, the different moiety is
an immunoeffector or immunoregulator.
[0104] In an embodiment, the target is protein. In an embodiment,
the target is an EGF receptor, a HER2, a VEGF receptor, a CD20
antigen, a CD11a, an IgE immunoglobulin, a glycoprotein IIa
receptor, a glycoprotein IIIa receptor, a TNF alpha, or a TNF
receptor, a gp120. In an embodiment, each of the first and second
stretch of consecutive amino acids comprises the amino acid
sequence of any one of TNFRSF1a, TNFRSF1b, VEGFR1, VEGFR6, VEGFR3,
human Erb1, human Erb2, human Erb6, human Erb3, or human Erb4. In
an embodiment, the first or second stretch of consecutive amino
acids comprises the amino acid sequence set forth of any one of
TNFRSF1a, TNFRSF1b, VEGFR1, VEGFR6, VEGFR3, human Erb1, human Erb2,
human Erb6, human Erb3, or human Erb4).
[0105] This invention provides a composition comprising any of the
instant compounds in an amount effective to bind the target, and a
carrier. In an embodiment, the compound is in an amount effective
to bind the target, and a carrier. In an embodiment, the compound
is a multimer and is in an amount effective to bind the target, and
a carrier. In an embodiment, the multimer is also present in an
amount effective to bind the different moiety. In an embodiment,
the carrier is a pharmaceutically acceptable carrier. In an
embodiment the carrier is a phosphate-buffered saline. Such a
composition may be lyophilized.
[0106] This invention provides a method of affecting the activity
of a target comprising contacting the target with the composition
of one or more of the instant compounds, under conditions such that
the compound binds to and affects the activity of the target. In
one embodiment, the binding of the composition to the target
increases the activity of the target. In one embodiment, the
binding of the composition to the target decreases the activity of
the target. In one embodiment, the target is an EGF receptor, a
HER2 protein, a VEGF receptor, a CD20 antigen, a CD11a, an IgE
immunoglobulin, a glycoprotein IIa receptor, a glycoprotein IIIa
receptor, a gp40, a gp120, a TNF alpha, or a TNF receptor.
[0107] This invention provides a complex comprising the composition
of any of the instant compounds and a third stretch of consecutive
amino acids, wherein the third stretch of consecutive amino acids
is bound to the one of the first or second stretch of consecutive
amino acids by one or more bonds. In an embodiment the one or more
bonds comprise van der Waals forces. In one embodiment the one or
more bonds comprise a hydrogen bond. In one embodiment the one or
bonds comprise a covalent bond. In one embodiment the one or bonds
comprise a disulfide bond. In an embodiment the at least one bond
is a disulfide bond. In an embodiment the disulfide bond is between
two non-terminal amino acid residues. In an embodiment the
disulfide bond is between two amino acid residues, at least one of
which is a non-terminal amino acid residue.
[0108] This invention provides a process of making one of the
instant compounds comprising: [0109] (a) transfecting a cell with a
recombinant nucleic acid which comprises (i) a first portion, the
sequence of which is a N-terminal signal sequence, contiguous with
(ii) a second portion, the sequence of which encodes a stretch of
consecutive amino acids contiguous with (iii) a third portion, the
sequence of which encodes a C-terminal intein-containing binding
domain, under conditions permitting synthesis of a chimeric
polypeptide comprising the stretch of consecutive amino acids
contiguous with the C-terminal intein-containing binding domain;
[0110] (b) isolating the chimeric polypeptide produced in step (a);
[0111] (c) treating the chimeric polypeptide so as to cause
thio-mediated cleavage of the C-terminal intein-containing binding
domain from the stretch of consecutive amino acids and its
replacement with a C-terminal thioester; [0112] (d) treating the
product of step (c) to permit the attachment of a cysteine residue
to the product so as to form product with a C-terminal cysteine;
and [0113] (e) oxidizing the product of step (e) in the presence of
another product of step (e) under conditions permitting formation
of the compound.
[0114] In one embodiment, the recombinant nucleic acid has the
sequence set forth in any one of SEQ ID NOs. 1-8. In one
embodiment, the C-terminal intein-containing binding domain is an
intein-chitin binding domain. In one embodiment, the C-terminal
intein-containing binding domain is an Mth RIR1 intein-chitin
binding domain. In one embodiment, the chimeric polypeptide is
isolated in step b) by affinity chromatography. In one embodiment,
the chimeric polypeptide is isolated in step b) by exposure of the
product to a chitin-derivatized resin. In one embodiment, the
oxidizing conditions permit formation of a disulfide bond between
the C-terminal cysteine of each of the products.
[0115] This invention provides a compound comprising an
independently folding protein domain fused to a second
independently folding protein domain by non-peptide bond. This
invention provides a compound comprising a first polypeptide chain,
comprising a terminal cysteine residue, fused at its S-terminus to
a S-terminus of a second polypeptide chain comprising a terminal
cysteine residue. This invention provides a compound comprising a
first polypeptide chain, comprising a terminal selenocysteine
residue, fused at its Se-terminus to a S-terminus of a second
polypeptide chain comprising a terminal cysteine residue. This
invention provides a compound comprising a first polypeptide chain,
comprising a terminal selenocysteine residue, fused at its
Se-terminus to a Se-terminus of a second polypeptide chain
comprising a terminal selenocysteine residue. This invention
provides a multimer comprising two or more identical compounds
according to any one of claims 66-69 joined together by at least
one bond.
[0116] This invention provides a method of making a stretch of
consecutive amino acids comprising an N-terminal cysteine
comprising: [0117] (a) transfecting a cell with a recombinant
nucleic acid which comprises (i) a first portion, the sequence of
which encodes a N-terminal signal sequence contiguous with (ii) a
second portion, the sequence of which encodes a stretch of
consecutive amino acids comprising a N-terminal cysteine residue,
under conditions permitting (i) synthesis of a chimeric polypeptide
which comprises the N-terminal signal sequence joined by a peptide
bond at its C-terminus to the N-terminal cysteine of the stretch of
consecutive amino acids and (ii) cleavage of the N-terminal signal
sequence from the chimeric polypeptide within the cell so as to
produce a stretch of consecutive amino acids comprising an
N-terminal cysteine; [0118] (b) recovering the stretch of
consecutive amino acids produced in step (a).
[0119] In one embodiment of the methods disclosed herein, the
stretch of consecutive amino acids is isolated in step (b).
[0120] In one embodiment, the stretch of consecutive amino acids
comprises an immunoglobulin Fc polypeptide. In one embodiment, the
immunoglobulin Fc polypeptide is a human immunoglobulin Fc
polypeptide. In one embodiment, the N-terminal cysteine residue is
a cys-5 residue of the human immunoglobulin Fc polypeptide. In one
embodiment, the cell is a 293 human embryonic cell or a CHO-K1
hamster ovary cell. In one embodiment, the transfection is
performed with a plasmid pSA. In one embodiment, N-terminal signal
sequence is selected from a protein having a N-terminal cysteine.
In one embodiment, the signal peptide is sonic hedgehog, interferon
alpha-2 or cholesterol ester transferase. In one embodiment, the
stretch of consecutive amino acids is recovered by affinity
chromatography. In one embodiment, the cleavage of the chimeric
polypeptide within the cell is effected by a cellular signal
peptidase.
[0121] This invention provides a method of making a stretch of
consecutive amino acids comprising an N-terminal cysteine or
selenocysteine comprising: [0122] (a) transfecting a cell with a
recombinant nucleic acid which comprises (i) a first portion, the
sequence of which encodes a N-terminal signal sequence contiguous
with (ii) a second portion, the sequence of which encodes a stretch
of consecutive amino acids comprising a N-terminal cysteine
residue, under conditions permitting (i) synthesis of a chimeric
polypeptide comprising the N-terminal signal sequence joined at its
C-terminus to the N-terminal cysteine of the Fc polypeptide and
(ii) cleavage of the N-terminal signal sequence from the chimeric
polypeptide within the cell so as to produce a stretch of
consecutive amino acids comprising an N-terminal cysteine; [0123]
(b) ligating the N-terminal of the stretch of consecutive amino
acids produced in step (a) with a C-terminal of a peptide
comprising the amino acid sequence cys-asp-lys-thr-his-thr or with
a peptide comprising the amino acid sequence
sec-asp-lys-thr-his-thr so as to thereby produce the stretch of
consecutive amino acids comprising an N-terminal cysteine or
selenocysteine; and [0124] (c) recovering the stretch of
consecutive amino acids produced in step (b).
[0125] In an embodiment, the stretch of consecutive amino acids
comprises an immunoglobulin Fc polypeptide. In an embodiment, the
immunoglobulin Fc polypeptide is a human immunoglobulin Fc
polypeptide. In an embodiment, the N-terminal cysteine residue is a
cys-5 residue of the human immunoglobulin Fc polypeptide. In one
embodiment, the N-terminal cysteine residue is a cys-11 residue of
the human immunoglobulin Fc polypeptide. In an embodiment, the
peptide in step (b) comprises the amino acid sequence
cys-asp-lys-thr-his-thr and the stretch of consecutive amino acids
produced comprises an N-terminal cysteine. In an embodiment,
peptide in step (b) comprises the amino acid sequence
sec-asp-lys-thr-his-thr and the stretch of consecutive amino acids
produced comprises an N-terminal selenocysteine. In an embodiment,
the peptide in step (b) is protected with a N-terminal Msc
protecting group prior to ligation. In an embodiment, the cell is a
293 human embryonic cell or a CHO-K1 hamster ovary cell. In an
embodiment, the transfection is performed with a plasmid pSA. In an
embodiment, the N-terminal signal sequence is selected from a
protein having a N-terminal cysteine. In an embodiment, the signal
peptide is sonic hedgehog, interferon alpha-2 or cholesterol ester
transferase. In an embodiment, the stretch of consecutive amino
acids is recovered by affinity chromatography. In an embodiment,
the cleavage of the chimeric polypeptide within the cell is
effected by a cellular signal peptidase. In embodiments other short
peptide sequences with a N-terminal cysteine or selenocysteine are
employed in place of those set forth above.
[0126] This invention provides process of making a stretch of
consecutive amino acids comprising a C-terminal cysteine or a
C-terminal selenocysteine, comprising: [0127] (a) transfecting a
cell with a recombinant nucleic acid which comprises (i) a first
portion, the sequence of which encodes a N-terminal signal
sequence, contiguous with (ii) a second portion, the sequence of
which encodes a stretch of consecutive amino acids contiguous with
(iii) a third portion, the sequence of which encodes a C-terminal
intein-containing binding domain, under conditions permitting (i)
synthesis of a chimeric polypeptide comprising the N-terminal
signal sequence contiguous with the stretch of consecutive amino
acids contiguous with the C-terminal intein-containing binding
domain and (ii) cleavage of the N-terminal signal sequence from the
chimeric polypeptide so as to produce a second chimeric polypeptide
having a N-terminal lysine residue and comprising the stretch of
consecutive amino acids contiguous with the C-terminal
intein-containing binding domain; [0128] (b) isolating the second
chimeric polypeptide produced in step (a); [0129] (c) treating the
second chimeric polypeptide so as to cause thio-mediated cleavage
of the C-terminal intein-containing binding domain from the stretch
of consecutive amino acids so as to form a C-terminal thioester;
[0130] (d) ligating the product of step (c) with a cysteine residue
or selenocysteine residue at its C-terminal so as to form product
with a C-terminal cysteine or a C-terminal selenocysteine; and
[0131] (e) recovering the product of step (d).
[0132] In an embodiment, the stretch of consecutive amino acids
contiguous comprises an IgG immunoglobulin Fc polypeptide and an
IgG M1 exon. In an embodiment, the IgG immunoglobulin is a human
IgG immunoglobulin. In an embodiment, the stretch of consecutive
amino acids contiguous comprises a CD4 extracellular domain. In an
embodiment, the N-terminal signal sequence is selected from a
protein having a N-terminal lysine. In an embodiment, the
N-terminal signal sequence is a CD2 T-cell surface glycoprotein or
a CD4 T-cell surface glycoprotein. In an embodiment, the cell is a
293 human embryonic cell or a CHO-K1 hamster ovary cell. In an
embodiment, the transfection is performed with a plasmid pSA. In an
embodiment, the C-terminal intein-containing binding domain is an
intein-chitin binding domain. In an embodiment, the C-terminal
intein-containing binding domain is an Mth RIR1 intein-chitin
binding domain. In an embodiment, the C-terminal intein-containing
binding domain is a self-splicing intein-containing binding domain.
In an embodiment, the chimeric polypeptide is isolated in step b)
by exposure of the product to a chitin-derivatized resin. In an
embodiment, the cleavage of the second chimeric polypeptide within
the cell is effected by a cellular signal peptidase. In an
embodiment, the product of step (c) is ligated with a cysteine
residue. In an embodiment, the product of step (c) is ligated with
a selenocysteine residue.
[0133] This invention provides a process of making a stretch of
consecutive amino acids comprising a N-terminal cysteine and a
C-terminal cysteine or selenocysteine comprising: [0134] (a)
transfecting a cell with a recombinant nucleic acid which comprises
(i) a first portion, the sequence of which encodes a N-terminal
signal sequence, contiguous with (ii) a second portion, the
sequence of which encodes a stretch of consecutive amino acids
contiguous with (iii) a third portion, the sequence of which
encodes a C-terminal intein-containing binding domain, under
conditions permitting (i) synthesis of a chimeric polypeptide
comprising the N-terminal signal sequence contiguous with the
stretch of consecutive amino acids contiguous with the C-terminal
intein-containing binding domain and (ii) cleavage of the
N-terminal signal sequence from the chimeric polypeptide so as to
produce a second chimeric polypeptide having a N-terminal cysteine
residue and comprising the stretch of consecutive amino acids
contiguous with the C-terminal intein-containing binding domain;
[0135] (b) isolating the second chimeric polypeptide produced in
step (a); [0136] (c) treating the second chimeric polypeptide so as
to cause thio-mediated cleavage of the C-terminal intein-containing
binding domain from the stretch of consecutive amino acids so as to
form a C-terminal thioester; [0137] (d) ligating the product of
step (c) with a cysteine residue or selenocysteine residue at its
C-terminal so as to form product with a C-terminal cysteine or a
C-terminal selenocysteine; and [0138] (e) recovering the product of
step (d).
[0139] In an embodiment, the stretch of consecutive amino acids
contiguous comprises an IgG immunoglobulin Fc polypeptide and an
IgG M1 exon. In an embodiment, the IgG immunoglobulin is a human
IgG immunoglobulin. In an embodiment, the N-terminal signal
sequence is selected from a protein having a N-terminal cysteine.
In an embodiment, the N-terminal cysteine residue is a cys-5
residue of the human immunoglobulin Fc polypeptide. In an
embodiment, the N-terminal cysteine residue is a cys-11 residue of
the human immunoglobulin Fc polypeptide. In an embodiment, the
N-terminal signal is a sonic hedgehog, interferon alpha-2 or
cholesterol ester transferase. In an embodiment, the cell is a 293
human embryonic cell or a CHO-K1 hamster ovary cell. In an
embodiment, the transfection is performed with a plasmid pSA. In an
embodiment, the C-terminal intein-containing binding domain is an
intein-chitin binding domain. In an embodiment, the C-terminal
intein-containing binding domain is an Mth RIR1 intein-chitin
binding domain. In an embodiment, the C-terminal intein-containing
binding domain is a self-splicing intein-containing binding domain
In an embodiment, the second chimeric polypeptide is isolated in
step b) by exposure of the product to a chitin-derivatized resin.
In an embodiment, the product is recovered in step e) by affinity
chromatography. In an embodiment, the cleavage of the chimeric
polypeptide within the cell is effected by a cellular signal
peptidase. In an embodiment, the product of step (c) is ligated
with a cysteine residue. In an embodiment, the product of step (c)
is ligated with a selenocysteine residue.
[0140] This invention provides a process of making a stretch of
consecutive amino acids comprising a N-terminal cysteine or
selenocysteine and a C-terminal cysteine or selenocysteine
comprising: [0141] (a) transfecting a cell with a recombinant
nucleic acid which comprises (i) a first portion, the sequence of
which encodes a N-terminal signal sequence, contiguous with (ii) a
second portion, the sequence of which encodes a stretch of
consecutive amino acids contiguous with (iii) a third portion, the
sequence of which encodes a C-terminal intein-containing binding
domain, under conditions permitting (i) synthesis of a chimeric
polypeptide comprising the N-terminal signal sequence contiguous
with the stretch of consecutive amino acids contiguous with the
C-terminal intein-containing binding domain and (ii) cleavage of
the N-terminal signal sequence from the chimeric polypeptide so as
to produce a second chimeric polypeptide having a N-terminal
cysteine residue and comprising the stretch of consecutive amino
acids contiguous with the C-terminal intein-containing binding
domain; [0142] (b) isolating the second chimeric polypeptide
produced in step (a); [0143] (c) (i) ligating the N-terminal of the
stretch of consecutive amino acids produced in step (a) with a
C-terminal of a peptide comprising the amino acid sequence
cys-asp-lys-thr-his-thr or with a peptide comprising the amino acid
sequence sec-asp-lys-thr-his-thr so as to thereby produce the
stretch of consecutive amino acids comprising a N-terminal cysteine
or a N-terminal selenocysteine, respectively; [0144] (ii) treating
the chimeric polypeptide so as to cause thio-mediated cleavage of
the C-terminal intein-containing binding domain from the stretch of
consecutive amino acids and its replacement with a C-terminal
thioester; [0145] (iii) ligating the product of step (c) with a
cysteine residue or selenocysteine residue at its C-terminal so as
to form product with a C-terminal cysteine pr with a C-terminal
selenocysteine; and [0146] (d) recovering the product of step
(c)(iii).
[0147] In an embodiment, the stretch of consecutive amino acids
contiguous comprises an IgG immunoglobulin Fc polypeptide and an
IgG M1 exon. In an embodiment, the IgG immunoglobulin is a human
IgG immunoglobulin. In an embodiment, the N-terminal signal
sequence is selected from a protein having a N-terminal cysteine.
In an embodiment, the N-terminal cysteine residue is a cys-11
residue of the human immunoglobulin Fc polypeptide. In an
embodiment, N-terminal signal sequence is selected from a protein
having a N-terminal cysteine. In an embodiment, the N-terminal
signal is a sonic hedgehog, interferon alpha-2 or cholesterol ester
transferase. In an embodiment, the cell is a 293 human embryonic
cell or a CHO-K1 hamster ovary cell. In an embodiment, the
transfection is performed with a plasmid pSA. In an embodiment, the
C-terminal intein-containing binding domain is an intein-chitin
binding domain. In an embodiment, the C-terminal intein-containing
binding domain is an Mth RIR1 intein-chitin binding domain. In an
embodiment, the chimeric polypeptide is isolated in step (b) by
exposure of the product to a chitin-derivatized resin.
[0148] In an embodiment, the immunoglobulin Fc polypeptide is a
human immunoglobulin Fc polypeptide. In an embodiment, the
N-terminal cysteine residue of the Fc polypeptide is a cys-11
residue. In an embodiment, the peptide in step (c) (i) comprises
the amino acid sequence cys-asp-lys-thr-his-thr and the stretch of
consecutive amino In an embodiment, the peptide in step (c) (i)
comprises the amino acid sequence sec-asp-lys-thr-his-thr and the
stretch of consecutive amino acids produced comprises an N-terminal
selenocysteine. In an embodiment, the peptide in step (c) is
protected with a N-terminal Msc protecting group prior to ligation.
In an embodiment, in the cell is a 293 human embryonic cell or a
CHO-K1 hamster ovary cell. In an embodiment, the transfection is
performed with a plasmid pSA. In an embodiment, the N-terminal
signal sequence is selected from a protein having a N-terminal
cysteine. In an embodiment, the signal peptide is sonic hedgehog,
interferon alpha-2 or cholesterol ester transferase. In an
embodiment, in the stretch of consecutive amino acids is recovered
by affinity chromatography.
[0149] In an embodiment, step (c) of this instant method is
performed in the order step (c) (i); step (c) (ii); step (c) (iii).
In an embodiment, step (c) is performed in the order step (c)(ii);
step (c)(iii); step (c)(i).
[0150] This invention provides a process for making a compound
comprising contacting a stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which comprises a binding site for a target with a
second stretch of consecutive amino acids, each of which is joined
to the preceding amino acid by a peptide bond and the sequence of
which is identical to the sequence of the first stretch of
consecutive amino acids and which comprises an identical binding
site for the target, wherein each of the first stretch of amino
acids and the second stretch of amino acids has at a predefined end
thereof a cysteine residue or a selenocysteine residue, under
reducing conditions so as to make the compound.
[0151] This invention provides a process for making a compound
comprising contacting a stretch of consecutive amino acids, each of
which is joined to the preceding amino acid by a peptide bond and
the sequence of which comprises a binding site for a target with a
second stretch of consecutive amino acids, each of which is joined
to the preceding amino acid by a peptide bond and the sequence of
which is different to the sequence of the first stretch of
consecutive amino acids and which comprises an identical binding
site for the target, wherein each of the first stretch of amino
acids and the second stretch of amino acids has at a predefined end
thereof a cysteine residue or a selenocysteine residue, under
reducing conditions so as to make the compound.
[0152] In an embodiment of the instant processes, the reducing
conditions do not denature the stretches of consecutive amino
acids. In an embodiment, the reducing conditions comprise exposing
the stretches of consecutive amino acids to a buffer comprising
Tris-HCL and mercaptoethanol. In an embodiment, the buffer is
between pH 7.6 and 8.4. In an embodiment, the buffer is pH 8. In an
embodiment, the method further comprises exchanging the product
into oxidation buffer. In an embodiment, the stretches of
consecutive amino acids comprises a CD4 extracellular domain. In an
embodiment, the stretches of consecutive amino acids comprises a
sequence of an immunoglobulin Fc polypeptide. In an embodiment, the
immunoglobulin is a human immunoglobulin.
[0153] In embodiments, the transfected cells of the instant methods
are grown under conditions suitable to permit expression of the
polypeptide.
[0154] In an embodiment a compound is provided comprising a first
stretch of consecutive amino acids, each of which is joined to the
preceding amino acid by a peptide bond and the sequence of which
comprises a binding site for a target; and
a second stretch of consecutive amino acids, each of which is
joined to the preceding amino acid by a peptide bond and the
sequence of which is identical to the sequence of the first stretch
of consecutive amino acids and which comprises an identical binding
site for the target; wherein each of the first stretch of amino
acids and the second stretch of amino acids has at a predefined end
thereof, independently, a natural amino acid or non-natural amino
having a linear aliphatic side-chain acid comprising a sulfur (S)
or a selenium (Se) and wherein such sulfur (S) or a selenium (Se)
are joined by a bond having the structure:
##STR00007##
wherein each X is a sulfur (S) or a selenium (Se) and each (C)
represents a carbon of the linear aliphatic side-chain of one of
such natural or non-natural amino acid and wherein n and m are,
independently, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
[0155] In an embodiment, the natural amino acid is homocysteine of
homoselenocysteine. In an embodiment, the first stretch and second
stretch of amino acids have a homocysteine at the predefined end
thereof. In an embodiment, the first stretch and second stretch of
amino acids have a homoselenocysteine at the predefined end
thereof. In an embodiment, the first stretch and second stretch of
amino acids have a homocysteine at the predefined end thereof. In
an embodiment, the predefined end is a C-terminus.
[0156] A compound is provided which comprises a first stretch of
consecutive amino acids, each of which is joined to the preceding
amino acid by a peptide bond and the sequence of which comprises a
binding site for a target; and
a second stretch of consecutive amino acids, each of which is
joined to the preceding amino acid by a peptide bond and the
sequence of which is different from the sequence of the first
stretch of consecutive amino acids and which comprises a binding
site for a different moiety; wherein each of the first stretch of
amino acids and the second stretch of amino acids has at a
predefined end thereof, independently, a natural amino acid or
non-natural amino having a linear aliphatic side-chain acid
comprising a sulfur (S) or a selenium (Se) and wherein such sulfur
(S) or a selenium (Se) are joined by a bond having the
structure:
##STR00008##
wherein each X may be the same or different and represents a sulfur
(S) or a selenium (Se) and each (C) represents a carbon of the
linear aliphatic side-chain of one of such natural or non-natural
amino acid and wherein n and m are, independently, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10.
[0157] In an embodiment, the natural amino acid is homocysteine of
homoselenocysteine. In an embodiment, the first stretch and second
stretch of amino acids have a homocysteine at the predefined end
thereof. In an embodiment, the first stretch and second stretch of
amino acids have a homoselenocysteine at the predefined end
thereof. In an embodiment, the first stretch and second stretch of
amino acids have a homocysteine at the predefined end thereof. In
an embodiment, the predefined end is a C-terminus.
[0158] A method is provided of producing a protein which comprises
a first polypeptide contiguous with an intein, which intein is
contiguous with a second polypeptide comprising a binding domain,
the method comprising transfecting an animal cell with a nucleic
acid, which nucleic acid comprises (i) a first portion which
encodes the polypeptide contiguous with (ii) a second portion which
encodes the intein, contiguous with a and the third portion of
which encodes the binding domain, under conditions such that the
animal cell expresses and secretes the protein. In an embodiment,
the animal cell is a derived from mammal. In an embodiment, the
binding domain is a chitin-binding domain.
[0159] A composition is provided comprising a polypeptide attached
to a solid surface through a terminal disulfide bond. In an
embodiment, the solid surface is a chip or a bead.
[0160] A compound is provided comprising:
a first stretch of consecutive amino acids each of which is joined
to the preceding amino acid by a peptide bond and which first
stretch of consecutive amino acids comprises an amino acid residue
having a chalcogen functional group-containing side chain; and a
second stretch of consecutive amino acids, comprising at least 100
amino acids, each of which is joined to the preceding amino acid by
a peptide bond, wherein at least 90 consecutive amino acids of the
second stretch of consecutive amino acids have a sequence identical
to portion of a human immunoglobulin constant region polypeptide,
and wherein the second stretch of consecutive amino acids comprises
an amino acid residue having a chalcogen functional
group-containing side chain at a predefined terminus thereof,
wherein said amino acid residue having a chalcogen functional
group-containing side chain of the first stretch of consecutive
amino acids and said amino acid residue having a chalcogen
functional group-containing side chain of the second stretch of
consecutive amino acids are joined by a bond having the
structure:
##STR00009##
wherein each X represents, independently, a chalcogen, and wherein
C.sub.1 represents a side chain carbon of the amino acid residue
having a chalcogen functional group-containing side chain of the
first stretch of consecutive amino acids and C.sub.2 represents a
side chain carbon of the second stretch of consecutive amino
acids.
[0161] In embodiments at least wherein at least 91, 92, 93, 94, 95,
96, 97, 98, 99 or 100 consecutive amino acids of the second stretch
of consecutive amino acids have a sequence identical to portion of
a human immunoglobulin constant region polypeptide.
[0162] In an embodiment of the compound, at least one of C.sub.1
and C.sub.2 is a beta carbon of amino acid. In an embodiment of the
compound the bond has the structure:
##STR00010##
wherein S is sulfur. In an embodiment of the compound the bond has
the structure:
##STR00011## [0163] wherein S is sulfur and Se is selenium.
[0164] In an embodiment of the compound the amino acid residue
having a chalcogen functional group-containing side chain at the
predefined terminus of the second stretch of amino acids is a
cysteine. In an embodiment of the compound the amino acid residue
having a chalcogen functional group-containing side chain at the
predefined terminus of the second stretch of amino acids is a
selenocysteine, homocysteine or homoseleneocysteine. In an
embodiment of the compound the amino acid residue having a
chalcogen functional group-containing side chain of the first
stretch of consecutive amino acids is a cysteine. In an embodiment
of the compound the amino acid residue having a chalcogen
functional group-containing side chain of the first stretch of
amino acids is a selenocysteine, homocysteine or
homoseleneocysteine. In an embodiment of the compound the amino
acid residue having a chalcogen functional group-containing side
chain of the first stretch of consecutive amino acids is a terminal
residue.
[0165] In an embodiment, the amino acid residue having a chalcogen
functional group-containing side chain of the first stretch of
consecutive amino acids is a penultimate, antepenultimate, or
pre-antepenultimate terminal residue.
[0166] In an embodiment, the second stretch of consecutive amino
acids has a sequence identical to a human immunoglobulin constant
region. In an embodiment, the second stretch of consecutive amino
acids is a portion of a human immunoglobulin constant region. In an
embodiment, the first stretch of consecutive amino acids has a
sequence identical to a human immunoglobulin constant region.
[0167] In an embodiment, the human immunoglobulin constant region
polypeptide is a human IgG1, human IgG2, human IgG3, or human IgG4.
A compound is provided wherein the side chain of at least one of
amino acid residues having a chalcogen functional group-containing
side chain comprises a C1-C10 alkylene.
[0168] A composition is provided comprising two of the instant
compounds bonded together via at least one disulfide bond between
the second stretch of consecutive amino acids of each of the
compounds.
[0169] A composition comprising a polypeptide is provided
comprising consecutive amino acids having the sequence set forth in
one of SEQ ID NOS:35 through 46, or having the sequence set forth
in one of SEQ ID NOS:53 through 67, or having the sequence set
forth in one of SEQ ID NOS:74 through 82, or having the sequence
set forth in one of SEQ ID NOS:89 through 97, wherein the
polypeptide does not consist of a naturally-occurring
immunoglobulin polypeptide, including enzyme-cleaved fragments
thereof.
[0170] A composition comprising a polypeptide is provided
consisting of consecutive amino acids having the sequence set forth
in one of SEQ ID NOS:35 through 46, or having the sequence set
forth in one of SEQ ID NOS:53 through 67, or having the sequence
set forth in one of SEQ ID NOS:74 through 82, or having the
sequence set forth in one of SEQ ID NOS:89 through 97.
[0171] A composition comprising the instant polypeptide is provided
and a carrier. In an embodiment of the compound, the carrier is
phosphate-buffered saline.
[0172] A composition is provided comprising two of the instant
independently chosen polypeptides, joined by a non-peptide bond. In
one embodiment the bond is a di-chalcogenide bond. In one
embodiment the bond is a disulfide bond.
[0173] A composition comprising two of the instant polypeptides
bonded together via at least one disulfide bond between the two
polypeptides is provided.
[0174] A composition is provided comprising a polypeptide is
provided consisting of consecutive amino acids having a sequence
identical to a portion of the sequence set forth in SEQ ID NO:44,
SEQ ID NO:64, SEQ ID NO:81 or SEQ ID NO:96, wherein at least one of
the terminal residues of the polypeptide has a chalcogen functional
group-containing side chain.
[0175] In an embodiment of the compound the terminal residue having
a chalcogen functional group-containing side chain is a cysteine or
analog thereof.
[0176] The various N-terminal signal sequences, plasmids,
expression vecots, recombinant nucleic acids, stretches of
consecutive amino acids, intein binding domains, cell types,
recovery/isolation methods, etc set forth hereinabove are
non-limiting examples, further of which are set forth in the
Examples below.
DEFINITION OF TERMS
[0177] Stretch of consecutive amino acids: a plurality of amino
acids arranged in a chain, each of which is joined to a preceding
amino acid by a peptide bond, excepting that the first amino acid
in the chain is not joined to a preceding amino acid. The amino
acids of the chain may be naturally or non-naturally occurring, or
may comprise a mixture thereof. The amino acids, unless otherwise
indicated, may be genetically encoded, naturally-occurring but not
genetically encoded, or non-naturally occurring, and any selection
thereof.
[0178] In an embodiment, a stretch of consecutive amino acids has
biological activity, including, but not limited to, target-binding
activity or an immunoeffector activity, which biological activity
is retained on the bonding of the stretch of consecutive amino
acids to another stretch of consecutive amino acids by an --X--X--
bond (e.g. --S--S--, --S--Se--, --Se--Se--, or --Se--S-- bond). A
"segment of consecutive amino acids" is an alternative description
of "a stretch of amino acids".
[0179] N-terminal amino acid residue: the terminal residue of a
stretch of two or more consecutive amino acids having a free
.alpha.-amino (NH.sub.2) functional group, or a derivative of an
.alpha.-amino (NH.sub.2) functional group.
[0180] N-terminus: the free .alpha.-amino (NH.sub.2) group (or
derivative thereof) of a N-terminal amino acid residue.
[0181] C-terminal amino acid residue: the terminal residue of a
stretch of two or more consecutive amino acids having a free
.alpha.-carboxyl (COOH) functional group, or a derivative of a
.alpha.-carboxyl (COOH) functional group.
[0182] C-terminus: the free .alpha.-carboxyl (COOH) group (or
derivative thereof) of a C-terminal amino acid residue.
[0183] S-terminal cysteine residue: a cysteine which is the N-
and/or C-terminal residue(s) of a stretch of consecutive amino
acids, and which has a free .beta.-sulfhydryl (SH) functional
group, or a derivative of a .beta.-sulfhydryl (SH) functional
group.
[0184] S-terminus: the free .beta.-sulfhydryl (SH) group (or
derivative thereof) of a S-terminal cysteine residue.
[0185] Se-terminal selenocysteine residue: a selenocysteine which
is the N- and/or C-terminal residue(s) of a stretch of consecutive
amino acids, and which has a free .beta.-selenohydryl (SeH)
functional group, or a derivative of a .beta.-selenohydryl (SeH)
functional group.
[0186] Se-terminus: the free .beta.-selenohydryl (SeH) group (or
derivative thereof) of a Se-terminal selenocysteine residue.
[0187] X-terminal amino acid residue: a cysteine (or cysteine
derivative) or homocysteine (or homocysteine derivative),
selenocysteine (or selenocysteine derivative) or homoselenocysteine
(or homoselenocysteine derivative), which is the N- and/or
C-terminal residue(s) of a stretch of consecutive amino acids, and
which has a free .beta.-sulfhydryl (SH) or .beta.-selenohydryl
(SeH) functional group, respectively, or a sulfur-containing or
selenium-containing derivative thereof.
[0188] X-terminus: the free .beta.-sulfhydryl (SH) or
.beta.-selenohydryl (SeH) group of a S-terminal cysteine/cysteine
derivative residue or of a Se-terminal
selenocysteine/selenocysteine derivative residue, respectively. In
addition, an X-terminus can be the free .beta.-sulfhydryl (SH) or
.beta.-selenohydryl (SeH) group of a S-terminal homocysteine
residue or Se-terminal homoselenocysteine residue.
[0189] Target: an entity which binds to a discrete selection of a
stretch of consecutive amino acids or a portion of a tertiary
structure thereof and includes, but is not limited to, receptors,
carrier proteins, hormones, cellular adhesive proteins,
tissue-specific adhesion factors, growth factors, and enzymes.
Specific examples of targets include a human EGF receptor, a HER2
protein, a VEGF receptor, a human CD20 antigen, a human CD11a, a
human IgE immunoglobulin, a human glycoprotein IIa receptor, a
human glycoprotein IIIa receptor, a human TNF alpha, and a TNF
receptor.
[0190] A "bond", unless otherwise specified, or contrary to
context, is understood to include a covalent bond, a dipole-dipole
interaction such as a hydrogen bond, and intermolecular
interactions such as van der Waals forces.
[0191] A "Signal Sequence" is a short (3-60 amino acids long)
peptide chain that directs the post-translational transport of a
polypeptide.
[0192] "Amino acid" as used herein, in one embodiment, means a L or
D isomer of the genetically encoded amino acids, i.e. isoleucine,
alanine, leucine, asparagine, lysine, aspartate, methionine,
cysteine, phenylalanine, glutamate, threonine, glutamine,
tryptophan, glycine, valine, proline, arginine, serine, histidine,
tyrosine, selenocysteine, pyrolysine and also includes homocysteine
and homoselenocysteine.
[0193] Other examples of amino acids include an L or D isomer of
taurine, gaba, dopamine, lanthionine, 2-aminoisobutyric acid,
dehydroalanine, ornithine and citrulline, as well as non-natural
homologues and synthetically modified forms thereof including amino
acids having alkylene chains shortened or lengthened by up to two
carbon atoms, amino acids comprising optionally substituted aryl
groups, and amino acids comprising halogenated groups, including
halogenated alkyl and aryl groups as well as beta or gamma amino
acids, and cyclic analogs.
[0194] Due to the presence of ionizable amino and carboxyl groups,
the amino acids in these embodiments may be in the form of acidic
or basic salts, or may be in neutral forms. Individual amino acid
residues may also be modified by oxidation or reduction. Other
contemplated modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, and methylation of the alpha-amino groups of lysine,
arginine, and histidine side chains.
[0195] Covalent derivatives may be prepared by linking particular
functional groups to the amino acid side chains or at the N- or
C-termini.
[0196] "Chalcogen" as used herein is limited to sulfur, selenium,
tellurium and polonium only, i.e. as used herein, "chalcogen"
excludes oxygen and ununhexium.
[0197] "Chalcogen functional group containing side chain" as used
herein is an amino acid residue side chain containing a terminally
reactive non-oxygen, non-ununhexium chalcogen atom. By way of
non-limiting example, an amino acid having a chalcogen functional
group containing side chain would be cysteine, selenocysteine,
homocysteine etc., but would not include methionine, for example,
which contains a chalcogen atom (S), but not a terminally reactive
chalcogen atom.
[0198] Compounds comprising amino acids with R-group substitutions
are within the scope of the invention. It is understood that
substituents and substitution patterns on the compounds of the
instant invention can be selected by one of ordinary skill in the
art to provide compounds that are chemically stable from readily
available starting materials.
[0199] "Natural amino acid" as used herein means a L or D isomer of
the genetically encoded amino acids, i.e. isoleucine, alanine,
leucine, asparagine, lysine, aspartate, methionine, cysteine,
phenylalanine, glutamate, threonine, glutamine, tryptophan,
glycine, valine, proline, arginine, serine, histidine, tyrosine,
selenocysteine, pyrolysine and homocysteine and
homoselenocysteine.
[0200] "Non-natural amino acid" as used herein means a chemically
modified L or D isomer of isoleucine, alanine, leucine, asparagine,
lysine, aspartate, methionine, cysteine, phenylalanine, glutamate,
threonine, glutamine, tryptophan, glycine, valine, proline,
arginine, serine, histidine, tyrosine, selenocysteine, pyrolysine,
homocysteine, homoselenocysteine, taurine, gaba, dopamine,
lanthionine, 2-aminoisobutyric acid, dehydroalanine, ornithine or
citrulline, including cysteine and selenocysteine derivatives
having C.sub.3-C.sub.10 aliphatic side chains between the alpha
carbon and the S or Se. In one embodiment the aliphatic side chain
is an alkylene. In another embodiment, the aliphatic side chain is
an alkenylene or alkynylene.
[0201] In addition to the stretches of consecutive amino acid
sequences described herein, it is contemplated that variants
thereof can be prepared by introducing appropriate nucleotide
changes into the encoding DNA, and/or by synthesis of the desired
consecutive amino acid sequences. Those skilled in the art will
appreciate that amino acid changes may alter post-translational
processes of the stretches of consecutive amino acids described
herein when expression is the chosen method of synthesis (rather
than chemical synthesis for example), such as changing the number
or position of glycosylation sites or altering the membrane
anchoring characteristics.
[0202] Variations in the sequences described herein, can be made,
for example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the consecutive amino acid sequence of interest that results in a
change in the amino acid sequence as compared with the native
sequence. Optionally the variation is by substitution of at least
one amino acid with any other amino acid in one or more of the
domains. Guidance in determining which amino acid residue may be
inserted, substituted or deleted without adversely affecting the
desired activity may be found by comparing the sequence with that
of homologous known protein molecules and minimizing the number of
amino acid sequence changes made in regions of high homology. Amino
acid substitutions can be the result of replacing one amino acid
with another amino acid having similar structural and/or chemical
properties, such as the replacement of a leucine with a serine,
i.e., conservative amino acid replacements. Insertions or deletions
may optionally be in the range of about 1 to 5 amino acids. The
variation allowed may be determined by systematically making
insertions, deletions or substitutions of amino acids in the
sequence and testing the resulting variants for activity exhibited
by the full-length or mature native sequence. It is understood that
any terminal variations are made within the context of the
invention disclosed herein.
[0203] Amino acid sequence variants of the binding partner are
prepared with various objectives in mind, including increasing the
affinity of the binding partner for its ligand, facilitating the
stability, purification and preparation of the binding partner,
modifying its plasma half life, improving therapeutic efficacy, and
lessening the severity or occurrence of side effects during
therapeutic use of the binding partner.
[0204] Amino acid sequence variants of these sequences are also
contemplated herein including insertional, substitutional, or
deletional variants. Such variants ordinarily can prepared by
site-specific mutagenesis of nucleotides in the DNA encoding the
target-binding monomer, by which DNA encoding the variant is
obtained, and thereafter expressing the DNA in recombinant cell
culture. Fragments having up to about 100-150 amino acid residues
can also be prepared conveniently by in vitro synthesis. Such amino
acid sequence variants are predetermined variants and are not found
in nature. The variants exhibit the qualitative biological activity
(including target-binding) of the nonvariant form, though not
necessarily of the same quantative value. While the site for
introducing an amino acid sequence variation is predetermined, the
mutation per se need not be predetermined. For example, in order to
optimize the performance of a mutation at a given site, random or
saturation mutagenesis (where all 20 possible residues are
inserted) is conducted at the target codon and the expressed
variant is screened for the optimal combination of desired
activities. Such screening is within the ordinary skill in the
art.
[0205] Amino acid insertions usually will be on the order of about
from 1 to 10 amino acid residues; substitutions are typically
introduced for single residues; and deletions will range about from
1 to 30 residues. Deletions or insertions preferably are made in
adjacent pairs, i.e. a deletion of 2 residues or insertion of 2
residues. It will be amply apparent from the following discussion
that substitutions, deletions, insertions or any combination
thereof are introduced or combined to arrive at a final
construct.
[0206] In an aspect, the invention concerns a compound comprising a
stretch of consecutive amino acids having at least about 80%
sequence identity, preferably at least about 81% sequence identity,
more preferably at least about 82% sequence identity, yet more
preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to an amino acid
sequence disclosed in the specification, a figure, a SEQ ID NO. or
a sequence listing of the present application.
[0207] The % amino acid sequence identity values can be readily
obtained using, for example, the WU-BLAST-2 computer program
(Altschul et al., Methods in Enzymology 266:460-480 (1996)).
[0208] Fragments of native sequences are provided herein. Such
fragments may be truncated at the N-terminus or C-terminus, or may
lack internal residues, for example, when compared with a full
length native protein. Again, it is understood that any terminal
variations are made within the context of the invention disclosed
herein. Certain fragments lack amino acid residues that are not
essential for a desired biological activity of the sequence of
interest.
[0209] Any of a number of conventional techniques may be used.
Desired peptide fragments or fragments of stretches of consecutive
amino acids may be chemically synthesized. An alternative approach
involves generating fragments by enzymatic digestion, e.g. by
treating the protein with an enzyme known to cleave proteins at
sites defined by particular amino acid residues, or by digesting
the DNA with suitable restriction enzymes and isolating the desired
fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding a desired polypeptide/sequence
fragment, by polymerase chain reaction (PCR). Oligonucleotides that
define the desired termini of the DNA fragment are employed at the
5' and 3' primers in the PCR.
[0210] In particular embodiments, conservative substitutions of
interest are shown in Table 1 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 1, or as further described below
in reference to amino acid classes, are introduced and the products
screened.
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Ala (A) val;
leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg
gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp
Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu;
val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val;
met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile
leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) ala ala Ser (S) thr
thr Thr (T) ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser
phe Val (V) ile; leu; met; phe; ala; norleucine leu
[0211] Substantial modifications in function or immunological
identity of the sequence are accomplished by selecting
substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide backbone in the
area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn,
gln, his, lys, arg; (5) residues that influence chain orientation:
gly, pro; (6) aromatic: trp, tyr, phe.
[0212] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0213] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
(Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et
al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or
other known techniques can be performed on the cloned DNA to
produce the variant DNA.
[0214] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant (Cunningham and Wells, Science, 244:1081-1085
(1989)). Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions (Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0215] Covalent modifications: The stretches of consecutive amino
acids may be covalently modified. One type of covalent modification
includes reacting targeted amino acid residues with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues that are not involved in an
-x-x- bond. Derivatization with bifunctional agents is useful, for
instance, for crosslinking to a water-insoluble support matrix or
surface for use in the method for purifying anti-sequence of
interest antibodies, and vice-versa. Commonly used crosslinking
agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide esters, for example, esters
with 4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-((p-azidophenyl)dithio)propioimidate.
[0216] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains (T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)), acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0217] Another type of covalent modification comprises altering the
native glycosylation pattern of the consecutive stretch of amino
acids or of a polypeptide. "Altering the native glycosylation
pattern" is intended for purposes herein to mean deleting one or
more carbohydrate moieties found in amino acid sequences (either by
removing the underlying glycosylation site or by deleting the
glycosylation by chemical and/or enzymatic means), and/or adding
one or more glycosylation sites that are not present in the native
sequence. In addition, the phrase includes qualitative changes in
the glycosylation of the native proteins, involving a change in the
nature and proportions of the various carbohydrate moieties
present.
[0218] Addition of glycosylation sites to the amino acid sequence
may be accomplished by altering the amino acid sequence. The
alteration may be made, for example, by the addition of, or
substitution by, one or more serine or threonine residues to the
native sequence (for O-linked glycosylation sites). The amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the amino acid
sequence at preselected bases such that codons are generated that
will translate into the desired amino acids.
[0219] Another means of increasing the number of carbohydrate
moieties on the amino acid sequence is by chemical or enzymatic
coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO 87/05330 published Sep. 11, 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0220] Removal of carbohydrate moieties present on the amino acid
sequence may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enzymol., 138:350 (1987).
[0221] Another type of covalent modification comprises linking the
amino acid sequence to one of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337,
or to a tag polypeptide which provides an epitope to which an
anti-tag antibody can selectively bind. Various tag polypeptides
and their respective antibodies are well known in the art. Examples
include poly-histidine (poly-his) or poly-histidine-glycine
(poly-his-gly) tags; the flu HA tag polypeptide and its antibody
12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)); the
c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies
thereto (Evan et al., Molecular and Cellular Biology, 5:3610-3616
(1985); and the Herpes Simplex virus glycoprotein D (gD) tag and
its antibody (Paborsky et al., Protein Engineering, 3(6):547-553
(1990)). Other tag polypeptides include the Flag-peptide (Hopp et
al., BioTechnology, 6:1204-1210 (1988)); the KT3 epitope peptide
(Martin et al., Science, 255:192-194 (1992)); an alpha-tubulin
epitope peptide (Skinner et al., J. Biol. Chem., 266:15163-15166
(1991)); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et
al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)).
Salts
[0222] Salts of the compounds disclosed herein are within the scope
of the invention. As used herein, a "salt" is salt of the instant
compounds which has been modified by making acid or base salts of
the compounds.
Pharmaceuticals
[0223] The salt can be pharmaceutically acceptable. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxcylic
acids. The salts can be made using an organic or inorganic acid.
Such acid salts are chlorides, bromides, sulfates, nitrates,
phosphates, sulfonates, formates, tartrates, maleates, malates,
citrates, benzoates, salicylates, ascorbates, and the like.
Carboxylate salts are the alkaline earth metal salts, sodium,
potassium or lithium.
[0224] Pharmaceutically acceptable salts of the compounds disclosed
here can be prepared in any conventional manner, for example by
treating a solution or suspension of the corresponding free base or
acid with one chemical equivalent of a pharmaceutically acceptable
acid or base. Conventional concentration or crystallization
techniques can be employed to isolate the salts. Illustrative of
suitable acids are acetic, lactic, succinic, maleic, tartaric,
citric, gluconic, ascorbic, benzoic, cinnamic, fumaric, sulfuric,
phosphoric, hydrochloric, hydrobromic, hydroiodic, sulfamic,
sulfonic acids such as methanesulfonic, benzene sulfonic,
p-toluenesulfonic, and related acids. Illustrative bases are
sodium, potassium, and calcium.
[0225] The term "pharmaceutically acceptable carrier" is understood
to include excipients, carriers or diluents. The particular
carrier, diluent or excipient used will depend upon the means and
purpose for which the active ingredient is being applied.
[0226] The compounds of this invention may be administered alone or
in combination with one or more pharmaceutically acceptable
carriers, in either single or multiple doses. Suitable
pharmaceutical carriers include inert solid diluents or fillers,
sterile aqueous solutions and various organic solvents. The
pharmaceutical compositions disclosed herein can be readily
administered in a variety of dosage forms such as injectable
solutions, tablets, powders, lozenges, syrups, and the like. These
pharmaceutical compositions can, if desired, contain additional
ingredients such as flavorings, binders, excipients and the like.
Additionally, lubricating agents such as magnesium stearate, sodium
lauryl sulfate and talc may be used for tabletting purposes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard filled gelatin capsules. Preferred materials for this
include lactose or milk sugar and high molecular weight
polyethylene glycols. When aqueous suspensions or elixirs are
desired for oral administration, the essential active ingredient
therein may be combined with various sweetening or flavoring
agents, coloring matter or dyes and, if desired, emulsifying or
suspending agents, together with diluents such as water, ethanol,
propylene glycol, glycerin and combinations thereof.
[0227] For parenteral administration, solutions containing a
compound of this invention or a pharmaceutically acceptable salt
thereof in sterile aqueous solution may be employed. Such aqueous
solutions should be suitably buffered if necessary and the liquid
diluent first rendered isotonic with sufficient saline or glucose.
These particular aqueous solutions are especially suitable for
intravenous, intramuscular, subcutaneous and intraperitoneal
administration. The sterile aqueous media employed are all readily
available by standard techniques known to those skilled in the
art.
[0228] The final pharmaceutical composition can be processed into a
unit dosage form (e.g., powdered or lyophilized in a vial, a
solution in a vial, tablet, capsule or sachet) and then packaged
for distribution. The processing step will vary depending upon the
particular unit dosage form. For example, a tablet is generally
compressed under pressure into a desired shape and a capsule or
sachet employs a simple fill operation. Those skilled in the art
are well aware of the procedures used for manufacturing the various
unit dosage forms.
[0229] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Some compositions are in the form of injectable or infusible
solutions. A mode of administration is parenteral (e.g.,
intravenous, subcutaneous, intraperitoneal, intramuscular). In an
embodiment, the compound is administered by intravenous infusion or
injection. In another embodiment, the compound is administered by
intramuscular or subcutaneous injection.
[0230] Therapeutic compositions as contemplated herein typically
must be sterile and stable under the conditions of manufacture and
storage. The composition can be formulated as a solution,
microemulsion, dispersion, liposome, or other ordered structure
suitable to high drug concentration. Sterile injectable solutions
can be prepared by incorporating the compound in the required
amount in an appropriate solvent with one or a combination of
ingredients as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0231] In certain embodiments, the active compound may be prepared
with a carrier that will protect the compound against rapid
release, such as a controlled release formulation, including
implants, transdermal patches, and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art. See, e.g., Sustained and
Controlled Release Drug Delivery Systems, J. R. Robinson, ed.,
Marcel Dekker, Inc., New York, 1978. In certain embodiments, the
compounds of the invention may be orally administered, for example,
with an inert diluent or an assimilable edible carrier. The
compound (and other ingredients, if desired) may also be enclosed
in a hard or soft shell gelatin capsule, compressed into tablets,
or incorporated directly into the subject's diet. For oral
therapeutic administration, the compounds may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. In administering a compound of the invention by other
than parenteral administration the compound may be coated with, or
co-administered with, a material to prevent its inactivation.
[0232] Supplementary active compounds can also be incorporated into
the compositions, for example a chemotherapeutic agent, an
antineoplastic agent or an anti-tumor agent. IN addition, the
compounds of the invention may be coformulated and/or
coadministered with one or more additional therapeutic agents.
These agents include, without limitation, antibodies that bind
other targets (e.g., antibodies that bind one or more growth
factors or cytokines, their cell surface receptors), binding
proteins, antineoplastic agents, chemotherapeutic agents,
anti-tumor agents, antisense oligonucleotides, growth factors. In
one embodiment, a pharmaceutical composition of the compounds
disclosed herein may comprise one or more additional therapeutic
agent.
[0233] For therapeutic use, the compositions disclosed here can be
administered in various manners, including soluble form by bolus
injection, continuous infusion, sustained release from implants,
oral ingestion, local injection (e.g. intracrdiac, intramuscular),
systemic injection, or other suitable techniques well known in the
pharmaceutical arts. Other methods of pharmaceutical administration
include, but are not limited to oral, subcutaneously, transdermal,
intravenous, intramuscular and parenteral methods of
administration. Typically, a soluble composition will comprise a
purified compound in conjunction with physiologically acceptable
carriers, excipients or diluents. Such carriers will be nontoxic to
recipients at the dosages and concentrations employed. The
preparation of such compositions can entail combining a compound
with buffers, antioxidants, carbohydrates including glucose,
sucrose or dextrins, chelating agents such as EDTA, glutathione and
other stabilizers and excipients. Neutral buffered saline or saline
mixed with conspecific serum albumin are exemplary appropriate
diluents. The product can be formulated as a lyophilizate using
appropriate excipient solutions (e.g., sucrose) as diluents.
[0234] Other derivatives comprise the compounds/compositions of
this invention covalently bonded to a nonproteinaceous polymer. The
bonding to the polymer is generally conducted so as not to
interfere with the preferred biological activity of the compound,
e.g. the binding activity of the compound to a target. The
nonproteinaceous polymer ordinarily is a hydrophilic synthetic
polymer, i.e., a polymer not otherwise found in nature. However,
polymers which exist in nature and are produced by recombinant or
in vitro methods are useful, as are polymers which are isolated
from nature. Hydrophilic polyvinyl polymers fall within the scope
of this invention, e.g. polyvinylalcohol and polyvinylpyrrolidone.
Particularly useful are polyalkylene ethers such as polyethylene
glycol, polypropylene glycol, polyoxyethylene esters or methoxy
polyethylene glycol; polyoxyalkylenes such as polyoxyethylene,
polyoxypropylene, and block copolymers of polyoxyethylene and
polyoxypropylene (Pluronics); polymethacrylates; carbomers;
branched or unbranched polysaccharides which comprise the
saccharide monomers D-mannose, D- and L-galactose, fucose,
fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid,
D-galacturontc acid, D-mannuronic acid (e.g. polymannuronic acid,
or alginic acid), D-glucosamine, D-galactosamine, D-glucose and
neuraminic acid including homopolysaccharides and
heteropolysaccharides such as lactose, amylopectin, starch,
hydroxyethyl starch, amylose, dextran sulfate, dextran, dextrins,
glycogen, or the polysaccharide subunit of acid
mucopolysaccharides, e.g. hyaluronic acid; polymers of sugar
alcohols such as polysorbitol and polymannitol; as well as heparin
or heparon.
[0235] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a compound of the invention. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the
desired therapeutic result. A therapeutically effective amount of
the compound may vary according to factors such as the disease
state, age, sex, and weight of the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the compound are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
Examples of Pharmaceutical Compositions
[0236] Non-limiting examples of such compositions and dosages are
set forth as follows:
[0237] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of bevacizumab (e.g. Avastin) may comprise
trehalose dihydrate, sodium phosphate (monobasic, monohydrate),
sodium phosphate (dibasic, anhydrous), polysorbate 20, and Water
for Injection, USP. The composition may also be lyophilized, to
which the water can be added for reconstitution. In an embodiment
the composition has a pH of 6.2 or about 6.2. In one embodiment the
compound can be administered combination with a chemotherapeutic
such as intravenous 5-fluorouracil for treatment of patients with
metastatic carcinoma of the colon or rectum. In one embodiment the
compound is administered in a dose of between 0.1 and 10 mg/kg
given once every 14 days as an IV infusion. In a further embodiment
the dose is 5 mg/kg given once every 14 days. In an embodiment the
dose is between 1.0 and 2.0 mg/kg given once every 14 days. In an
embodiment the dose is between 0.01 and 1.5 mg/kg given once every
14 days. In an embodiment the dose is between 0.001 and 10 mg/kg
given once every 1-21 days.
[0238] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of trastuzumab (e.g. Herceptin) may comprise
trehalose dihydrate, L-histidine HCl, L-histidine, and polysorbate
20, USP. This can be reconstituted with Bacteriostatic Water for
Injection (BWFI), USP, or equivalents thereof, containing 1.1%
benzyl alcohol as a preservative or equivalents thereof. In an
embodiment the composition has a pH of about 6.0. The composition
may also be lyophilized, to which the water can be added for
reconstitution. In one embodiment the compound can be administered
to subjects with metatastic breast cancer whose tumor overexpresses
HER2 protein. In an embodiment the subject has received/is
receiving chemotherapy. In another embodiment the compound is
administered in combination with paclitaxel to subjects with
metastatic breast cancer whose tumors overexpress the HER2 protein
and who have not received chemotherapy for their metastatic
disease. In one embodiment the compound is administered in an
initial dose of between 0.1 and 10 mg/kg in a continuous 45-120
minute infusion IV infusion. In a further embodiment the compound
is administered in a dose of 4 mg/kg in a 90 minute infusion. In an
embodiment a weekly maintenance dose is administered to the subject
at a dose of 2 mg/kg in a 30 minute infusion. In an embodiment the
compound is administered in a dose of 0.5 to 1.5 mg/kg in a 90
minute infusion. In an embodiment a weekly maintenance dose is
administered to the subject at a dose of 0.5-1.0 mg/kg in a 30
minute infusion. In an embodiment the compound is administered in a
dose of 0.04 to 0.5 mg/kg in a 90 minute infusion. In an embodiment
the compound is administered in a dose of between 0.001 to 10 mg/kg
in a 90 minute infusion.
[0239] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of rituximab (e.g. Rituxin) may comprise sodium
chloride, sodium citrate dihydrate, polysorbate 80, and Water for
Injection (USP), or equivalents thereof. In an embodiment the pH of
the composition is adjusted to 6.5. The composition may also be
lyophilized, to which the water can be added for reconstitution. In
one embodiment the compound is administered to a subject for the
treatment of relapsed or refractory, low-grade or follicular,
CD20-positive, B-cell non-Hodgkin's lymphoma. In one embodiment the
compound is administered at a dose of 250-500 mg/m2 IV infusion
once weekly for 4 or 8 doses. In a further embodiment the compound
is administered at 375 mg/m2 IV infusion once weekly for 4 or 8
doses. In one embodiment the compound is administered at a dose of
150-250 mg/m2 IV infusion once weekly for 4 or 8 doses. In one
embodiment the compound is administered at a dose of 1.5 to 5 mg/m2
IV infusion once weekly for 4 or 8 doses. In one embodiment the
compound is administered at a dose of between 1.0 and 500 mg/m2 IV
infusion once weekly for 4 or 8 doses
[0240] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of efalizumab (e.g. Raptiva) may comprise
sucrose, L-histidine hydrochloride monohydrate, L-histidine and
polysorbate 20. Such a composition may be diluted to an
approporiate dosage form with sterile non-USP water, or Sterile
Water for Injection, USP, or equivalents thereof. The composition
may also be lyophilized, to which the water can be added for
reconstitution. In one embodiment the compound is administered to a
subject for the treatment of chronic moderate to severe plaque
psoriasis. Such a subject may be a candidate for systemic therapy
or phototherapy. In an embodiment the compound is administered in a
single 0.1-1.1 mg/kg subcutaneous (SC) conditioning dose followed
by weekly SC doses of 0.8-1.5 mg/kg (maximum single dose not to
exceed a total of 250 mg). In a further embodiment the compound is
administered in a single 0.7 mg/kg SC conditioning dose followed by
weekly SC doses of 1 mg/kg (maximum single dose not to exceed a
total of 200 mg). In an embodiment the compound is administered in
a single 0.001-1.0 mg/kg subcutaneous (SC) conditioning dose
followed by weekly SC doses of 0.008-0.015 mg/kg.
[0241] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of omalizumab (e.g. Xolair) may comprise
sucrose, L-histidine hydrochloride monohydrate, L-histidine, and
polysorbate 20. Such a composition may lyophilized. In an
embodiment the composition is diluted to an appropriate dosage form
with Sterile Water for Injection, USP, or equivalents thereof. In
one embodiment the compound is administered to a subject for the
treatment of moderate to severe persistent asthma. In another
embodiment the compound is administered to a subject who has a
positive skin test or in vitro reactivity to a perennial
aeroallergen and whose symptoms are inadequately controlled with
inhaled corticosteroids in order to decrease the incidence of
asthma exacerbations. In an embodiment the compound is administered
at a dose of 100 to 400 mg subcutaneously every 2 or 4 weeks. In a
further embodiment the compound is administered at a dose of 150 to
375 mg SC every 2 or 4 weeks. In an embodiment the compound is
administered at a dose of 25 to 150 mg subcutaneously every 2 or 4
weeks. In an embodiment the compound is administered at a dose of 1
to 4 mg subcutaneously every 2 or 4 weeks.
[0242] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of etanercept (e.g. Enbrel) may comprise
mannitol, sucrose, and tromethamine. In an embodiment, the
composition is in the form of a lyophilizate. In an embodiment, the
composition is reconstituted with, for example, Sterile
Bacteriostatic Water for Injection (BWFI), USP (containing 0.9%
benzyl alcohol). In an embodiment the compound is administered to a
subject for reducing signs and symptoms, inducing major clinical
response, inhibiting the progression of structural damage, and
improving physical function in subjects with moderately to severely
active rheumatoid arthritis. The compound may be initiated in
combination with methotrexate (MTX) or used alone. In an embodiment
the compound is administered to a subject for reducing signs and
symptoms of moderately to severely active polyarticular-course
juvenile rheumatoid arthritis in subjects who have had an
inadequate response to one or more DMARDs. In an embodiment the
compound is administered to a subject for reducing signs and
symptoms, inhibiting the progression of structural damage of active
arthritis, and improving physical function in subjects with
psoriatic arthritis. In an embodiment the compound is administered
to a subject for reducing signs and symptoms in subjects with
active ankylosing spondylitis. In an embodiment the compound is
administered to a subject for the treatment of chronic moderate to
severe plaque psoriasis. In an embodiment wherein the subject has
rheumatoid arthritis, psoriatic arthritis, or ankylosing
spondylitis the compound is administered at 25-75 mg per week given
as one or more subcutaneous (SC) injections. In a further
embodiment the compound is administered at 50 mg per week in a
single SC injection. In an embodiment wherein the subject has
plaque psoriasis the compound is administered at 25-75 mg twice
weekly or 4 days apart for 3 months followed by a reduction to a
maintenance dose of 25-75 mg per week. In a further embodiment the
compound is administered at a dose of at 50 mg twice weekly or 4
days apart for 3 months followed by a reduction to a maintenance
dose of 50 mg per week. In an embodiment the dose is between
2.times. and 10.times. less than the doses set forth herein. In an
embodiment wherein the subject has active polyarticular-course JRA
the compound may be administered at a dose of 0.2-1.2 mg/kg per
week (up to a maximum of 75 mg per week). In a further embodiment
the compound is administered at a dose of 0.8 mg/kg per week (up to
a maximum of 50 mg per week). In some embodiments the dose is
between 2.times. and 100.times. less than the doses set forth
hereinabove.
[0243] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of infliximab (e.g. Remicade) may comprise
sucrose, polysorbate 80, monobasic sodium phosphate, monohydrate,
and dibasic sodium phosphate, dihydrate. Preservatives are not
present in one embodiment. In an embodiment, the composition is in
the form of a lyophilizate. In an embodiment, the composition is
reconstituted with, for example, Water for Injection (BWFI), USP.
In an embodiment the pH of the composition is 7.2 or is about 7.2.
In one embodiment the compound is administered is administered to a
subject with rheumatoid arthritis in a dose of 2-4 mg/kg given as
an intravenous infusion followed with additional similar doses at 2
and 6 weeks after the first infusion then every 8 weeks thereafter.
In a further embodiment the compound is administered in a dose of 3
mg/kg given as an intravenous infusion followed with additional
similar doses at 2 and 6 weeks after the first infusion then every
8 weeks thereafter. In an embodiment the dose is adjusted up to 10
mg/kg or treating as often as every 4 weeks. In an embodiment the
compound is administered in combination with methotrexate. In one
embodiment the compound is administered is administered to a
subject with Crohn's disease or fistulizing Crohn's disease at dose
of 2-7 mg/kg given as an induction regimen at 0, 2 and 6 weeks
followed by a maintenance regimen of 4-6 mg/kg every 8 weeks
thereafter for the treatment of moderately to severely active
Crohn's disease or fistulizing disease. In a further embodiment the
compound is administered at a dose of 5 mg/kg given as an induction
regimen at 0, 2 and 6 weeks followed by a maintenance regimen of 5
mg/kg every 8 weeks thereafter for the treatment of moderately to
severely active Crohn's disease or fistulizing disease. In an
embodiment the dose is adjusted up to 10 mg/kg. In one embodiment
the compound is administered to a subject with ankylosing
spondylitis at a dose of 2-7 mg/kg given as an intravenous infusion
followed with additional similar doses at 2 and 6 weeks after the
first infusion, then every 6 weeks thereafter. In a further
embodiment the compound is administered at a dose of 5 mg/kg given
as an intravenous infusion followed with additional similar doses
at 2 and 6 weeks after the first infusion, then every 6 weeks
thereafter. In one embodiment the compound is administered to a
subject with psoriatic arthritis at a dose of 2-7 mg/kg given as an
intravenous infusion followed with additional similar doses at 2
and 6 weeks after the first infusion then every 8 weeks thereafter.
In a further embodiment the compound is administered at a dose of 5
mg/kg given as an intravenous infusion followed with additional
similar doses at 2 and 6 weeks after the first infusion then every
8 weeks thereafter. In an embodiment the compound is administered
with methotrexate. In one embodiment the compound is administered
to a subject with ulcerative colitis at a dose of 2-7 mg/kg given
as an induction regimen at 0, 2 and 6 weeks followed by a
maintenance regimen of 2-7 mg/kg every 8 weeks thereafter for the
treatment of moderately to severely active ulcerative colitis. In a
further embodiment the compound is administered to a subject with
ulcerative colitis at a dose of 5 mg/kg given as an induction
regimen at 0, 2 and 6 weeks followed by a maintenance regimen of 5
mg/kg every 8 weeks thereafter. In some embodiments the dose is
between 2.times. and 100.times. less than the doses set forth
hereinabove for treating the indivisual diseases.
[0244] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of cetuximab (e.g. Erbitux) may comprise a
preservative-free composition including sodium chloride, sodium
phosphate dibasic heptahydrate, sodium phosphate monobasic
monohydrate. In an embodiment, the composition is in the form of a
lyophilizate. In an embodiment, the composition is reconstituted
with, for example, Water for Injection, USP. In an embodiment the
pH of the composition is in the range of about 7.0 to about 7.4. In
one embodiment the compound is administered to a subject for the
treatment of EGFR-expressing, metastatic colorectal carcinoma. In
another embodiment the compound is used in combination with
irinotecan for the treatment in patients who are refractory to
irinotecan-based chemotherapy. In an embodiment the compound is
administered at a dose of 300-500 mg/m2 as an initial loading dose
(first infusion) administered as a 120-minute IV infusion (maximum
infusion rate 5 mL/min). In a further embodiment the compound is
administered at a dose of 400 mg/m2 as an initial loading dose
(first infusion) administered as a 120-minute IV infusion (maximum
infusion rate 5 mL/min). In an embodiment the weekly maintenance
dose (all other infusions) is 200-300 mg/m2 infused over 60 minutes
(maximum infusion rate 5 mL/min). In a further embodiment the
weekly maintenance dose (all other infusions) is 250 mg/m2 infused
over 60 minutes (maximum infusion rate 5 mL/min). In some
embodiments the dose is between 2.times. and 100.times. less than
the doses set forth hereinabove.
[0245] Compositions comprising a compound comprising a stretch of
consecutive amino acids which comprises consecutive amino acids
having the sequence of abciximab (e.g. Reopro) may comprise, a
preservative-free composition including sodium phosphate, sodium
chloride and polysorbate 80. In an embodiment, the composition is
in the form of a lyophilizate. In an embodiment, the composition is
diluted in, or reconstituted with, for example, Water for
Injection, USP. In an embodiment the pH of the composition is 7.2
or is about 7.2. In one embodiment, the composition is used as an
adjunct to percutaneous coronary intervention (PCI). In such a use
the compositions may be administered as an intravenous bolus of
0.15 to 0.35 mg/kg at 10-60 minutes before the start of PCI. In a
further embodiment the dose is 0.2 mg/kg. In an embodiment, the
bolus is followed by continuous intravenous infusion of 0.1 to 0.15
g/kg/min for up to 12 hours. In a further embodiment the dose is
0.125 g/kg/min for up to 12 hours. In one embodiment, the
composition is used as an adjunct to percutaneous coronary
intervention (PCI) in subjects suffering from unstable angina. In
one embodiment of such a use the compositions may be administered
as an intravenous bolus at 0.1 to 0.4 mg/kg before the start of PCI
followed by continuous intravenous infusion of 5-15 g/min for up to
24 hours, concluding 1 hour after the PCI. In a further embodiment
the composition is administered as an intravenous bolus at 0.25
mg/kg before the start of PCI followed by continuous intravenous
infusion of 10 g/min for up to 24 hours, concluding 1 hour after
the PCI. In some embodiments the dose is between 2.times. and
100.times. less than the doses set forth hereinabove.
[0246] In each of the embodiments of the compositions described
herein, the compositions, when in the form of a lyophilizate, may
be reconstituted with, for example, sterile aqueous solutions,
sterile water, Sterile Water for Injections (USP), Sterile
Bacteriostatic Water for Injections (USP), and equivalents thereof
known to those skilled in the art.
[0247] It is understood that in administration of any of the
instant compounds, the compound may be administered in isolation,
in a carrier, as part of a pharmaceutical composition, or in any
appropriate vehicle.
Dosage
[0248] It is understood that where a dosage range is stated herein,
e.g. 1-10 mg/kg per week, the invention disclosed herein also
contemplates each integer dose, and tenth thereof, between the
upper and lower limits. In the case of the example given,
therefore, the invention contemplates 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 etc. mg/kg up to 10
mg/kg.
[0249] In embodiments, the compounds of the present invention can
be administered as a single dose or may be administered as multiple
doses.
[0250] In general, the daily dosage for treating a disorder or
condition according to the methods described above will generally
range from about 0.01 to about 10.0 mg/kg body weight of the
subject to be treated.
[0251] Variations based on the aforementioned dosage ranges may be
made by a physician of ordinary skill taking into account known
considerations such as the weight, age, and condition of the person
being treated, the severity of the affliction, and the particular
route of administration chosen.
[0252] It is also expected that the compounds disclosed will effect
cooperative binding with attendant consequences on effective
dosages required.
Kits
[0253] Another aspect of the present invention provides kits
comprising the compounds disclosed herein and the pharmaceutical
compositions comprising these compounds. A kit may include, in
addition to the compound or pharmaceutical composition, diagnostic
or therapeutic agents. A kit may also include instructions for use
in a diagnostic or therapeutic method. In a diagnostic embodiment,
the kit includes the compound or a pharmaceutical composition
thereof and a diagnostic agent. In a therapeutic embodiment, the
kit includes the antibody or a pharmaceutical composition thereof
and one or more therapeutic agents, such as an additional
antineoplastic agent, anti-tumor agent or chemotherapeutic
agent.
Subjects
[0254] In some embodiments the subject is a human. In an embodiment
the subject is 18 years or older. In another embodiment the subject
is less than 18 years old.
[0255] All combinations of the various elements disclosed herein
are within the scope of the invention.
[0256] This invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific examples detailed are
only illustrative of the invention as described more fully in the
claims which follow thereafter.
SymmetroAdhesins
[0257] This patent specification describes genetic devices which
are devices comprising functional stretches of consecutive amino
acids joined in a novel manner. Until now, proteins have been
genetically engineered to create new functions by virtue of their
adopting a novel fixed structure or conformation. In contrast to
such previously genetically engineered proteins, the genetic
devices disclosed herein comprise two or more distinguishable
protein domains that are connected by novel chemical bonds in a
manner permitting relative motion to occur between the domains. The
relative motion between protein domains constitutes the moving
parts of the genetic devices, and the useful work which they are
able to thereby carry out. Input energy for this work is provided
by the kinetic and rotational energies intrinsic to the protein
domains themselves, as well as the energy of mechanical
interactions between the protein domains and solvent molecules, and
the like.
[0258] This disclosure describes "symmetroadhesins", a specific
class of genetic devices. Symmetroadhesins comprise two or more
chemically-bonded adhesins (independently folding polypeptide
binding domains or stretches of consecutive amino acids). These
chemically-bonded adhesins are capable of relative motion with
respect to one another, resulting in the formation of two or more
symmetrically-oriented binding domains useful in binding dimeric
ligands, trimeric ligands, tetrameric ligands, and the like, with
greatly increased affinity. Symmetroadhesin-Fc hybrid proteins are
a particularly useful embodiment of the genetic devices.
[0259] The importance of symmetry in the effectiveness of
therapeutic proteins is due to the fact that most protein disease
targets themselves display such a higher-order symmetric structure.
For example, if the disease target consists of two proteins it is
said to be a dimer, if it consists of three proteins it is said to
be a trimer, and so forth. If a protein therapeutic binds to the
protein disease target only on a one-at-a-time basis, then the
strength of the binding is typically on the order of the nano-molar
range. At this ordinary level of binding, which is typical of
present-day therapeutic proteins, it becomes necessary to flood the
body with an enormous and wasteful excess of the therapeutic
protein relative to the protein disease target (one million-to-one
basis).
[0260] Each symmetroadhesin is engineered using a simple set of
rules that makes proteins into more useful therapeutics by making
them more symmetric. For a symmetroadhesin designed to bind to the
protein disease target on a two-at-a-time basis, the strength of
binding will be on the order of the nano H nano-molar range.
Cooperative binding permits such an extraordinary level of binding.
At this extraordinary level of binding, it is only necessary to
administer much less of the therapeutic protein relative to the
protein disease target (one to one basis).
Peptide Bond Chemistry
[0261] All proteins consist of one or more polypeptide chains,
stretches of consecutive amino acids, each of which is joined to
the preceding amino acid by a peptide bond. Due to resonance
between the single bond form (--C--N--) and double bond form
(--C.dbd.N--) which occur in amides, the peptide bond has a
significant degree of double bond character
(--C.dbd.N--C.sub..alpha.--C.dbd.N--). As a consequence, peptide
bonds in proteins are approximately planar. Although the adjoining
N--C.sub..alpha. and C.sub..alpha.--C bonds are relatively free to
rotate, the rigidly of the peptide bond reduces the degrees of
freedom of the folded polypeptide chain to the point where it
behaves like a single, static object.
[0262] Consider a first stretch of N consecutive amino acids and a
second stretch of P consecutive amino acids, AA.sub.1-[ ]-AA.sub.n
and AA.sub.1-[ ]-AA.sub.p, with the peptide bonding shown in 1:
C.sub..alpha.(1)--C.dbd.N--[ ]--C.dbd.N--C.sub..alpha.(N) &
C.sub..alpha.(1)--C.dbd.N--[ ]--C.dbd.N--C.sub..alpha.(P) 1
[0263] Joining of the first stretch of consecutive amino acids at
its N-terminus by a new peptide bond to the C-terminus of the
second stretch of consecutive amino acids will form a chimeric
polypeptide, AA.sub.1-[ ]-AA.sub.n-AA.sub.n+1-[ ]-AA.sub.n+p, with
the peptide bonding shown in 2:
C.sub..alpha.(1)--C.dbd.N--[
]-C.dbd.N--C.sub..alpha.(N)--C.dbd.N--C.sub..alpha.(N+1)--C.dbd.N--[
]--C.dbd.N--C.sub..alpha.(N+P) 2
[0264] The chimeric polypeptide, like either of its progenitors, is
a single stretch of consecutive amino acids, each of which is also
joined to the preceding amino acid by a peptide bond. The folded
chimeric polypeptide chain will thus also generally behave like a
single, static object.
[0265] This invention provides for novel protein-like molecules,
herein termed genetic devices, which like mechanical devices are
dynamic objects with two or more moving parts that are
interconnected in a manner to permit relative movement. Each part,
or domain, in a genetic device is a stretch of consecutive amino
acids, and each interconnection is made by a non-peptide bond
joining a predefined end of each stretch of consecutive amino
acids. Preferably, the domains in a genetic device are binding
domains. Three distinct types of genetic devices are distinguished
by the topology of their interconnections as shown in 3, 4, and
5:
C.sub..alpha.(1)--C.dbd.N--[
]--C.dbd.N--C.sub..alpha.(N)--X.sub.c--X.sub.n--C.sub..alpha.(N+1)--C.dbd-
.N--[ ]--C.dbd.N--C.sub..alpha.(N+P) 3
C.sub..alpha.(1)--C.dbd.N--[
]--C.dbd.N--C.sub..alpha.(N)--X.sub.c--X.sub.c--C.sub..alpha.(P)--N.dbd.C-
--[ ]--N.dbd.C--C.sub..alpha.(1) 4
C.sub..alpha.(N)--N.dbd.C--[
]N.dbd.C--C.sub..alpha.(1)--X.sub.n--X.sub.n--C.sub..alpha.(1)--C.dbd.N---
[ ]--C.dbd.N--C.sub..alpha.(P) 5
[0266] Genetic device with two identical binding domains that are
connected at distinct termini (6) have an asymmetric
configuration,
C.sub..alpha.(1)--C.dbd.N--[
]--C.dbd.N--C.sub..alpha.(N)--X.sub.c--X.sub.n--C.sub..alpha.(N+1)--C.dbd-
.N--[ ]--C.dbd.N--C.sub..alpha.(2N) 6
[0267] Genetic device with two identical binding domains that are
connected at identical termini (7 and 8) have a configuration with
point symmetry,
C.sub..alpha.(1)--C.dbd.N--[
]--C.dbd.N--C.sub..alpha.(N)--X.sub.c--X.sub.c--C.sub..alpha.(N)--N.dbd.C-
--[ ]--N.dbd.C--C.sub..alpha.(1) 7
C.sub..alpha.(N)--N.dbd.C--[
]--N.dbd.C--C.sub..alpha.(1)--X.sub.n--X.sub.n--C.sub..alpha.(1)--C.dbd.N-
--[ ]--C.dbd.N--C.sub..alpha.(N) 8
[0268] The genetic devices shown in 7 and 8 are herein termed
hemi-symmetroadhesins. Although hemi-symmetroadhesins are have
structures with point symmetry they are not capable of rotating
both their binding domains independently, and thus, they are
generally not able to bind symmetrically to more than one binding
site in a symmetric target.
[0269] This invention also provides protein-like molecules capable
of binding symmetrically to two or more binding sites in a
symmetric target (i.e., cooperatively). Genetic devices that are
capable of binding symmetric targets symmetrically are termed
herein symmetroadhesins. Like a human body grasping a oversized,
yet symmetric object (e.g., a medicine ball) with two hands instead
of one, the ability of symmetroadhesins to bind symmetric targets
is generally much greater than proteins.
Symmetroadhesin Subtypes
[0270] Tables 2-11 set forth various non-limiting embodiments of
different symmetroadhesins. For example, Table 2, describing
CD4-symmetroadhesins, shows in the top line the configuration of a
CD4 hemi-symmetroadhesin, i.e. a CD4 domain with a C-terminal
X-terminus, e.g. a stretch of consecutive amino acids which is a
CD4 domain with a C-terminal cysteine or selenosyteine residue
bonded through a non-peptide link (for example a cysteine-cysteine
disulfide bond or a selenocysteine-selenocysteine diselenide bond)
to a second stretch of consecutive amino acids which is a CD4
domain with a C-terminal cysteine or selenosyteine residue bonded
through a non-peptide link, generically described as
[CD4-Xc-Xc-CD4]. A dimer of the CD4 hemi-symmetroadhesin and a Fc
hemisymmetroadhesin to form an immuno-symmetroadhesin is set forth
in the second row of Table 2, for example, described as
[CD4-Xc-Sn-Fc].sub.2. In each of the tables Xc represents a
C-terminal X-terminus; Xn a N-terminal X-terminus, Sn a N-terminal
cysteine residue
TABLE-US-00002 TABLE 2 CD4 Symmetroadhesins Stretches of
Consecutive Amino Acids Class Symmetroadhesin Configuration
CD4-X.sub.c hemi- [CD4-X.sub.c-X.sub.c-CD4] CD4-X.sub.c +
S.sub.n-Fc immuno [CD4-X.sub.c-S.sub.n-Fc].sub.2 CD4-X.sub.c +
X.sub.n-Fc immuno [CD4-X.sub.c-X.sub.n-Fc].sub.2 CD4-X.sub.c +
Fc-X.sub.c immuno [Fc-X.sub.c-X.sub.c-CD4].sub.2 CD4-X.sub.c +
S.sub.n-Fc-X.sub.c bi-
[CD4-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-CD4].sub.2 CD4-X.sub.c +
X.sub.n-Fc-X.sub.c bi-
[CD4-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-CD4].sub.2
TABLE-US-00003 TABLE 3 TNR Hemi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
TNR.sub.1-X.sub.c 1 [TNR.sub.1-X.sub.c-X.sub.c-TNR.sub.1]
TNR.sub.2-X.sub.c 1 [TNR.sub.2-X.sub.c-X.sub.c-TNR.sub.2]
TNR.sub.Fab-X.sub.c 1 [TNR.sub.Fab-X.sub.c-X.sub.c-TNR.sub.Fab]
TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c 3
[(TNR.sub.1/2)-X.sub.c-X.sub.c-(TNR.sub.1/2)] TNR.sub.1-X.sub.c +
TNR.sub.Fab-X.sub.c 3
[(TNR.sub.1/Fab)-X.sub.c-X.sub.c-(TNR.sub.1/Fab)] TNR.sub.2-X.sub.c
+ TNR.sub.Fab-X.sub.c 3
[(TNR.sub.2/Fab)-X.sub.c-X.sub.c-(TNR.sub.2/Fab)] TNR.sub.1-X.sub.c
+ TNR.sub.2-X.sub.c + 6
[(TNR.sub.1/2/Fab)-X.sub.c-X.sub.c-(TNR.sub.1/2/Fab)]
TNR.sub.Fab-X.sub.c
TABLE-US-00004 TABLE 4 TNR ImmunoSymmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
TNR.sub.1-X.sub.c + S.sub.n-Fc 1
[TNR.sub.1-X.sub.c-S.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c + X.sub.n-Fc
1 [TNR.sub.1-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-TNR.sub.1].sub.2 TNR.sub.2-X.sub.c
+ S.sub.n-Fc 1 [TNR.sub.2-X.sub.c-S.sub.n-Fc].sub.2
TNR.sub.2-X.sub.c + X.sub.n-Fc 1
[TNR.sub.2-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.2-X.sub.c + Fc-X.sub.c
1 [Fc-X.sub.c-X.sub.c-TNR.sub.2].sub.2 TNR.sub.Fab-X.sub.c +
S.sub.n-Fc 1 [TNR.sub.Fab-X.sub.c-S.sub.n-Fc].sub.2
TNR.sub.Fab-X.sub.c + X.sub.n-Fc 1
[TNR.sub.Fab-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.Fab-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-TNR.sub.Fab].sub.2
TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c + S.sub.n-Fc 3
[(TNR.sub.1/2)-X.sub.c-S.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.2-X.sub.c + X.sub.n-Fc 3
[(TNR.sub.1/2)-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.2-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(TNR.sub.1/2)].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.Fab-X.sub.c + S.sub.n-Fc 3
[(TNR.sub.1/Fab)-X.sub.c-S.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.Fab-X.sub.c + X.sub.n-Fc 3
[(TNR.sub.1/Fab)-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.Fab-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(TNR.sub.1/Fab)].sub.2 TNR.sub.2-X.sub.c +
TNR.sub.Fab-X.sub.c + S.sub.n-Fc 3
[(TNR.sub.2/Fab)-X.sub.c-S.sub.n-Fc].sub.2 TNR.sub.2-X.sub.c +
TNR.sub.Fab-X.sub.c + X.sub.n-Fc 3
[(TNR.sub.2/Fab)-X.sub.c-X.sub.n-Fc].sub.2 TNR.sub.2-X.sub.c +
TNR.sub.Fab-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(TNR.sub.2/Fab)].sub.2 TNR.sub.1-X.sub.c +
TNR.sub.2-X.sub.c + 6 [(TNR.sub.1/2/Fab)-X.sub.c-S.sub.n-Fc].sub.2
TNR.sub.Fab-X.sub.c + S.sub.n-Fc TNR.sub.1-X.sub.c +
TNR.sub.2-X.sub.c + 6 [(TNR.sub.1/2/Fab)-X.sub.c-X.sub.n-Fc].sub.2
TNR.sub.Fab-X.sub.c + X.sub.n-Fc TNR.sub.1-X.sub.c +
TNR.sub.2-X.sub.c + 6 [Fc-X.sub.c-X.sub.c-(TNR.sub.1/2/Fab)].sub.2
TNR.sub.Fab-X.sub.c + Fc-X.sub.c
TABLE-US-00005 TABLE 5 TNR Bi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
TNR.sub.1-X.sub.c + S.sub.n-Fc-X.sub.c 1
[TNR.sub.1-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.1].sub.2
TNR.sub.1-X.sub.c + X.sub.n-Fc-X.sub.c 1
[TNR.sub.1-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.1].sub.2
TNR.sub.2-X.sub.c + S.sub.n-Fc-X.sub.c 1
[TNR.sub.2-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.2].sub.2
TNR.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 1
[TNR.sub.2-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.2].sub.2
TNR.sub.Fab-X.sub.c + S.sub.n-Fc-X.sub.c 1
[TNR.sub.Fab-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.Fab].sub.2
TNR.sub.Fab-X.sub.c + X.sub.n-Fc-X.sub.c 1
[TNR.sub.Fab-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-TNR.sub.Fab].sub.2
TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(TNR.sub.1/2)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/2)].sub.2
TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(TNR.sub.1/2)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/2)].sub.2
TNR.sub.1-X.sub.c + TNR.sub.Fab-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(TNR.sub.1/Fab)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/Fab)].sub.-
2 TNR.sub.1-X.sub.c + TNR.sub.Fab-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(TNR.sub.1/Fab)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/Fab)].sub.-
2 TNR.sub.2-X.sub.c + TNR.sub.Fab-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(TNR.sub.2/Fab)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.2/Fab)].sub.-
2 TNR.sub.2-X.sub.c + TNR.sub.Fab-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(TNR.sub.2/Fab)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.2/Fab)].sub.-
2 TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c + TNR.sub.Fab-X.sub.c +
S.sub.n-Fc-X.sub.c 63
[(TNR.sub.1/2/Fab)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/2/Fab)].-
sub.2 TNR.sub.1-X.sub.c + TNR.sub.2-X.sub.c + TNR.sub.Fab-X.sub.c +
S.sub.n-Fc-X.sub.c 63
[(TNR.sub.1/2/Fab)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(TNR.sub.1/2/Fab)].-
sub.2
TABLE-US-00006 TABLE 6 VGFR Hemi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
VGFR.sub.1-X.sub.c 1 [VGFR.sub.1-X.sub.c-X.sub.c-VGFR.sub.1]
VGFR.sub.2-X.sub.c 1 [VGFR.sub.2-X.sub.c-X.sub.c-VGFR.sub.2]
VGFR.sub.3-X.sub.c 1 [VGFR.sub.3-X.sub.c-X.sub.c-VGFR.sub.3]
VGFR.sub.1-X.sub.c + VGFR.sub.2-X.sub.c 3
[(VGFR.sub.1/2)-X.sub.c-X.sub.c-(VGFR.sub.1/2)] VGFR.sub.1-X.sub.c
+ VGFR.sub.3-X.sub.c 3
[(VGFR.sub.1/3)-X.sub.c-X.sub.c-(VGFR.sub.1/3)] VGFR.sub.2-X.sub.c
+ VGFR.sub.3-X.sub.c 3
[(VGFR.sub.2/3)-X.sub.c-X.sub.c-(VGFR.sub.2/3)] VGFR.sub.1-X.sub.c
+ VGFR.sub.2-X.sub.c + 6
[(VGFR.sub.1/2/3)-X.sub.c-X.sub.c-(VGFR.sub.1/2/3)]
VGFR.sub.3-X.sub.c
TABLE-US-00007 TABLE 7 VGFR ImmunoSymmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
VGFR.sub.1-X.sub.c + S.sub.n-Fc 1
[VGFR.sub.1-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
X.sub.n-Fc 1 [VGFR.sub.1-X.sub.c-X.sub.n-Fc].sub.2
VGFR.sub.1-X.sub.c + Fc-X.sub.c 1
[Fc-X.sub.c-X.sub.c-VGFR.sub.1].sub.2 VGFR.sub.2-X.sub.c +
S.sub.n-Fc 1 [VGFR.sub.2-X.sub.c-S.sub.n-Fc].sub.2
VGFR.sub.2-X.sub.c + X.sub.n-Fc 1
[VGFR.sub.2-X.sub.c-X.sub.n-Fc].sub.2 VGFR.sub.2-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-VGFR.sub.2].sub.2
VGFR.sub.3-X.sub.c + S.sub.n-Fc 1
[VGFR.sub.3-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.3-X.sub.c +
X.sub.n-Fc 1 [VGFR.sub.3-X.sub.c-X.sub.n-Fc].sub.2
VGFR.sub.3-X.sub.c + Fc-X.sub.c 1
[Fc-X.sub.c-X.sub.c-VGFR.sub.3].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + S.sub.n-Fc 3
[(VGFR.sub.1/2)-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + X.sub.n-Fc 3
[(VGFR.sub.1/2)-X.sub.c-X.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2)].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.3-X.sub.c + S.sub.n-Fc 3
[(VGFR.sub.1/3)-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.3-X.sub.c + X.sub.n-Fc 3
[(VGFR.sub.1/3)-X.sub.c-X.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.3-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(VGFR.sub.1/3)].sub.2 VGFR.sub.2-X.sub.c +
VGFR.sub.3-X.sub.c + S.sub.n-Fc 3
[(VGFR.sub.2/3)-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.2-X.sub.c +
VGFR.sub.3-X.sub.c + X.sub.n-Fc 3
[(VGFR.sub.2/3)-X.sub.c-X.sub.n-Fc].sub.2 VGFR.sub.2-X.sub.c +
VGFR.sub.3-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(VGFR.sub.2/3)].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c + S.sub.n-Fc 6
[(VGFR.sub.1/2/3)-X.sub.c-S.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c + X.sub.n-Fc 6
[(VGFR.sub.1/2/3)-X.sub.c-X.sub.n-Fc].sub.2 VGFR.sub.1-X.sub.c +
VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c + Fc-X.sub.c 6
[Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2/3)].sub.2
TABLE-US-00008 TABLE 8 VGFR Bi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
VGFR.sub.1-X.sub.c + S.sub.n-Fc-X.sub.c 1
[VGFR.sub.1-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.1].sub.2
VGFR.sub.1-X.sub.c + X.sub.n-Fc-X.sub.c 1
[VGFR.sub.1-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.1].sub.2
VGFR.sub.2-X.sub.c + S.sub.n-Fc-X.sub.c 1
[VGFR.sub.2-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.2].sub.2
VGFR.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 1
[VGFR.sub.2-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.2].sub.2
VGFR.sub.3-X.sub.c + S.sub.n-Fc-X.sub.c 1
[VGFR.sub.3-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.3].sub.2
VGFR.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 1
[VGFR.sub.3-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-VGFR.sub.3].sub.2
VGFR.sub.1-X.sub.c + VGFR.sub.2-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(VGFR.sub.1/2)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2)].sub.2
VGFR.sub.1-X.sub.c + VGFR.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(VGFR.sub.1/2)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2)].sub.2
VGFR.sub.1-X.sub.c + VGFR.sub.3-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(VGFR.sub.1/3)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/3)].sub.2
VGFR.sub.1-X.sub.c + VGFR.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(VGFR.sub.1/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/3)].sub.2
VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c + S.sub.n-Fc-X.sub.c 12
[(VGFR.sub.2/3)-X.sub.c-S.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.2/3)].sub.2
VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(VGFR.sub.2/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.2/3)].sub.2
VGFR.sub.1-X.sub.c + VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c +
S.sub.n-Fc-X.sub.c 63
[(VGFR.sub.1/2/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2/3)].su-
b.2 VGFR.sub.1-X.sub.c + VGFR.sub.2-X.sub.c + VGFR.sub.3-X.sub.c +
S.sub.n-Fc-X.sub.c 63
[(VGFR.sub.1/2/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(VGFR.sub.1/2/3)].su-
b.2
TABLE-US-00009 TABLE 9 ErbB Hemi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
ErbB.sub.1-X.sub.c 1 [ErbB.sub.1-X.sub.c-X.sub.c-ErbB.sub.1]
ErbB.sub.2-X.sub.c 1 [ErbB.sub.2-X.sub.c-X.sub.c-ErbB.sub.2]
ErbB.sub.3-X.sub.c 1 [ErbB.sub.3-X.sub.c-X.sub.c-ErbB.sub.3]
ErbB.sub.4-X.sub.c 1 [ErbB.sub.4-X.sub.c-X.sub.c-ErbB.sub.4]
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c 3
[(ErbB.sub.1/2)-X.sub.c-X.sub.c-(ErbB.sub.1/2)] ErbB.sub.1-X.sub.c
+ ErbB.sub.3-X.sub.c 3
[(ErbB.sub.1/3)-X.sub.c-X.sub.c-(ErbB.sub.1/3)] ErbB.sub.1-X.sub.c
+ ErbB.sub.4-X.sub.c 3
[(ErbB.sub.1/4)-X.sub.c-X.sub.c-(ErbB.sub.1/4)] ErbB.sub.2-X.sub.c
+ ErbB.sub.3-X.sub.c 3
[(ErbB.sub.2/3)-X.sub.c-X.sub.c-(ErbB.sub.2/3)] ErbB.sub.2-X.sub.c
+ ErbB.sub.4-X.sub.c 3
[(ErbB.sub.2/4)-X.sub.c-X.sub.c-(ErbB.sub.2/4)] ErbB.sub.3-X.sub.c
+ ErbB.sub.4-X.sub.c 3
[(ErbB.sub.3/4)-X.sub.c-X.sub.c-(ErbB.sub.3/4)] ErbB.sub.1-X.sub.c
+ ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c 6
[(ErbB.sub.1/2/3)-X.sub.c-X.sub.c-(ErbB.sub.1/2/3)]
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.4-X.sub.c 6
[(ErbB.sub.1/2/4)-X.sub.c-X.sub.c-(ErbB.sub.1/2/4)]
ErbB.sub.1-X.sub.c + ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c 6
[(ErbB.sub.1/3/4)-X.sub.c-X.sub.c-(ErbB.sub.1/3/4)]
ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c 6
[(ErbB.sub.2/3/4)-X.sub.c-X.sub.c-(ErbB.sub.2/3/4)]
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c +
ErbB.sub.4-X.sub.c 10
[(ErbB.sub.1/2/3/4)-X.sub.c-X.sub.c-(ErbB.sub.1/2/3/4)]
TABLE-US-00010 TABLE 10 ErbB ImmunoSymmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
ErbB.sub.1-X.sub.c + X.sub.n-Fc 1
[ErbB.sub.1-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-ErbB.sub.1].sub.2
ErbB.sub.2-X.sub.c + X.sub.n-Fc 1
[ErbB.sub.2-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.2-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-ErbB.sub.2].sub.2
ErbB.sub.3-X.sub.c + X.sub.n-Fc 1
[ErbB.sub.3-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.3-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-ErbB.sub.3].sub.2
ErbB.sub.4-X.sub.c + X.sub.n-Fc 1
[ErbB.sub.4-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.4-X.sub.c +
Fc-X.sub.c 1 [Fc-X.sub.c-X.sub.c-ErbB.sub.4].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.1/2)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.3-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.1/3)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.3-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/3)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.4-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.1/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.4-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/4)].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.3-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.2/3)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.3-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.2/3)].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.4-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.2/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.4-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.2/4)].sub.2 ErbB.sub.3-X.sub.c +
ErbB.sub.4-X.sub.c + X.sub.n-Fc 3
[(ErbB.sub.3/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.3-X.sub.c +
ErbB.sub.4-X.sub.c + Fc-X.sub.c 3
[Fc-X.sub.c-X.sub.c-(ErbB.sub.3/4)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + X.sub.n-Fc 6
[(ErbB.sub.1/2/3)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + Fc-X.sub.c 6
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/3)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc 6
[(ErbB.sub.1/2/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + ErbB.sub.4-X.sub.c + Fc-X.sub.c 6
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/4)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc 6
[(ErbB.sub.1/3/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c + Fc-X.sub.c 6
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/3/4)].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc 6
[(ErbB.sub.2/3/4)-X.sub.c-X.sub.n-Fc].sub.2 ErbB.sub.2-X.sub.c +
ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c + Fc-X.sub.c 6
[Fc-X.sub.c-X.sub.c-(ErbB.sub.2/3/4)].sub.2 ErbB.sub.1-X.sub.c +
ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c +
X.sub.n-Fc 10 [(ErbB.sub.1/2/3/4)-X.sub.c-X.sub.n-Fc].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c +
ErbB.sub.4-X.sub.c + Fc-X.sub.c 10
[Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/3/4)].sub.2
TABLE-US-00011 TABLE 11 ErbB Bi-Symmetroadhesins Stretches of
Consecutive Amino Acids # Symmetroadhesin Configurations
ErbB.sub.1-X.sub.c + X.sub.n-Fc-X.sub.c 1
[ErbB.sub.1-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-ErbB.sub.1].sub.2
ErbB.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 1
[ErbB.sub.2-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-ErbB.sub.2].sub.2
ErbB.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 1
[ErbB.sub.3-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-ErbB.sub.3].sub.2
ErbB.sub.4-X.sub.c + X.sub.n-Fc-X.sub.c 1
[ErbB.sub.4-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-ErbB.sub.4].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.1/2)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2)].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.1/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/3)].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.1/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/4)].sub.2
ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.2/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.2/3)].sub.2
ErbB.sub.2-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.2/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.2/4)].sub.2
ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c + X.sub.n-Fc-X.sub.c 12
[(ErbB.sub.3/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.3/4)].sub.2
ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c +
X.sub.n-Fc-X.sub.c 63
[(ErbB.sub.1/2/3)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/3)].su-
b.2 ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.4-X.sub.c +
X.sub.n-Fc-X.sub.c 63
[(ErbB.sub.1/2/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/4)].su-
b.2 ErbB.sub.1-X.sub.c + ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c +
X.sub.n-Fc-X.sub.c 63
[(ErbB.sub.1/3/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/3/4)].su-
b.2 ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c + ErbB.sub.4-X.sub.c +
X.sub.n-Fc-X.sub.c 63
[(ErbB.sub.2/3/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.2/3/4)].su-
b.2 ErbB.sub.1-X.sub.c + ErbB.sub.2-X.sub.c + ErbB.sub.3-X.sub.c +
ErbB.sub.4-X.sub.c + X.sub.n-Fc-X.sub.c
[(ErbB.sub.1/2/3/4)-X.sub.c-X.sub.n-Fc-X.sub.c-X.sub.c-(ErbB.sub.1/2/3/4)-
].sub.2
Stretches of Consecutive Amino Acids
[0271] Examples of stretches of consecutive amino acids as referred
to herein include, but are not limited to, consecutive amino acids
including binding domains such as secreted or transmembrane
proteins, intracellular binding domains and antibodies (whole or
portions thereof) and modified versions thereof. The following are
some non-limiting examples:
1) Immunoglobulins
[0272] Immunoglobulins are molecules containing polypeptide chains
held together by intra-chain disulfide bonds, wherein at least one
of the bonded amino acids is not a terminus residue, typically
having two light chains and two heavy chains. In each chain, one
domain (V) has a variable amino acid sequence depending on the
antibody specificity of the molecule. The other domains (C) have a
rather constant sequence common among molecules of the same class.
The domains are numbered in sequence from the amino-terminal
end.
[0273] The immunoglobulin gene superfamily consists of molecules
with immunoglobulin-like domains. Members of this family include
class I and class II major histocompatibility antigens,
immunoglobulins, T-cell receptor alpha, beta, gamma and delta
chains, CD1, CD2, CD4, CD8, CD28, the gamma, delta and epsilon
chains of CD3, OX-2, Thy-1, the intercellular or neural cell
adhesion molecules (1-CAM or N-CAM), lymphocyte function associated
antigen-3 (LFA-3), neurocytoplasmic protein (NCP-3), poly-Ig
receptor, myelin-associated glycoprotein (MAG), high affinity IgE
receptor, the major glycoprotein of peripheral myelin (Po),
platelet derived growth factor receptor, colony stimulating
factor-1 receptor, macrophage Fc receptor, Fc gamma receptors and
carcinoembryonic antigen.
[0274] It is known that one can substitute variable domains
(including hypervariable regions) of one immunoglobulin for
another, and from one species to another. See, for example, EP 0
173 494; EP 0 125 023; Munro, Nature 312 (13 Dec. 1984); Neuberger
et al., Nature 312: (13 Dec. 1984); Sharon et al., Nature 309 (May
24, 1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855
(1984); Morrison et al. Science 229:1202-1207 (1985); and Boulianne
et al., Nature 312:643-646 (Dec. 13, 1984).
[0275] Morrison et al., Science 229:1202-1207 (1985) teaches the
preparation of an immunoglobulin chimera having a variable region
from one species fused to an immunoglobulin constant region from
another species.
[0276] It has also been shown that it is possible to substitute
immunoglobulin variable-like domains from two members of the
immunoglobulin gene superfamily-CD4 and the T cell receptor--for a
variable domain in an immunoglobulin; see e.g. Capon et al., Nature
337:525-531, 1989, Traunecker et al., Nature 339:68-70, 1989,
Gascoigne et al., Proc. Nat. Acad. Sci. 84:2936-2940, 1987, and
published European application EPO 0 325 224 A2.
[0277] U.S. Pat. No. 5,116,964 (Capon et al., May 26, 1992) hereby
incorporated by reference, describes hybrid immunoglobulins
commonly referred to as immunoadhesins, which combine, for example,
the adhesive and targeting properties of a ligand binding partner
with immunoglobulin effector functions. U.S. Pat. No. 5,336,603
(Capon et al., Aug. 9, 1994), hereby incorporated by reference,
describes a heterofunctional immunoadhesin comprising a fusion
protein in which a polypeptide comprising a human CD4 antigen
variable (V) region is fused at its C-terminus to the N-terminus of
a polypeptide comprising a constant region of an immunoglobulin
chain disulfide bonded to a companion immunoglobulin heavy
chain-light chain pair bearing a antibody combining site capable of
binding a predetermined antigen.
[0278] "Components" of immunoglobulins include antibody fragments
comprise a portion of an intact antibody, preferably the antigen
binding or variable region of the intact antibody. Examples of
antibody fragments include Fab, Fab', F(ab').sub.2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein
Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0279] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0280] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0281] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear a free thiol group.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known.
[0282] The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa and lambda, based on the amino acid sequences
of their constant domains.
[0283] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0284] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0285] Thus, linking of the various components of immunoglobulins
and other biologically active molecules has allowed production of
hybrid molecules which retain the functionalities of the individual
components.
[0286] In an embodiment, the invention described herein provides
novel hybrid molecules which include one or more immunoglobulin
components.
2) Extracellular Proteins
[0287] Extracellular proteins play important roles in, among other
things, the formation, differentiation and maintenance of
multicellular organisms. A discussion of various intracellular
proteins of interest is set forth in U.S. Pat. No. 6,723,535,
Ashkenazi et al., issued Apr. 20, 2004, hereby incorporated by
reference.
[0288] The fate of many individual cells, e.g., proliferation,
migration, differentiation, or interaction with other cells, is
typically governed by information received from other cells and/or
the immediate environment. This information is often transmitted by
secreted polypeptides (for instance, mitogenic factors, survival
factors, cytotoxic factors, differentiation factors, neuropeptides,
and hormones) which are, in turn, received and interpreted by
diverse cell receptors or membrane-bound proteins. These secreted
polypeptides or signaling molecules normally pass through the
cellular secretory pathway to reach their site of action in the
extracellular environment.
[0289] Secreted proteins have various industrial applications,
including as pharmaceuticals, diagnostics, biosensors and
bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons, interleukins, erythropoietines,
colony stimulating factors, and various other cytokines, are
secretory proteins. Their receptors, which are membrane proteins,
also have potential as therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new,
native secreted proteins. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted proteins. Examples of screening
methods and techniques are described in the literature (see, for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);
U.S. Pat. No. 5,536,637)).
[0290] Membrane-bound proteins and receptors can play important
roles in, among other things, the formation, differentiation and
maintenance of multicellular organisms. The fate of many individual
cells, e.g., proliferation, migration, differentiation, or
interaction with other cells, is typically governed by information
received from other cells and/or the immediate environment. This
information is often transmitted by secreted polypeptides (for
instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and hormones) which are, in
turn, received and interpreted by diverse cell receptors or
membrane-bound proteins. Such membrane-bound proteins and cell
receptors include, but are not limited to, cytokine receptors,
receptor kinases, receptor phosphatases, receptors involved in
cell-cell interactions, and cellular adhesin molecules like
selectins and integrins. For instance, transduction of signals that
regulate cell growth and differentiation is regulated in part by
phosphorylation of various cellular proteins. Protein tyrosine
kinases, enzymes that catalyze that process, can also act as growth
factor receptors. Examples include fibroblast growth factor
receptor and nerve growth factor receptor.
[0291] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interactions. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0292] Examples of such proteins include EGF and growth
factors.
[0293] Epidermal growth factor (EGF) is a conventional mitogenic
factor that stimulates the proliferation of various types of cells
including epithelial cells and fibroblasts. EGF binds to and
activates the EGF receptor (EGFR), which initiates intracellular
signaling and subsequent effects. The EGFR is expressed in neurons
of the cerebral cortex, cerebellum, and hippocampus in addition to
other regions of the central nervous system (CNS). In addition, EGF
is also expressed in various regions of the CNS. Therefore, EGF
acts not only on mitotic cells, but also on postmitotic neurons. In
fact, many studies have indicated that EGF has neurotrophic or
neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on cultured cerebral cortical and
cerebellar neurons, enhancing neurite outgrowth and survival. On
the other hand, EGF also acts on other cell types, including septal
cholinergic and mesencephalic dopaminergic neurons, indirectly
through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the mechanisms of action remain
essentially unknown. EGF-induced signaling in mitotic cells is
better understood than in postmitotic neurons. Studies of cloned
pheochromocytoma PC12 cells and cultured cerebral cortical neurons
have suggested that the EGF-induced neurotrophic actions are
mediated by sustained activation of the EGFR and mitogen-activated
protein kinase (MAPK) in response to EGF. The sustained
intracellular signaling correlates with the decreased rate of EGFR
down-regulation, which might determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor
that acts upon various types of cells including mitotic cells and
postmitotic neurons.
[0294] EGF is produced by the salivary and Brunner's glands of the
gastrointestinal system, kidney, pancreas, thyroid gland, pituitary
gland, and the nervous system, and is found in body fluids such as
saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,
prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman,
Peptides 12:653-663 (1991).
[0295] EGF is mediated by its membrane specific receptor, which
contains an intrinsic tyrosine kinase. Stoscheck et al., J. Cell
Biochem. 31:135-152 (1986). EGF is believed to function by binding
to the extracellular portion of its receptor which induces a
transmembrane signal that activates the intrinsic tyrosine
kinase.
[0296] Purification and sequence analysis of the EGF-like domain
has revealed the presence of six conserved cysteine residues which
cross-bind to create three peptide loops, Savage et al., J. Biol.
Chem. 248:7669-7672 (1979). It is now generally known that several
other peptides can react with the EGF receptor which share the same
generalized motif X.sub.n CX.sub.7 CX.sub.4/5 CX.sub.10 CXCX.sub.5
GX.sub.2 CX.sub.n, where X represents any non-cysteine amino acid,
and n is a variable repeat number. Non isolated peptides having
this motif include TGF-alpha, amphiregulin, schwannoma-derived
growth factor (SDGF), heparin-binding EGF-like growth factors and
certain virally encoded peptides (e.g., Vaccinia virus, Reisner,
Nature 313:801-803 (1985), Shope fibroma virus, Chang et al., Mol
Cell Biol. 7:535-540 (1987), Molluscum contagiosum, Porter and
Archard, J. Gen. Virol. 68:673-682 (1987), and Myxoma virus, Upton
et al., J. Virol. 61:1271-1275 (1987), Prigent and Lemoine, Prog.
Growth Factor Res. 4:1-24 (1992).
[0297] EGF-like domains are not confined to growth factors but have
been observed in a variety of cell-surface and extracellular
proteins which have interesting properties in cell adhesion,
protein-protein interaction and development, Laurence and
Gusterson, Tumor Biol. 11:229-261 (1990). These proteins include
blood coagulation factors (factors VI, IX, X, XII, protein C,
protein S, protein Z, tissue plasminogen activator, urokinase),
extracellular matrix components (laminin, cytotactin, entactin),
cell surface receptors (LDL receptor, thrombomodulin receptor) and
immunity-related proteins (complement C1r, uromodulin).
[0298] Even more interesting, the general structure pattern of
EGF-like precursors is preserved through lower organisms as well as
in mammalian cells. A number of genes with developmental
significance have been identified in invertebrates with EGF-like
repeats. For example, the notch gene of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF,
Wharton et al., Cell 43:557-581 (1985). Hydropathy plots indicate a
putative membrane spanning domain, with the EGF-related sequences
being located on the extracellular side of the membrane. Other
homeotic genes with EGF-like repeats include Delta, 95F and 5ZD
which were identified using probes based on Notch, and the nematode
gene Lin-12 which encodes a putative receptor for a developmental
signal transmitted between two specified cells.
[0299] Specifically, EGF has been shown to have potential in the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions, Konturek et al., Eur.
J. Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the
treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration gastrointestinal ulcerations and
congenital microvillus atrophy, Guglietta and Sullivan, Eur. J.
Gastroenterol Hepatol, 7(10), 945-50 (1995). Additionally, EGF has
been implicated in hair follicle differentiation; du Cros, J.
Invest. Dermatol. 101 (1 Suppl.), 106S-113S (1993), Hillier, Clin.
Endocrinol. 33(4), 427-28 (1990); kidney function, Hamm et al.,
Semin. Nephrol. 13(1): 109-15 (1993), Harris, Am. J. Kidney Dis.
17(6): 627-30 (1991); tear fluid, van Setten et al., Int. Opthalmol
15(6); 359-62 (1991); vitamin K mediated blood coagulation, Stenflo
et al., Blood 78(7): 1637-51 (1991). EGF is also implicated various
skin disease characterized by abnormal keratinocyte
differentiation, e.g., psoriasis, epithelial cancers such as
squamous cell carcinomas of the lung, epidermoid carcinoma of the
vulva and gliomas. King et al., Am. J. Med. Sci. 296:154-158
(1988).
[0300] Of great interest is mounting evidence that genetic
alterations in growth factors signaling pathways are closely linked
to developmental abnormalities and to chronic diseases including
cancer. Aaronson, Science 254: 1146-1153 (1991). For example,
c-erb-2 (also known as HER-2), a proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in
human breast cancer. King et al., Science 229:974-976 (1985);
Gullick, Hormones and their actions, Cooke et al., eds, Amsterdam,
Elsevier, pp 349-360 (1986).
[0301] Growth factors are molecular signals or mediators that
enhance cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factor (TGF-beta) is highly pleiotropic and can stimulate
proliferation in some cells, especially connective tissue, while
being a potent inhibitor of proliferation in others, such as
lymphocytes and epithelial cells.
[0302] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules involves
comprehends autocrine (same cell), juxtacrine (neighbor cell), and
paracrine (adjacent cells) pathways. Peptide growth factors are
elements of a complex biological language, providing the basis for
intercellular communication. They permit cells to convey
information between each other, mediate interaction between cells
and change gene expression. The effect of these multifunctional and
pluripotent factors is dependent on the presence or absence of
other peptides.
[0303] FGF-8 is a member of the fibroblast growth factors (FGFs)
which are a family of heparin-binding, potent mitogens for both
normal diploid fibroblasts and established cell lines,
Gospodarowicz et al. (1984), Proc. Nat. Acad. Sci. USA 81:6963. The
FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2
(FGF-3), K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8)
among others. All FGFs have two conserved cysteine residues and
share 30-50% sequence homology at the amino acid level. These
factors are mitogenic for a wide variety of normal diploid
mesoderm-derived and neural crest-derived cells, including
granulosa cells, adrenal cortical cells, chondrocytes, myoblasts,
corneal and vascular endothelial cells (bovine or human), vascular
smooth muscle cells, lens, retina and prostatic epithelial cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and
osteoblasts.
[0304] Fibroblast growth factors can also stimulate a large number
of cell types in a non-mitogenic manner. These activities include
promotion of cell migration into wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird & Bohlen, Handbook of Exp.
Pharmacol. 95(1): 369418, Springer, (1990). These properties
provide a basis for using fibroblast growth factors in therapeutic
approaches to accelerate wound healing, nerve repair, collateral
blood vessel formation, and the like. For example, fibroblast
growth factors have been suggested to minimize myocardium damage in
heart disease and surgery (U.S. Pat. No. 4,378,347).
[0305] FGF-8, also known as androgen-induced growth factor (AIGF),
is a 215 amino acid protein which shares 30-40% sequence homology
with the other members of the FGF family. FGF-8 has been proposed
to be under androgenic regulation and induction in the mouse
mammary carcinoma cell line SC3. Tanaka et al., Proc. Natl. Acad.
Sci. USA 89:8928-8932 (1992); Sato et al., J. Steroid Biochem.
Molec. Biol. 47:91-98 (1993). As a result, FGF-8 may have a local
role in the prostate, which is known to be an androgen-responsive
organ. FGF-8 can also be oncogenic, as it displays transforming
activity when transfected into NIH-3T3 fibroblasts. Kouhara et al.,
Oncogene 9 455-462 (1994). While FGF-8 has been detected in heart,
brain, lung, kidney, testis, prostate and ovary, expression was
also detected in the absence of exogenous androgens. Schmitt et
al., J. Steroid Biochem. Mol. Biol. 57 (34): 173-78 (1996).
[0306] FGF-8 shares the property with several other FGFs of being
expressed at a variety of stages of murine embryogenesis, which
supports the theory that the various FGFs have multiple and perhaps
coordinated roles in differentiation and embryogenesis. Moreover,
FGF-8 has also been identified as a protooncogene that cooperates
with Wnt-1 in the process of mammary tumorigenesis (Shackleford et
al., Proc. Natl. Acad. Sci. USA 90, 740-744 (1993); Heikinheimo et
al., Mech. Dev. 48:129-138 (1994)).
[0307] In contrast to the other FGFs, FGF-8 exists as three protein
isoforms, as a result of alternative splicing of the primary
transcript. Tanaka et al., supra. Normal adult expression of FGF-8
is weak and confined to gonadal tissue, however northern blot
analysis has indicated that FGF-8 mRNA is present from day 10
through day 12 or murine gestation, which suggests that FGF-8 is
important to normal development. Heikinheimo et al., Mech Dev.
48(2): 129-38 (1994). Further in situ hybridization assays between
day 8 and 16 of gestation indicated initial expression in the
surface ectoderm of the first bronchial arches, the frontonasal
process, the forebrain and the midbrain-hindbrain junction. At days
10-12, FGF-8 was expressed in the surface ectoderm of the forelimb
and hindlimb buds, the nasal its and nasopharynx, the infundibulum
and in the telencephalon, diencephalon and metencephalon.
Expression continues in the developing hindlimbs through day 13 of
gestation, but is undetectable thereafter. The results suggest that
FGF-8 has a unique temporal and spatial pattern in embryogenesis
and suggests a role for this growth factor in multiple regions of
ectodermal differentiation in the post-gastrulation embryo.
[0308] The TGF-beta supergene family, or simply TGF-beta
superfamily, a group of secreted proteins, includes a large number
of related growth and differentiation factors expressed in
virtually all phyla. Superfamily members bind to specific cell
surface receptors that activate signal transduction mechanisms to
elicit their multifunctional cytokine effects. Kolodziejczyk and
Hall, Biochem. Cell. Biol., 74:299-314 (1996); Attisano and Wrana,
Cytokine Growth Factor Rev., 7:327-339 (1996); and Hill, Cellular
Signaling, 8:533-544 (1996).
[0309] Members of this family include five distinct forms of
TGF-beta (Sporn and Roberts, in Peptide Growth Factors and Their
Receptors, Sporn and Roberts, eds. (Springer-Verlag: Berlin, 1990)
pp. 419-472), as well as the differentiation factors vg1 (Weeks and
Melton, Cell, 51:861-867 (1987)) and DPP-C polypeptide (Padgett et
al., Nature, 325:81-84 (1987)), the hormones activin and inhibin
(Mason et al., Nature 318-659-663 (1985); Mason et al., Growth
Factors, 1:77-88 (1987)), the Mullerian-inhibiting substance (MIS)
(Cate et al., Cell, 45: 685-698 (1986)), the bone morphogenetic
proteins (BMPs) (Wozney et al., Science, 242:1528-1534 (1988); PCT
WO 88/00205 published Jan. 14, 1988; U.S. Pat. No. 4,877,864 issued
Oct. 31, 1989), the developmentally regulated proteins Vgr-1 (Lyons
et al., Proc. Natl. Acad. Sci. USA. 86:45544558 (1989)) and Vgr-2
(Jones et al., Molec. Endocrinol., 6:1961-1968 (1992)), the mouse
growth differentiation factor (GDF), such as GDF-3 and GDF-9
(Kingsley, Genes Dev., 8:133-146 (1994); McPherron and Lee, J.
Biol. Chem., 268:3444-3449 (1993)), the mouse lefty/Stral (Meno et
al., Nature, 381:151-155 (1996); Bouillet et al., Dev. Biol., 170:
420-433 (1995)), glial cell line-derived neurotrophic factor (GDNF)
(Lin et al., Science, 260:1130-1132 (1993), neurturin (Kotzbauer et
al., Nature, 384:467-470 (1996)), and endometrial
bleeding-associated factor (EBAF) (Kothapalli et al., J. Clin.
Invest., 99:2342-2350 (1997)). The subset BMP-2A and BMP-2B is
approximately 75% homologous in sequence to DPP-C and may represent
the mammalian equivalent of that protein.
[0310] The proteins of the TGF-beta superfamily are
disulfide-linked homo- or heterodimers encoded by larger precursor
polypeptide chains containing a hydrophobic signal sequence, a long
and relatively poorly conserved N-terminal pro region of several
hundred amino acids, a cleavage site (usually polybasic), and a
shorter and more highly conserved C-terminal region. This
C-terminal region corresponds to the processed mature protein and
contains approximately 100 amino acids with a characteristic
cysteine motif, i.e. the conservation of seven of the nine cysteine
residues of TGF-beta among all known family members. Although the
position of the cleavage site between the mature and pro regions
varies among the family members, the C-terminus of all of the
proteins is in the identical position, ending in the sequence
Cys-X-Cys-X, but differing in every case from the TGF-beta
consensus C-terminus of Cys-Lys-Cys-Ser. Sporn and Roberts, 1990,
supra.
[0311] There are at least five forms of TGF-beta currently
identified, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, and
TGF-beta5. The activated form of TGF-beta1 is a homodimer formed by
dimerization of the carboxy-terminal 112 amino acids of a 390 amino
acid precursor. Recombinant TGF-beta1 has been cloned (Derynck et
al., Nature, 316:701-705 (1985)) and expressed in Chinese hamster
ovary cells (Gentry et al., Mol. Cell. Biol. 7:3418-3427 (1987)).
Additionally, recombinant human TGF-beta2 (deMartin et al., EMBO
J., 6:3673 (1987)), as well as human and porcine TGF-beta3 (Derynck
et al., EMBO J., 7:3737-3743 (1988); ten Dijke et al., Proc. Natl.
Acad. Sci. USA, 85:4715 (1988)) have been cloned. TGF-beta2 has a
precursor form of 414 amino acids and is also processed to a
homodimer from the carboxy-terminal 112 amino acids that shares
approximately 70% homology with the active form of TGF-beta1
(Marquardt et al., J. Biol. Chem., 262:12127 (1987)). See also EP
200,341; 169,016; 268,561; and 267,463; U.S. Pat. No. 4,774,322;
Cheifetz et al., Cell, 48:409-415 (1987); Jakowlew et al.,
Molecular Endocrin., 2:747-755 (1988); Derynck et al., J. Biol.
Chem., 261:4377-4379 (1986); Sharples et al., DNA, 6:239-244
(1987); Derynck et al., Nucl. Acids. Res., 15:3188-3189 (1987);
Derynck et al., Nucl. Acids. Res. 15:3187 (1987); Seyedin et al.,
J. Biol. Chem., 261:5693-5695 (1986); Madisen et al., DNA 7:1-8
(1988); and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A.), 85:79-82
(1988).
[0312] TGF-beta4 and TGF-beta5 were cloned from a chicken
chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol.,
2:1186-1195 (1988)) and from a frog oocyte cDNA library,
respectively.
[0313] The pro region of TGF-beta associates non-covalently with
the mature TGF-beta dimer (Wakefield et al., J. Biol. Chem.,
263:7646-7654 (1988); Wakefield et al., Growth Factors, 1:203-218
(1989)), and the pro regions are found to be necessary for proper
folding and secretion of the active mature dimers of both TGF-beta
and activin (Gray and Mason, Science, 247:1328-1330 (1990)). The
association between the mature and pro regions of TGF-beta masks
the biological activity of the mature dimer, resulting in formation
of an inactive latent form. Latency is not a constant of the
TGF-beta superfamily, since the presence of the pro region has no
effect on activin or inhibin biological activity.
[0314] A unifying feature of the biology of the proteins from the
TGF-beta superfamily is their ability to regulate developmental
processes. TGF-beta has been shown to have numerous regulatory
actions on a wide variety of both normal and neoplastic cells.
TGF-beta is multifunctional, as it can either stimulate or inhibit
cell proliferation, differentiation, and other critical processes
in cell function (Sporn and Roberts, supra).
[0315] One member of the TGF-beta superfamily, EBAF, is expressed
in endometrium only in the late secretory phase and during abnormal
endometrial bleeding. Kothapalli et al., J. Clin. Invest.,
99:2342-2350 (1997). Human endometrium is unique in that it is the
only tissue in the body that bleeds at regular intervals. In
addition, abnormal endometrial bleeding is one of the most common
manifestations of gynecological diseases, and is a prime indication
for hysterectomy. In situ hybridization showed that the mRNA of
EBAF was expressed in the stroma without any significant mRNA
expression in the endometrial glands or endothelial cells.
[0316] The predicted protein sequence of EBAF showed a strong
homology to the protein encoded by mouse lefty/stra3 of the
TGF-beta superfamily. A motif search revealed that the predicted
EBAF protein contains most of the cysteine residues which are
conserved among the TGF-beta-related proteins and which are
necessary for the formation of the cysteine knot structure. The
EBAF sequence contains an additional cysteine residue, 12 amino
acids upstream from the first conserved cysteine residue. The only
other family members known to contain an additional cysteine
residue are TGF-betas, inhibins, and GDF-3. EBAF, similar to LEFTY,
GDF-3/Vgr2, and GDF-9, lacks the cysteine residue that is known to
form the intermolecular disulfide bond. Therefore, EBAF appears to
be an additional member of the TGF-beta superfamily with an
unpaired cysteine residue that may not exist as a dimer. However,
hydrophobic contacts between the two monomer subunits may promote
dimer formation. Fluorescence in situ hybridization showed that the
ebaf gene is located on human chromosome 1 at band q42.1.
[0317] Further examples of such extracellular proteins are well
known in the art, for example see U.S. Pat. No. 6,723,535.
Conjugates
[0318] The invention also pertains to conjugates of
symmtroadhesins/immunosymmetroadhesins. Thus the instant
compositions can be conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0319] Chemotherapeutic agents useful in the generation of such
immunoconjugates are well known in the art. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPH, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.113In,
.sup.90Y, and .sup.186Re.
[0320] Conjugates of the compositions of the invention including
cytotoxic agent are made using a variety of bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), iminothiolane (IT), bifunctional derivatives of
imidoesters (such as dimethyl adipimidate HCL), active esters (such
as disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94111026.
Synthesis of Genetic Devices
[0321] The genetic devices disclosed herein may be synthesized by
various routes. One particular route is to synthesize components in
vivo by recombinant DNA technology and then to chemically modify
the secreted or procured products under conditions so as to form
the compounds. Alternative routes include solid-state
synthesis.
General Techniques
[0322] The description below relates primarily to production of
stretches of consecutive amino acids or polypeptides of interest by
culturing cells transformed or transfected with a vector containing
an encoding nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed. For instance, the amino acid sequence, or portions
thereof, may be produced by direct peptide synthesis using
solid-phase techniques (see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
stretches of consecutive amino acids or polypeptides of interest
may be chemically synthesized separately and combined using
chemical or enzymatic methods to produce the full-length stretches
of consecutive amino acids or polypeptides of interest.
1. Isolation of DNA Encoding Stretches of Consecutive Amino Acids
or Polypeptides of Interest
[0323] Encoding DNA may be obtained from a cDNA library prepared
from tissue believed to possess the mRNA of interest and to express
it at a detectable level. Accordingly, human DNA can be
conveniently obtained from a cDNA library prepared from human
tissue, and so forth. An encoding gene may also be obtained from a
genomic library or by known synthetic procedures (e.g., automated
nucleic acid synthesis).
[0324] Libraries can be screened with probes (such as antibodies to
the stretch of consecutive amino acids or oligonucleotides of at
least about 20-80 bases) designed to identify the gene of interest
or the protein encoded by it. Screening the cDNA or genomic library
with the selected probe may be conducted using standard procedures,
such as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989). An alternative means to isolate the encoding gene is to use
PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR
Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press,
1995)).
[0325] The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0326] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0327] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
2. Selection and Transformation of Host Cells
[0328] Host cells are transfected or transformed with expression or
cloning vectors described herein for production and cultured in
conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled
artisan without undue experimentation. In general, principles,
protocols, and practical techniques for maximizing the productivity
of cell cultures can be found in Mammalian Cell Biotechnology: a
Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook
et al., supra.
[0329] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0330] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonAptr3phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r, E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued
Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g., PCR
or other nucleic acid polymerase reactions, are suitable.
[0331] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for encoding vectors. Saccharomyces cerevisiae is a commonly used
lower eukaryotic host microorganism. Others include
Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981);
EP 139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat.
No. 4,943,529; Fleer et al., Bio/Technology, 9:968-975 (1991)) such
as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al.,
J. Bacteriol., 737 (1983)), K. fragilis (ATCC 12,424), K.
bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii
(ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published Oct. 31, 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published Jan. 10, 1991), and
Aspergillus hosts such as A. nidulans (Ballance et al., Biochem.
Biophys. Res. Commun., 112:284-289 (1983); Tilburn et al., Gene,
26:205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA,
81:1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J.,
4:475479 (1985)). Methylotropic yeasts are suitable herein and
include, but are not limited to, yeast capable of growth on
methanol selected from the genera consisting of Hansenula, Candida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A
list of specific species that are exemplary of this class of yeasts
may be found in C. Anthony, The Biochemistry of Methylotrophs, 269
(1982).
[0332] Suitable host cells for the expression of glycosylated
stretches of consecutive amino acids or polypeptides of interest
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
3. Selection and Use of a Replicable Vector
[0333] The nucleic acid (e.g., cDNA or genomic DNA) encoding the
stretch of consecutive amino acids or polypeptides of interest may
be inserted into a replicable vector for cloning (amplification of
the DNA) or for expression. Various vectors are publicly available.
The vector may, for example, be in the form of a plasmid, cosmid,
viral particle, or phage. The appropriate nucleic acid sequence may
be inserted into the vector by a variety of procedures. In general,
DNA is inserted into an appropriate restriction endonuclease
site(s) using techniques known in the art. Vector components
generally include, but are not limited to, one or more of a signal
sequence, an origin of replication, one or more marker genes, an
enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable vectors containing one or more
of these components employs standard ligation techniques which are
known to the skilled artisan.
[0334] The stretches of consecutive amino acids or polypeptides of
interest may be produced recombinantly not only directly, but also
as a fusion polypeptide with a heterologous polypeptide, which may
be a signal sequence or other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces alpha-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C. albicans glucoamylase leader (EP 362,179
published Apr. 4, 1990), or the signal described in WO 90/13646
published Nov. 15, 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0335] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the 2mu
plasmid origin is suitable for yeast, and various viral origins
(SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning
vectors in mammalian cells.
[0336] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0337] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the encoding nucleic acid, such as DHFR or thymidine
kinase. An appropriate host cell when wild-type DHFR is employed is
the CHO cell line deficient in DHFR activity, prepared and
propagated as described by Urlaub et al., Proc. Natl. Acad. Sci.
USA, 77:4216 (1980). A suitable selection gene for use in yeast is
the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al.,
Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);
Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides a
selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1
(Jones, Genetics, 85:12 (1977)).
[0338] Expression and cloning vectors usually contain a promoter
operably linked to the encoding nucleic acid sequence to direct
mRNA synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the beta-lactamase and lactose promoter systems
(Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter
system (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and
hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)). Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the encoding DNA.
[0339] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase (Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic
enzymes (Hess et al., J. Adv. Enzyme Re.g., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0340] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0341] Transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0342] Transcription of a DNA encoding the stretches of consecutive
amino acids or polypeptides of interest by higher eukaryotes may be
increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to
300 bp, that act on a promoter to increase its transcription. Many
enhancer sequences are now known from mammalian genes (globin,
elastase, albumin, alpha-fetoprotein, and, insulin). Typically,
however, one will use an enhancer from a eukaryotic cell virus.
Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or 3' to the coding sequence, but is
preferably located at a site 5' from the promoter.
[0343] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
stretches of consecutive amino acids or polypeptides of
interest.
[0344] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of stretches of consecutive amino acids
or polypeptides in recombinant vertebrate cell culture are
described in Gething et al., Nature 293:620-625 (1981); Mantei et
al., Nature, 281:4046 (1979); EP 117,060; and EP 117,058.
4. Detecting Gene Amplification/Expression
[0345] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA (Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0346] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence stretches of consecutive amino acids or
polypeptides of interest or against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence
fused to DNA encoding a stretch of consecutive amino acids or
polypeptide of interest and encoding a specific antibody
epitope.
5. Purification of Polypeptide
[0347] Forms of the stretches of consecutive amino acids or
polypeptides of interest may be recovered from culture medium or
from host cell lysates. If membrane-bound, it can be released from
the membrane using a suitable detergent solution (e.g. Triton-X
100) or by enzymatic cleavage. Cells employed in expression of the
stretches of consecutive amino acids or polypeptides of interest
can be disrupted by various physical or chemical means, such as
freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents.
[0348] It may be desired to purify the stretches of consecutive
amino acids or polypeptides of interest from recombinant cell
proteins or polypeptides. The following procedures are exemplary of
suitable purification procedures: by fractionation on an
ion-exchange column; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation-exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
gel filtration using, for example, Sephadex G-75; protein A
Sepharose columns to remove contaminants such as IgG; and metal
chelating columns to bind epitope-tagged forms. Various methods of
protein purification may be employed and such methods are known in
the art and described for example in Deutscher, Methods in
Enzymology, 182 (1990); Scopes, Protein Purification: Principles
and Practice, Springer-Verlag, New York (1982). The purification
step(s) selected will depend, for example, on the nature of the
production process used and the particular stretches of consecutive
amino acids or polypeptides of interest produced.
Example of Expression of Stretch of Consecutive Amino Acids or
Polypeptide Component of Interest in E. coli
[0349] The DNA sequence encoding the desired amino acid sequence of
interest or polypeptide is initially amplified using selected PCR
primers. The primers should contain restriction enzyme sites which
correspond to the restriction enzyme sites on the selected
expression vector. A variety of expression vectors may be employed.
An example of a suitable vector is pBR322 (derived from E. coli;
see Bolivar et al., Gene, 2:95 (1977)) which contains genes for
ampicillin and tetracycline resistance. The vector is digested with
restriction enzyme and dephosphorylated. The PCR amplified
sequences are then ligated into the vector. The vector will
preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the specific amino acid sequence of interest/polypeptide
coding region, lambda transcriptional terminator, and an argU
gene.
[0350] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0351] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0352] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized amino acid sequence of interest or
polypeptide can then be purified using a metal chelating column
under conditions that allow tight binding of the protein.
[0353] The primers can contain restriction enzyme sites which
correspond to the restriction enzyme sites on the selected
expression vector, and other useful sequences providing for
efficient and reliable translation initiation, rapid purification
on a metal chelation column, and proteolytic removal with
enterokinase. The PCR-amplified, poly-His tagged sequences can be
ligated into an expression vector used to transform an E. coli host
based on, for example, strain 52 (W3110 fuhA(tonA) Ion galE
rpoHts(htpRts) clpP(lacIq). Transformants can first be grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into C RAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2 SO.sub.4, 0.71 g sodium citrate-2H.sub.2O, 1.07 g
KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500
mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7
mM MgSO.sub.4) and grown for approximately 20-30 hours at
30.degree. C. with shaking. Samples were removed to verify
expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell pellets were frozen until
purification and refolding.
[0354] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris,
pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added
to make final concentrations of 0.1M and 0.02 M, respectively, and
the solution was stirred overnight at 4.degree. C. This step
results in a denatured protein with all cysteine residues blocked
by sulfitolization. The solution was centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant was diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
Depending the clarified extract was loaded onto a 5 mil Qiagen
Ni-NTA metal chelate column equilibrated in the metal chelate
column buffer. The column was washed with additional buffer
containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The
protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein were pooled and stored at
4.degree. C. Protein concentration was estimated by its absorbance
at 280 nm using the calculated extinction coefficient based on its
amino acid sequence.
Expression of Consecutive Stretches of Amino Acids in Mammalian
Cells
[0355] This general example illustrates a preparation of a
glycosylated form of a desired amino acid sequence of interest or
polypeptide component by recombinant expression in mammalian
cells.
[0356] The vector pRK5 (see EP 307,247, published Mar. 15, 1989)
can be employed as the expression vector. Optionally, the encoding
DNA is ligated into pRK5 with selected restriction enzymes to allow
insertion of the DNA using ligation methods such as described in
Sambrook et al., supra.
[0357] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g of the ligated vector DNA is mixed with about 1 .mu.g DNA
encoding the VA RNA gene [Thimmappaya et al., Cell 31:543 (1982)]
and dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl.sub.2 To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0358] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of amino acid sequence of interest or polypeptide
component. The cultures containing transfected cells may undergo
further incubation (in serum free medium) and the medium is tested
in selected bioassays.
[0359] In an alternative technique, the nucleic acid amino acid
sequence of interest or polypeptide component may be introduced
into 293 cells transiently using the dextran sulfate method
described by Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575
(1981). 293 cells are grown to maximal density in a spinner flask
and 700 .mu.g of the ligated vector is added. The cells are first
concentrated from the spinner flask by centrifugation and washed
with PBS. The DNA-dextran precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for
90 seconds, washed with tissue culture medium, and re-introduced
into the spinner flask containing tissue culture medium, 5 .mu.g/ml
bovine insulin and 0.1 .mu.g/ml bovine transferrin. After about
four days, the conditioned media is centrifuged and filtered to
remove cells and debris. The sample containing expressed amino acid
sequence of interest or polypeptide component can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0360] In another embodiment, the amino acid sequence of interest
or polypeptide component can be expressed in CHO cells. The amino
acid sequence of interest or polypeptide component can be
transfected into CHO cells using known reagents such as CaPO.sub.4
or DEAE-dextran. As described above, the cell cultures can be
incubated, and the medium replaced with culture medium (alone) or
medium containing a radiolabel such as .sup.35S-methionine. After
determining the presence of amino acid sequence of interest or
polypeptide component, the culture medium may be replaced with
serum free medium. Preferably, the cultures are incubated for about
6 days, and then the conditioned medium is harvested. The medium
containing the expressed amino acid sequence of interest or
polypeptide component can then be concentrated and purified by any
selected method.
[0361] Epitope-tagged amino acid sequence of interest or
polypeptide component may also be expressed in host CHO cells. The
amino acid sequence of interest or polypeptide component may be
subcloned out of a pRK5 vector. The subclone insert can undergo PCR
to fuse in frame with a selected epitope tag such as a poly-his tag
into a Baculovirus expression vector. The poly-his tagged amino
acid sequence of interest or polypeptide component insert can then
be subcloned into a SV40 driven vector containing a selection
marker such as DHFR for selection of stable clones. Finally, the
CHO cells can be transfected (as described above) with the SV40
driven vector. Labeling may be performed, as described above, to
verify expression. The culture medium containing the expressed
poly-His tagged amino acid sequence of interest or polypeptide
component can then be concentrated and purified by any selected
method, such as by Ni.sup.2+-chelate affinity chromatography.
[0362] In an embodiment the amino acid sequence of interest or
polypeptide component are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0363] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used in expression in CHO cells is as described
in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses
the SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
Expression of Stretch of Consecutive Amino Acids in Yeast
[0364] The following method describes recombinant expression of a
desired amino acid sequence of interest or polypeptide component in
yeast.
[0365] First, yeast expression vectors are constructed for
intracellular production or secretion of a stretch of consecutive
amino acids from the ADH2/GAPDH promoter. DNA encoding a desired
amino acid sequence of interest or polypeptide component, a
selected signal peptide and the promoter is inserted into suitable
restriction enzyme sites in the selected plasmid to direct
intracellular expression of the amino acid sequence of interest or
polypeptide component. For secretion, DNA encoding the stretch of
consecutive amino acids can be cloned into the selected plasmid,
together with DNA encoding the ADH2/GAPDH promoter, the yeast
alpha-factor secretory signal/leader sequence, and linker sequences
(if needed) for expression of the stretch of consecutive amino
acids.
[0366] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0367] Recombinant amino acid sequence of interest or polypeptide
component can subsequently be isolated and purified by removing the
yeast cells from the fermentation medium by centrifugation and then
concentrating the medium using selected cartridge filters. The
concentrate containing the amino acid sequence of interest or
polypeptide component may further be purified using selected column
chromatography resins.
Expression of Stretches of Consecutive Amino Acids in
Baculovirus-Infected Insect Cells
[0368] The following method describes recombinant expression of
stretches of consecutive amino acids in Baculovirus-infected insect
cells.
[0369] The desired nucleic acid encoding the stretch of consecutive
amino acids is fused upstream of an epitope tag contained with a
baculovirus expression vector. Such epitope tags include poly-his
tags and immunoglobulin tags (like Fc regions of IgG). A variety of
plasmids may be employed, including plasmids derived from
commercially available plasmids such as pVL1393 (Novagen). Briefly,
the amino acid sequence of interest or polypeptide component or the
desired portion of the amino acid sequence of interest or
polypeptide component (such as the sequence encoding the
extracellular domain of a transmembrane protein) is amplified by
PCR with primers complementary to the 5' and 3' regions. The 5'
primer may incorporate flanking (selected) restriction enzyme
sites. The product is then digested with those selected restriction
enzymes and subcloned into the expression vector.
[0370] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression is performed as described by O'Reilley et al.,
Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford
University Press (1994).
[0371] Expressed poly-his tagged amino acid sequence of interest or
polypeptide component can then be purified, for example, by
Ni.sup.2+-chelate affinity chromatography as follows. Extracts are
prepared from recombinant virus-infected Sf9 cells as described by
Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are
washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5
mM MgCl.sub.2; 0.1 mM EDTA; 10% Glycerol; 0.1% NP40; 0.4 M KCl),
and sonicated twice for 20 seconds on ice. The sonicates are
cleared by centrifugation, and the supernatant is diluted 50-fold
in loading buffer (50 mM phosphate, 300 mM NaCl, 10% Glycerol, pH
7.8) and filtered through a 0.45 .mu.m filter. A Ni.sup.2+-NTA
agarose column (commercially available from Qiagen) is prepared
with a bed volume of 5 mL, washed with 25 mL of water and
equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged sequence are pooled and dialyzed against loading
buffer.
[0372] Alternatively, purification of the IgG tagged (or Fc tagged)
amino acid sequence can be performed using known chromatography
techniques, including for instance, Protein A or Protein G column
chromatography.
[0373] Immunoadhesin (Fc containing) constructs of proteins can be
purified from conditioned media as follows. The conditioned media
is pumped onto a 5 ml Protein A column (Pharmacia) which is
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 mL of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity of the
proteins is verified by SDS polyacrylamide gel (PEG)
electrophoresis and N-terminal amino acid sequencing by Edman
degradation.
Intein-Based C-Terminal Syntheses
[0374] As described, for example, in U.S. Pat. No. 6,849,428,
issued Feb. 1, 2005, inteins are the protein equivalent of the
self-splicing RNA introns (see Perler et al., Nucleic Acids Res.
22:1125-1127 (1994)), which catalyze their own excision from a
precursor protein with the concomitant fusion of the flanking
protein sequences, known as exteins (reviewed in Perler et al.,
Curr. Opin. Chem. Biol. 1:292-299 (1997); Perler, F. B. Cell
92(1):1-4 (1998); Xu et al., EMBO J. 15(19):5146-5153 (1996)).
[0375] Studies into the mechanism of intein splicing led to the
development of a protein purification system that utilized
thiol-induced cleavage of the peptide bond at the N-terminus of the
Sce VMA intein (Chong et al., Gene 192(2):271-281 (1997)).
Purification with this intein-mediated system generates a
bacterially-expressed protein with a C-terminal thioester (Chong et
al., (1997)). In one application, where it is described to isolate
a cytotoxic protein, the bacterially expressed protein with the
C-terminal thioester is then fused to a chemically-synthesized
peptide with an N-terminal cysteine using the chemistry described
for "native chemical ligation" (Evans et al., Protein Sci.
7:2256-2264 (1998); Muir et al., Proc. Natl. Acad. Sci. USA
95:6705-6710 (1998)).
[0376] This technique, referred to as "intein-mediated protein
ligation" (IPL), represents an important advance in protein
semi-synthetic techniques. However, because chemically-synthesized
peptides of larger than about 100 residues are difficult to obtain,
the general application of IPL was limited by the requirement of a
chemically-synthesized peptide as a ligation partner.
[0377] IPL technology was significantly expanded when an expressed
protein with a predetermined N-terminus, such as cysteine, was
generated, as described for example in U.S. Pat. No. 6,849,428.
This allows the fusion of one or more expressed proteins from a
host cell, such as bacterial, yeast or mammalian cells. In one
non-limiting example the intein a modified RIR1 Methanobacterium
thermoautotrophicum is that cleaves at either the C-terminus or
N-terminus is used which allows for the release of a bacterially
expressed protein during a one-column purification, thus
eliminating the need proteases entirely.
[0378] Intein technology is one example of one route to obtain
components. In one embodiment, the subunits of the compounds of the
invention are obtained by transfecting suitable cells, capable of
expressing and secreting mature chimeric polypeptides, wherein such
polypeptides comprise, for example, an adhesin domain contiguous
with an isolatable c-terminal intein domain (see U.S. Pat. No.
6,849,428, Evans et al., issued Feb. 1, 2005, hereby incorporated
by reference). The cells, such as mammalian cells or bacterial
cells, are transfected using known recombinant DNA techniques. The
secreted chimeric polypeptide can then be isolated, e.g. using a
chitin-derivatized resin in the case of an intein-chitin binding
domain (see U.S. Pat. No. 6,897,285, Xu et al., issued May 24,
2005, hereby incorporated by reference), and is then treated under
conditions permitting thiol-mediated cleavage and release of the
now C-terminal thioester-terminated adhesion subunit. The
thioester-terminated adhesion subunit is readily converted to a
C-terminal cysteine terminated subunit.
[0379] These subunits can be treated under oxidizing conditions to
permit formation of, for example, a disulfide bond between the two
terminal cysteine residues, thus forming a symmetroadhesin. In
addition, this technique can be used to make the symmetroadhesin-Fc
hybrid subunits by treating the individual adhesion-Fc heterodimers
under conditions permitting formation of bonds between the Fc
portions of the heterodimers.
EXAMPLE 1
Preparation of Immunoglobulin Fc with N-Terminal-S-Termini
(S-Fc)
[0380] Digestion of immunoglobulin (IgG) with papain yields two Fab
fragments and one Fc fragment (Porter (1959) Biochem. 73, 119-126).
The site of proteolysis in human IgG is the heavy chain hinge
region between the cys-5 and cys-11 residues, EPKSCDKTHTCPPCP,
(Fleischman et al., Biochem J. (1963) 88, 220-227; Edelman et al.
(1969) Proc. Natl. Acad. Sci. 63, 78-85). The cys-5 residue
normally forms a disulfide bond with the human IgG light chain,
which is easily cleaved under mild reducing conditions, making it
an ideal candidate for Fc-like molecules with N-terminal-S-termini
(S-Fc).
[0381] Accordingly, host cells were transfected with expression
vectors encoding IgG1 pre-Fc chimeric polypeptides consisting of a
signal peptide joined at its C-terminus by a peptide bond to the
N-terminus of an Fc domain beginning at the cys-5 residue,
CDKTHTCPPCP (FIG. 35A). The heterologous signal peptides used are
selected from proteins with N-terminal cysteines (part i). Thus,
cleavage by the cellular signal peptidase will provide a mature
S-Fc protein having cys-5 at the N-terminus (part ii).
[0382] The sequences of the IgG1 precursor polypeptides of FIG. 35A
are shown in SEQ ID NO: 32, SEQ ID NO:33, and SEQ ID NO:34. The
sequence of the mature IgG1 polypeptide of FIG. 35A is shown in SEQ
ID NO: 35. Other mature IgG1 polypeptides made by methods described
in EXAMPLES 1 to 5 are shown in SEQ ID NO: 36 through SEQ ID NO:
46.
[0383] Host cells are transfected with expression vectors encoding
IgG2 pre-Fc chimeric polypeptides consisting of a signal peptide
joined at its C-terminus by a peptide bond to the N-terminus of an
Fc domain beginning at the cys-4 residue, CCVECPPCP (FIG. 35B). The
heterologous signal peptides used are selected from proteins with
N-terminal cysteines (part i). Thus, cleavage by the cellular
signal peptidase will provide a mature S-Fc protein having cys-4 at
the N-terminus (part ii).
[0384] The sequences of the IgG2 precursor polypeptides of FIG. 35B
are shown in SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. The
sequence of the mature IgG2 polypeptide of FIG. 35B is shown in SEQ
ID NO:53. Other mature IgG2 polypeptides made by methods described
in EXAMPLES 1 to 5 are shown in SEQ ID NO:54 through SEQ ID
NO:67.
[0385] Host cells are transfected with expression vectors encoding
IgG3 pre-Fc chimeric polypeptides consisting of a signal peptide
joined at its C-terminus by a peptide bond to the N-terminus of an
Fc domain beginning at the cys-13 residue, CPRCP (FIG. 35C). The
heterologous signal peptides used are selected from proteins with
N-terminal cysteines (part i). Thus, cleavage by the cellular
signal peptidase will provide a mature S-Fc protein having cys-13
at the N-terminus (part ii).
[0386] The sequences of the IgG3 precursor polypeptides of FIG. 35C
are shown in SEQ ID NO:71, SEQ ID NO:72, and SEQ ID NO:73. The
sequence of the mature IgG3 polypeptide of FIG. 35C is shown in SEQ
ID NO:74. Other mature IgG2 polypeptides made by methods described
in EXAMPLES 1 to 5 are shown in SEQ ID NO:75 through SEQ ID
NO:82.
[0387] Host cells are transfected with expression vectors encoding
IgG4 pre-Fc chimeric polypeptides consisting of a signal peptide
joined at its C-terminus by a peptide bond to the N-terminus of an
Fc domain beginning at the cys-8 residue, CPSCP (FIG. 35D). The
heterologous signal peptides used are selected from proteins with
N-terminal cysteines (part i). Thus, cleavage by the cellular
signal peptidase will provide a mature S-Fc protein having cys-8 at
the N-terminus (part ii).
[0388] The sequences of the IgG4 precursor polypeptides of FIG. 35D
are shown in SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88. The
sequence of the mature IgG4 polypeptide of FIG. 35D is shown in SEQ
ID NO:89. Other mature IgG4 polypeptides made by methods described
in EXAMPLES 1 to 5 are shown in SEQ ID NO:90 through SEQ ID
NO:97.
[0389] Suitable host cells include 293 human embryonic cells (ATCC
CRL-1573) and CHO-K1 hamster ovary cells (ATCC CCL-61) obtained
from the American Type Culture Collection (Rockville, Md.). Cells
are grown at 37.degree. C. in an atmosphere of air, 95%; carbon
dioxide, 5%. 293 cells are maintained in Minimal essential medium
(Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain
1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and
1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10%. CHO-K1 cells
are maintained in Ham's F12K medium with 2 mM L-glutamine adjusted
to contain 1.5 g/L sodium bicarbonate, 90%; fetal bovine serum,
10%. Other suitable host cells include CV1 monkey kidney cells
(ATCC CCL-70), COS-7 monkey kidney cells (ATCC CRL-1651), VERO-76
monkey kidney cells (ATCC CRL-1587), HELA human cervical cells
(ATCC CCL-2), W138 human lung cells (ATCC CCL-75), MDCK canine
kidney cells (ATCC CCL-34), BRL3A rat liver cells (ATCC CRL-1442),
BHK hamster kidney cells (ATCC CCL-10), MMT060562 mouse mammary
cells (ATCC CCL-51), and human CD8.sup.+ T lymphocytes (described
in U.S. Ser. No. 08/258,152 incorporated herein in its entirety by
reference).
[0390] An example of a suitable expression vector is plasmid pSA
(SEQ ID NO:1). Plasmid pSA contains the following DNA sequence
elements: 1) pBluescriptIIKS(+) (nucleotides 912 to 2941/1 to 619,
GenBank Accession No. X52327), 2) a human cytomegalovirus promoter,
enhancer, and first exon splice donor (nucleotides 63 to 912,
GenBank Accession No. K03104), 3) a human alpha1-globin second exon
splice acceptor (nucleotides 6808 to 6919, GenBank Accession No.
J00153), 4) an SV40 T antigen polyadenylation site (nucleotides
2770 to 2533, Reddy et al. (1978) Science 200, 494-502), and 5) an
SV40 origin of replication (nucleotides 5725 to 5578, Reddy et al.,
ibid). For expression of the polypeptide of interest, an
EcoRI-BglII DNA fragment encoding the polypeptide is inserted into
plasmid pSA between the EcoRI and BglII restriction sites located
at positions 1,608 and 1,632, respectively. Other suitable
expression vectors include plasmids pSVeCD4DHFR and pRKCD4 (U.S.
Pat. No. 5,336,603), plasmid pIK.1.1 (U.S. Pat. No. 5,359,046),
plasmid pVL-2 (U.S. Pat. No. 5,838,464), plasmid pRT43.2F3
(described in U.S. Ser. No. 08/258,152 incorporated herein in its
entirety by reference), and plasmid pCDNA3.1(+) (Invitrogen,
Inc.).
[0391] Suitable selectable markers include the Tn5 transposon
neomycin phosphotransferase (NEO) gene (Southern and Berg (1982) J.
Mol. Appl. Gen. 1, 327-341), and the dihydrofolate reductase (DHFR)
cDNA (Lucas et al. (1996) Nucl. Acids Res. 24, 1774-1779). One
example of a suitable expression vector that incorporates a NEO
gene is plasmid pSA-NEO, which is constructed by ligating a first
DNA fragment, prepared by digesting SEQ ID NO:2 with EcoRI and
BglII, with a second DNA fragment, prepared by digesting SEQ ID
NO:1 with EcoRI and BglII. SEQ ID NO:2 incorporates a NEO gene
(nucleotides 1551 to 2345, Genbank Accession No. U00004) preceded
by a sequence for translational initiation (Kozak (1991) J. Biol.
Chem, 266, 19867-19870). Another example of a suitable expression
vector that incorporates a NEO gene and a DHFR cDNA is plasmid
pSVe-NEO-DHFR, which is constructed by ligating a first DNA
fragment, prepared by digesting SEQ ID NO:2 with EcoRI and BglII,
with a second DNA fragment, prepared by digesting pSVeCD4DHFR with
EcoRI and BglII. Plasmid pSVe-NEO-DHFR uses SV40 early
promoter/enhancers to drive expression of the NEO gene and the DHFR
cDNA. Other suitable selectable markers include the XPGT gene
(Mulligan and Berg (1980) Science 209, 1422-1427) and the
hygromycin resistance gene (Sugden et al. (1985) Mol. Cell. Biol.
5, 410-413).
[0392] Human IgG1 DNA sequences are described in Ellison et al.
(1982) Nuc. Acids Res. 10, 4071-4079) (Genbank Acc. No. Z17370)
[0393] Suitable examples of signal peptides are sonic hedgehog
(SHH) (GenBank Acc. No. NM.sub.--000193), interferona-2 (IFN)
(GenBank Acc. No. NP.sub.--000596), and cholesterol ester
transferase (CETP) (GenBank Accession No. NM.sub.--000078). Other
suitable examples include Indian hedgehog (Genbank Acc. No.
NM.sub.--002181), desert hedgehog (Genbank Acc. No.
NM.sub.--021044), IFN.alpha.-1 (Genbank Acc. No. NP.sub.--076918),
IFN.alpha.-4 (Genbank Acc. No. NM.sub.--021068), IFN.alpha.-5
(Genbank Acc. No. NM.sub.--002169), IFN.alpha.-6 (Genbank Acc. No.
NM.sub.--021002), IFN.alpha.-7 (Genbank Acc. No. NM.sub.--021057),
IFN.alpha.-8 (Genbank Acc. No. NM.sub.--002170), IFN.alpha.-10
(Genbank Acc. No. NM.sub.--002171), IFN.alpha.-13 (Genbank Acc. No.
NM.sub.--006900), IFN.alpha.-14 (Genbank Acc. No. NM.sub.--002172),
IFN.alpha.-16 (Genbank Acc. No. NM.sub.--002173), IFN.alpha.-17
(Genbank Acc. No. NM.sub.--021268) and IFN.alpha.-21 (Genbank Acc.
No. NM.sub.--002175).
[0394] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pSHH-Fc5 (SHH signal) is
constructed using SEQ ID NO:3 and SEQ ID NO:1, plasmid pIFN-Fc5
(IFN signal) is constructed using SEQ ID NO:4 and SEQ ID NO:1 and
plasmid pCETP-Fc5 (CETP signal) is constructed using SEQ ID NO:3
and SEQ ID NO:1. For amplified expression, plasmid PSHH-Fc5-DHFR is
constructed using SEQ ID NO:3 and pSVeCD4DHFR (U.S. Pat. No.
5,336,603), pIFN-Fc5-DHFR is constructed using SEQ ID NO:4 and
pSVeCD4DHFR, and pCETP-Fc5-DHFR is constructed using SEQ ID NO:5
and pSVeCD4DHFR.
[0395] Suitable expression vectors for human IgG1 Fc polypeptides
were constructed by ligation of an insert fragment prepared by
digestion with Hind III and EagI and a vector fragment prepared by
digestion with Hind III and PspOM1. Plasmid pCDNA3-SHH-IgG1-Fc (SHH
signal) was constructed using SEQ ID NO:29 and pCDNA3.1(+), plasmid
pCDNA3-IFN-IgG1-Fc (IFN signal) was constructed using SEQ ID NO:30
and pCDNA3.1(+), and plasmid pCDNA3-IgG1-Fc (CETP signal) was
constructed using SEQ ID NO:31 and pCDNA3.1(+).
[0396] Suitable expression vectors for human IgG2 Fc polypeptides
are constructed by ligation of an insert fragment prepared by
digestion with Hind III and EagI and a vector fragment prepared by
digestion with Hind III and PspOM1. Plasmid pCDNA3-SHH-IgG2-Fc (SHH
signal) is constructed using SEQ ID NO:47 and pCDNA3.1(+), plasmid
pCDNA3-IFN-IgG2-Fc (IFN signal) is constructed using SEQ ID NO:48
and pCDNA3.1(+), and plasmid pCDNA3-IgG2-Fc (CETP signal) is
constructed using SEQ ID NO:49 and pCDNA3.1(+).
[0397] Suitable expression vectors for human IgG3 Fc polypeptides
are constructed by ligation of an insert fragment prepared by
digestion with Hind III and EagI and a vector fragment prepared by
digestion with Hind III and PspOM1. Plasmid pCDNA3-SHH-IgG3-Fc (SHH
signal) is constructed using SEQ ID NO:68 and pCDNA3.1(+), plasmid
pCDNA3-IFN-IgG3-Fc (IFN signal) is constructed using SEQ ID NO:69
and pCDNA3.1(+), and plasmid pCDNA3-IgG3-Fc (CETP signal) is
constructed using SEQ ID NO:70 and pCDNA3.1(+).
[0398] Suitable expression vectors for human IgG4 Fc polypeptides
are constructed by ligation of an insert fragment prepared by
digestion with Hind III and EagI and a vector fragment prepared by
digestion with Hind III and PspOM1. Plasmid pCDNA3-SHH-IgG4-Fc (SHH
signal) is constructed using SEQ ID NO:83 and pCDNA3.1(+), plasmid
pCDNA3-IFN-IgG4-Fc (IFN signal) is constructed using SEQ ID NO:84
and pCDNA3.1(+), and plasmid pCDNA3-IgG4-Fc (CETP signal) is
constructed using SEQ ID NO:85 and pCDNA3.1(+).
[0399] In one embodiment, cells are transfected by the calcium
phosphate method of Graham et al. (1977) J. Gen. Virol. 36, 59-74.
A DNA mixture (10 micrograms) is dissolved in 0.5 ml of 1 mM
Tris-HCl, 0.1 mM EDTA, and 227 mM CaCl.sub.2. The DNA mixture
contains (in a ratio of 10:1:1) the expression vector DNA, the
selectable marker DNA, and a DNA encoding the VA RNA gene
(Thimmappaya et al. (1982) Cell 31, 543-551). To this mixture is
added, dropwise, 0.5 mL of 50 mM Hepes (pH 7.35), 280 mM NaCl, and
1.5 mM NaPO.sub.4. The DNA precipitate is allowed to form for 10
minutes at 25.degree. C., then suspended and added to cells grown
to confluence on 100 mm plastic tissue culture dishes. After 4
hours at 37.degree. C., the culture medium is aspirated and 2 ml of
20% glycerol in PBS is added for 0.5 minutes. The cells are then
washed with serum-free medium, fresh culture medium is added, and
the cells are incubated for 5 days.
[0400] In another embodiment, cells are transiently transfected by
the dextran sulfate method of Somparyrac et al. (1981) Proc. Nat.
Acad. Sci. 12, 7575-7579. Cells are grown to maximal density in
spinner flasks, concentrated by centrifugation, and washed with
PBS. The DNA-dextran precipitate is incubated on the cell pellet.
After 4 hours at 37.degree. C., the DEAE-dextran is aspirated and
20% glycerol in PBS is added for 1.5 minutes. The cells are then
washed with serum-free medium, and re-introduced into spinner
flasks containing fresh culture medium with 5 micrograms/ml bovine
insulin and 0.1 micrograms/ml bovine transferring, and the cells
are incubated for 4 days. Following transfection by either method,
the conditioned media is centrifuged and filtered to remove the
host cells and debris. The sample contained the S-Fc domain is then
concentrated and purified by any selected method, such as dialysis
and/or column chromatography (see below).
[0401] To identify the S-Fc in the cell culture supernatant, the
culture medium is removed 24 hours after transfection and replaced
with culture medium containing 200 microCi/ml each of
.sup.35S-methionine and .sup.35S-cysteine. After a 12 hour
incubation, the conditioned medium is collected by centrifugation
to remove the host cells and debris, concentrated on a spin
dialysis filter. The labeled supernatants is analyzed by
immunoprecipitation with protein A sepharose beads in the absence
of added antibodies. The precipitated proteins are analyzed on 7.5%
polyacrylamide-SDS gels either with or without reduction with
.beta.-mercaptoethanol. The processed gel is dried and exposed to
x-ray film to reveal the presence of the S-Fc domain.
[0402] For unamplified expression, plasmids pSHH-Fc-5, pIFN-Fc-5
and pCETP-Fc-5 are transfected into human 293 cells (Graham et al.,
J. Gen. Virol. 36:59 74 (1977)), using a high efficiency procedure
(Gorman et al., DNA Prot. Eng. Tech. 2:3 10 (1990)). Media is
changed to serum-free and harvested daily for up to five days. The
S-Fc proteins are purified from the cell culture supernatant using
protein A-Sepharose CL-4B (Pharmacia). The eluted S-Fc protein is
buffer-exchanged into PBS using a Centricon-30 (Amicon),
concentrated to 0.5 ml, sterile filtered using a Millex-GV
(Millipore) and stored at 4.degree. C.
[0403] For unamplified expression, plasmids pCDNA3-SHH-IgG1-Fc,
pCDA3-IFN-IgG1-Fc and pCDA-3-CETP-IgG1-Fc were transfected into
human 293 cells (Graham et al., J. Gen. Virol. 36:59 74 (1977)),
using a high efficiency procedure (Gorman et al., DNA Prot. Eng.
Tech. 2:3 10 (1990)). Media was changed to serum-free and harvested
daily for up to five days. The S-Fc proteins were purified from the
cell culture supernatant using protein A-Sepharose CL-4B
(Pharmacia). The eluted S-Fc protein was buffer-exchanged into PBS
using a Centricon-30 (Amicon), concentrated to 0.5 ml, sterile
filtered using a Millex-GV (Millipore) and stored at 4.degree.
C.
[0404] FIG. 52 shows expression in 293 kidney cells of human IgG1
Fc symmetroadhesin subunits with N-terminal-S-termini. Lanes 1-6
and lanes 7-12 show the IgG1 Fc polypeptides of FIG. 35A (ii) and
FIG. 36A (ii), respectively. Cell supernatants: lanes 1, 3, 5, 7, 9
and 11; cell lysates: lanes 2, 4, 6, 8, 10 and 12. Signal sequences
used: SHH (lanes 1, 2, 7 and 8); IFN.alpha. (lanes 3, 4, 9, 10);
CETP (lanes 5, 6, 11 and 12).
[0405] FIG. 53 shows expression in 293 kidney cells of human IgG1
Fc symmetroadhesin subunits. Lanes 1-2, 3-4 and 5-6 show the IgG1
Fc polypeptides of FIG. 35A (ii), FIG. 36A (ii) and FIG. 37B (ii),
respectively. Cell supernatants: (lanes 1-6). Signal sequences
used: SHH (lanes 1-6).
[0406] FIG. 54 shows Protein A purification of human IgG1 Fc
symmetroadhesin subunits expressed in 293 kidney cells. Lane 2 and
8 show the IgG1 Fc polypeptides of FIG. 36A and FIG. 35A,
respectively. Lanes 1-7: proteinA-sepharose column fractions for
the IgG1 Fc polypeptide of FIG. 36A.
[0407] FIG. 55 shows Thiol-sepharose binding of proteinA-purified
human IgG1 Fc symmetroadhesin subunits shown in FIG. 54. Lanes 1-3
and lanes 4-6 show the human IgG1 Fc polypeptides of FIG. 35A and
FIG. 36A, respectively. Lanes 1 and 4: starting material; lanes 2
and 5: thiol-sepharose flow-thru fraction; lanes 3 and 6:
thiol-sepharose bound fraction.
[0408] Analysis of the two IgG1 Fc polypeptides of FIG. 35A and
FIG. 36A by mass spectrometry (MALDI) reveals average molecular
masses of 54,552.85 and 53,173.43 daltons, respectively. Assuming
each molecular mass represents the corresponding Fc dimers, the
apparent difference in molecular mass between the two polypeptides
(1,379.4 daltons) is in good agreement (0.6% deviation) with the
predicted difference in molecular mass (1,371.5).
[0409] For amplified expression, Chinese hamster ovary (CHO) cells
are transfected with dicistronic vectors pSHH-Fc5-DHFR,
pIFN-Fc-5-DHFR and pCETP-Fc-5-DHFR which co-express a DHFR cDNA.
Plasmids are introduced into CHO-K1 DUX B11 cells developed by L.
Chasin (Columbia University) via lipofection and selected for
growth in GHT-free medium (Chisholm (1996) High efficiency gene
transfer in mammalian cells. In: Glover, D M, Hames, B D. DNA
Cloning vol 4. Mammalian systems. Oxford Univ. Press, pp 1-41).
Approximately 20 unamplified clones are randomly chosen and
reseeded into 96 well plates. Relative specific productivity of
each colony is monitored using an ELISA to quantitate the S-Fc
protein accumulated in each well after 3 days and a fluorescent
dye, Calcien AM, as a surrogate marker of viable cell number per
well. Based on these data, several unamplified clones are chosen
for further amplification in the presence of increasing
concentrations of methotrexate. Individual clones surviving at 10,
50, and 100 nM methotrexate are chosen and transferred to 96 well
plates for productivity screening. Suitable clones, which
reproducibly exhibit high specific productivity, are expanded in
T-flasks and used to inoculate spinner cultures. After several
passages, the suspension-adapted cells are used to inoculate
production cultures in GHT-containing, serum-free media
supplemented with various hormones and protein hydrolysates.
Harvested cell culture fluid containing the S-Fc protein is
purified using protein A-Sepharose CL-4B.
EXAMPLE 2
Preparation of Immunoglobulin Fc with N-Terminal-X-Termini
(X-Fc)
[0410] Selenocysteine (sec) is the 21.sup.st amino acid
incorporated during ribosome-mediated protein synthesis (Zinoni et
al. (1986) Proc. Natl. Acad. Sci. 83, 4650-4654; Chambers et al.
(1986) EMBO J. 5, 1221-1227). The process is complex and distinct
from cysteine incorporation, requiring an mRNA selenocysteine
insertion element in order to decode a UGA stop codon. Protein
semi-synthesis offers an alternative means for preparing Fc-like
molecules having N-terminal-X-termini (X-Fc) that begin at cysteine
(cys) and/or selenocysteine (sec).
[0411] Accordingly, host cells are transfected with constructs that
encode pre-Fc chimeric polypeptides consisting of a signal peptide
joined at its C-terminus by a peptide bond to the N-terminus of an
Fc domain beginning at cys-11, CDKTHTCPPCP (FIG. 36A) and cys-14
CDKTHTCPPCP (FIG. 36B). The heterologous signal peptides used are
selected from proteins with N-terminal cysteines (part i). Thus,
cleavage by the cellular signal peptidase will provide a mature
S-Fc protein having cys-11 at the N-terminus (part ii). Native
chemical ligation is then employed to prepare mature X-Fc proteins
having cys-5 or sec-5 at the N-terminus, XDKTHTCPPCP (part
iii).
[0412] The sequences of the IgG1 precursor polypeptides of FIG. 36A
are shown in SEQ ID NO: 101, SEQ ID NO:102, and SEQ ID NO:103. The
sequence of the mature IgG1 polypeptide of FIG. 35A is shown in SEQ
ID NO: 104. The sequences of the IgG1 precursor polypeptides of
FIG. 36B are shown in SEQ ID NO: 109, SEQ ID NO:110, and SEQ ID
NO:111. The sequence of the mature IgG1 polypeptide of FIG. 35B is
shown in SEQ ID NO: 112.
[0413] Accordingly, host cells are transfected with constructs that
encode pre-Fc chimeric polypeptides consisting of a signal peptide
joined at its C-terminus by a peptide bond to the N-terminus of an
Fc domain beginning at cys-14, CDKTHTCPPCP (FIG. 36B). The
heterologous signal peptides used are selected from proteins with
N-terminal cysteines (part i). Thus, cleavage by the cellular
signal peptidase will provide a mature S-Fc protein having cys-11
at the N-terminus (part ii). Native chemical ligation is then
employed to prepare mature X-Fc proteins having cys-11 or sec-11 at
the N-terminus, XPPCP (part iii).
[0414] The S-Fc proteins with cys-11 and cys-14 at the N-terminus
is first prepared using the procedures described in EXAMPLE 1.
Native chemical ligation is carried out with S-Fc protein and
peptide Fc-A (5-11: cys-asp-lys-thr-his-thr), or S-Fc protein and
peptide Fc-B (5-11: sec-asp-lys-thr-his-thr). Suitable expression
vectors are constructed by ligation of an insert and vector
fragment prepared by digestion with EcoRI and BglII. For
unamplified expression, plasmid pSHH-Fc11 (SHH signal) is
constructed using SEQ ID NO:6 and SEQ ID NO:1, plasmid pIFN-Fc11
(IFN signal) is constructed using SEQ ID NO 7 and SEQ ID NO:1 and
plasmid pCETP-Fc11 (CETP signal) is constructed using SEQ ID NO:8
and SEQ ID NO:1. For amplified expression, plasmid pSHH-Fc11-DHFR
is constructed using SEQ ID NO:6 and pSVeCD4DHFR, plasmid
pIFN-Fc11-DHFR is constructed using SEQ ID NO:7 and pSVeCD4DHFR,
and plasmid pCETP-Fc11-DHFR is constructed using SEQ ID NO:8 and
pSVeCD4DHFR.
[0415] Suitable expression vectors for human IgG1 Fc are
constructed by ligation of an insert fragment prepared by digestion
with Hind III and EagI and a vector fragment prepared by digestion
with Hind III and PspOM1. Plasmid pCDNA3-SHH-IgG1-Fc11 (SHH signal)
is constructed using SEQ ID NO:98 and pCDNA3.1(+), plasmid
pCDNA3-IFN-IgG1-Fc11 (IFN signal) is constructed using SEQ ID NO:99
and pCDNA3.1(+), and plasmid pCDNA3-IgG1-Fc11 (CETP signal) is
constructed using SEQ ID NO:100 and pCDNA3.1(+). Plasmid
pCDNA3-SHH-IgG1-Fc14 (SHH signal) is constructed using SEQ ID
NO:106 and pCDNA3.1(+), plasmid pCDNA3-IFN-IgG1-Fc14 (IFN signal)
is constructed using SEQ ID NO:107 and pCDNA3.1(+), and plasmid
pCDNA3-IgG1-Fc14 (CETP signal) is constructed using SEQ ID NO:108
and pCDNA3.1(+).
[0416] General principles of native chemical ligation are described
in U.S. Pat. No. 6,184,344, the whole of which is incorporated
herein by reference. Peptides Fc-A and Fc-B are synthesized using a
TAMPAL resin from which any desired thioester can be readily
obtained. After de-protection of side chain protecting groups, the
resulting C-terminal activated peptides are used in native chemical
ligation without further modification. To prevent the cyclization
or polymerization of bifunctional peptides, the sulfhydryl moiety
(peptide Fc-A) and the selenohydryl moiety (peptide Fc-B) are
reversibly blocked with Msc.
[0417] Peptide synthesis is carried out by manual a solid-phase
procedure using an in situ neutralization/HBTU activation procedure
for Boc chemistry (Schnolzer et al. (1992) Int. J. Pept. Protein
Res. 40, 180-193). After each coupling step, yields are determined
by measuring residual free amine with the quantitative ninhydrin
assay (Sarin et al. (1981) Anal. Biochem. 117, 147-157). Side chain
protected amino acids are Boc-Asp(O-cyclohexyl)-OH,
Boc-Cys(4-methylbenzyl)-OH, Boc-Lys(2-Cl-Z)-OH, and
Boc-Thr(benzyl)-OH. After chain assembly is completed, peptides are
deprotected and cleaved from the resin by treatment with anhydrous
HF for 1 hour at 0.degree. C. with 4% anisole as scavenger.
[0418] Peptides Fc-A and Fc-B are synthesized on trityl-associated
mercaptopropionic acid leucine (TAMPAL) resin to yield C-terminal
MPAL-activated thioesters (Hackeng et al. (1999) Proc. Natl. Acad.
Sci. 96, 10068-10073). The N-terminal cys/sec residues of the
thioester peptides are protected with 2-(methylsulfonyl)ethyl
carbonate (Msc) groups in a 2 hour reaction (10-fold excess) of
activated Msc nitrophenol ester dissolved in a minimal volume of
dimethylformamide/5% diisopropylethylamine. The thioester-activated
peptides are deprotected and cleaved from the TAMPAL resin,
HPLC-purified, lyophilized, and stored until use at -20.degree. C.
Preparative reversed-phase HPLC is performed using a Vydac C-18
column (10 micrometer, 1.0 cm.times.25 cm). The bound peptides are
eluted using a linear gradient of acetonitrile in H.sub.20/10%
trifluoroacetic acid.
[0419] Starting with the purified C-terminal S-Fc protein, native
chemical ligation is carried out with the
Msc-NH-cys.sup.5-thr.sup.10-.alpha.-thioester peptide (Fc-A) or the
Msc-NH-sec.sup.5-thr.sup.10-.alpha.-thioester peptide (Fc-B) under
non-denaturing conditions as previously described (Evans et al.
(1999) J. Biol. Chem. 274, 3923-3926). The thioester-activated
peptides are mixed in molar excess with freshly prepared S-Fc
protein (starting concentration, 1-200 micromolar). The solution is
concentrated with a Centriprep 3/30 apparatus (Millipore, Mass.),
then with a Centricon 3/10 apparatus to a final concentration of
0.15 to 1.2 mM for the S-Fc protein. Ligations are incubated
overnight at 4.degree. C. and visualized using SDS-page
electrophoresis. After native chemical ligation, the N-terminal Msc
protecting group is removed by a brief incubation (<5 minute) at
pH 13. The X-Fc product is purified to remove unreacted peptides by
affinity chromatography with protein A sepharose using the
procedure in EXAMPLE 1.
[0420] The sequence of the native ligation product of FIG. 36A is
shown in SEQ ID NO: 105. The sequence of the native ligation
product of FIG. 36B is shown in SEQ ID NO: 113.
EXAMPLE 3
Preparation of Immuoglobulin Fc with C-Terminal-X-Termini
(Fc-X)
[0421] IgG is expressed in two abundant forms, the soluble antibody
molecule and the cell-bound B-cell receptor. Both forms arise from
a single messenger RNA by alternative splicing with the result that
two additional exons are added to the IgG heavy chain coding region
(Tyler et al. (1982) Proc. Natl. Acad. Sci. 79, 2008-2012;
Yamawaki-Kataoka et al. (1982) Proc. Natl. Acad. Sci. 79,
2623-2627). The first exon added (M1 exon) encodes a stretch of 18
amino acids, ELQLEESCAEAQDGELDG, which flexibly tethers IgG to the
cell surface, making it a good choice for novel Fc-like molecules
with a C-terminal-X-terminus (Fc-X). The C-terminal gly-18 residue
of the M1 domain is also well-suited for preparing Fc-intein fusion
proteins used in generating a C-terminal activated thioester.
Following an intein autocleavage reaction, a thioester intermediate
is generated that permits the facile addition of cysteine or
selenocysteine to the C-terminus by native chemical ligation.
[0422] Accordingly, host cells are transfected with expression
vectors that encode pre-Fc-intein chimeric polypeptides containing
the M1 domain joined at its C-terminus, ELQLEESCAEAQDGELDG, by a
peptide bond to the N-terminus of a self-splicing intein at the
autocleavage site (FIG. 37A), or the Fc protein containing a
portion of the M1 domain, ELQLEESC (FIG. 37B). To ensure that the
Fc-X protein does not have an N-terminal-X-terminus, heterologous
signal peptides are used that are cleaved by the cellular signal
peptidase before lysine residues (part i). Thus, cleavage by the
cellular signal peptidase will provide a Fc-intein fusion protein
with lys-7, EPKSCDKTHTCPPCP, at the N-terminus (part ii). Excision
of the intein domain by protein splicing provides an Fc-thioester
intermediate (part iii). Finally, native chemical ligation of the
Fc-thioester with free cysteine and/or selenocysteine is employed
to prepare Fc-X proteins having the C-terminal-X-terminus,
ELQLEESCAEAQDGELDGX (part iv).
[0423] The sequences of the IgG1 precursor polypeptides of FIG. 37A
are shown in SEQ ID NO:116, and SEQ ID NO:117. The sequence of the
mature and modified IgG1 polypeptides of FIG. 37A is shown in SEQ
ID NO:118 to SEQ ID NO:120. The sequences of the IgG1 precursor
polypeptides of FIG. 37B are shown in SEQ ID NO:123, and SEQ ID
NO:124. The sequence of the mature IgG1 polypeptide of FIG. 37B is
shown in SEQ ID NO:125.
[0424] The Fc-intein fusion protein with lys-7 at the N-terminus is
prepared using the procedures described in EXAMPLE 1. The initial
purification step is carried out using affinity chromatography with
a chitin resin instead of protein A sepharose. After cleavage from
the resin, the activated Fc-thioester intermediate is used directly
for native ligation with cysteine and/or selenocysteine.
[0425] A suitable DNA sequence for the M1 membrane domain of human
IgG1 is described in Strausberg et. al. (2002) Proc. Natl. Acad.
Sci. 99, 16899-16903 (GenBank Acc. No. BC019046).
[0426] Suitable examples of signal peptides are the CD2 T-cell
surface glycoprotein (CD2) (GenBank Acc. No. NM.sub.--001767), and
the CD4 T-cell surface glycoprotein (CD4) (GenBank Acc. No.
NP.sub.--000616).
[0427] A suitable example of a self-splicing intein is found in the
Methanobacterium thermoautotrophicum ribonucleotide reductase large
subunit (MthRIR1) (Genbank Acc. No. AE000845). To limit the intein
autocleave reaction to the Fc-intein fusion junction, an MthRR1
intein variant with only N-terminal cleavage activity is prepared
by changing the pro at position -1 to gly and the C-terminal asn at
position 134 to ala (Evans et al. (1999) J. Biol. Chem. 274,
3923-3926). In addition, an Mth RRI intein sequence fused to a
Bacillus circulans chitin binding domain is used to facilitate the
purification of the Fc-intein chimeric polypeptide by affinity
chromatography. A suitable sequence for the modified MthRIR1 intein
is found in plasmid pTWIN-2 (New England BioLabs, MA).
[0428] Other suitable examples are found in the Mycobacterium
xenopi gyrase subunit A (Mxe GyrA) (Genbank Acc. No. MXU67876), and
the Saccharomyces cerevisiae vacuolar ATPase (Sce VMA1) (GenBank
Acc. No. NC.sub.--001136). Many other suitable examples of
self-splicing inteins are described in Inbase: the Intein Database
(Perler (2002) Nucl. Acids Res. 30, 383-384).
[0429] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pCD2-Fc7-Mth (CD2
signal) is constructed with SEQ ID NO:9 and SEQ ID NO:1, and
plasmid pCD4-Fc7-Mth (CD4 signal) is constructed with SEQ ID NO:10
and SEQ ID NO:1. For amplified expression, plasmid
pCD2-Fc7-Mth-DHFR is constructed with SEQ ID NO:9 and pSVeCD4DHFR,
and plasmid pCD4-Fc7-Mth-DHFR is constructed with SEQ ID NO:10 and
pSVeCD4DHFR.
[0430] Suitable expression vectors are constructed by ligation of
an insert fragment prepared by digestion with Hind III and EagI and
a vector fragment prepared by digestion with Hind III and PspOM1.
Plasmid pCDNA3-CD2-Fc7-Mth (CD2 signal) is constructed with SEQ ID
NO:114 and pCDNA3.1(+), and plasmid pCDNA3-CD4-Fc7-Mth (CD4 signal)
is constructed with SEQ ID NO:115 and pCDNA3.1(+). Plasmid
pCDNA3-CD2-Fc7-ELQLEESC (CD2 signal) is constructed with SEQ ID
NO:121 and pCDNA3.1(+), and plasmid pCDNA3-CD4-Fc7-ELQLEESC (CD4
signal) is constructed with SEQ ID NO:122 and pCDNA3.1(+).
[0431] General principles of chitin affinity purification and the
self-splicing intein autocleavage reaction are described in U.S.
Pat. No. 5,834,247, the whole of which is incorporated herein by
reference.
[0432] Following host cell transfection, cell culture supernatant
is applied to a column packed with chitin resin (New England
BioLabs, MA) that is equilibrated in buffer A (20 mM Tris-HCl, pH
7.5 containing 500 mM NaCl). Unbound protein is washed from the
column with 10 column volumes of buffer A. Thiol reagent-induced
cleavage is initiated by rapidly equilibrating the chitin resin in
buffer B (20 mM Tris-HCl, pH 8 containing 0.5 M NaCl and 0.1 M
2-mercaptoethane-sulfonic acid (MESNA)). The cleavage, which
simultaneously generates a C-terminal thioester on the target
protein, is carried out overnight at 4.degree. C. after which the
protein was eluted from the column.
[0433] Starting with the purified Fc-thioester intermediate, native
chemical ligation is carried out with cysteine or selenocysteine
using the procedure in EXAMPLE 2. The final Fc-X product is
purified to remove unreacted cysteine and selenocysteine by
affinity chromatography with protein A sepharose.
EXAMPLE 4
Preparation of Immunoglobulin Fc with N-Terminal-S-Termini and
C-Terminal-X-Termini (S-Fc-X)
[0434] The S-Fc and Fc-X proteins are useful in preparing
immunosymmetroadhesins having two binding domains joined to a
single Fc domain (see below). Binding domains are added to the
N-terminal-S-termini (S-Fc) or C-terminal-X-termini (Fc-X). S-Fc-X
domains that are useful in preparing bi-symmetroadhesins with four
binding domains joined to a single Fc domain are prepared using the
procedures described in EXAMPLE 1 and EXAMPLE 3.
[0435] Accordingly, host cells are transfected with expression
vectors that encode pre-Fc-intein chimeric polypeptides containing
the M1 domain joined at its C-terminus, ELQLEESCAEAQDGELDG, by a
peptide bond to the N-terminus of a self-splicing intein at the
autocleavage site (FIG. 38). Heterologous signal peptides are
selected from proteins with N-terminal cysteines (part i). Thus,
cleavage by the cellular signal peptidase will provide a mature
S-Fc-intein fusion protein having cys-5 at the N-terminus,
CDKTHTCPPCP (part ii). Excision of the intein by protein splicing
provides an Fc-thioester intermediate (part iii). Finally, native
ligation of the Fc-thioester with free cysteine and/or
selenocysteine is employed to prepare S-Fc-X proteins having the
C-terminal-X-terminus, ELQLEESCAEAQDGELDGX (part iv).
[0436] The sequences of the IgG1 precursor polypeptides of FIGS.
38A-38B are shown in SEQ ID NO:126, SEQ ID NO:127, and SEQ ID
NO:128. The sequence of the mature and modified IgG1 polypeptides
of FIGS. 38A-38B is shown in SEQ ID NO:129 to SEQ ID NO:131.
[0437] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, PSHH-Fc5-Mth is constructed with
SEQ ID NO:11 and SEQ ID NO:1, pIFN-Fc5-Mth is constructed with SEQ
ID NO:12 and SEQ ID NO:1, and pCETP-Fc5-Mth is constructed with SEQ
ID NO:13 and SEQ ID NO:1. For amplified expression,
pSHH-Fc5-Mth-DHFR is constructed with SEQ ID NO:11 and pSVeCD4DHFR,
pIFN-Fc5-Mth-DHFR is constructed with SEQ ID NO:12 and pSVeCD4DHFR,
and pCETP-Fc5-Mth-DHFR is constructed with SEQ ID NO:13 and
pSVeCD4DHFR.
[0438] The S-Fc-intein fusion protein is first purified from the
culture supernatant of transfected host cells using chitin affinity
chromatography. After cleavage from the chitin resin, the
Fc-thioester intermediate is directly applied to a protein A
Sepharose column in order to prevent cyclization or polymerization
side-reactions. The column bound activated S-Fc-thioester is used
directly for native ligation with cysteine and/or selenocysteine.
The column is then washed to remove excess amino acids, and the
S-Fc-X is eluted from the protein A sepharose.
EXAMPLE 5
Preparation of Immunoglobulin Fc with N-Terminal-X-Termini and
C-Terminal-X-Termini (X-Fc-X)
[0439] The X-Fc and Fc-X proteins are useful in preparing
immunosymmetroadhesins having two binding domains joined to a
single Fc domain (see below). Binding domains are added to the
N-terminal-X-termini (X-Fc) or C-terminal-X-termini (Fc-X). X-Fc-X
domains that are useful in preparing bi-symmetroadhesins with four
binding domains joined to a single Fc domain are prepared using the
procedures described in EXAMPLE 2 and EXAMPLE 3.
[0440] Accordingly, host cells are transfected with expression
vectors that encode pre-Fc-intein chimeric polypeptides containing
the M1 domain joined at its C-terminus, ELQLEESCAEAQDGELDG, by a
peptide bond to the N-terminus of a self-splicing intein at the
autocleavage site (FIG. 39). Heterologous signal peptides are
selected from proteins with N-terminal cysteines (part i). Thus,
cleavage by the cellular signal peptidase will provide a mature
S-Fc-intein fusion protein having cys-11 at the N-terminus (part
ii). Native chemical ligation is then employed to prepare
X-Fc-intein fusion proteins having cys-5 or sec-5 at the
N-terminus, XDKTHTCPPCP (part iii). Excision of the intein by
protein splicing provides an Fc-thioester intermediate (part iv).
Finally, in a second native chemical ligation reaction, the
Fc-thioester with is carried out with free cysteine and/or
selenocysteine to prepare X-Fc-X proteins having a
C-terminal-X-terminus ELQLEESCAEAQDGELDGX (part v). Alternately,
part iii is carried following parts iv and v.
[0441] The sequences of the IgG1 precursor polypeptides of FIGS.
39A-39B are shown in SEQ ID NO:132, SEQ ID NO:133, and SEQ ID
NO:134. The sequence of the mature and modified IgG1 polypeptides
of FIGS. 38A-38B is shown in SEQ ID NO:135 to SEQ ID NO:138.
[0442] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pSHH-Fc11-Mth is
constructed with SEQ ID NO:14 and SEQ ID NO:1, pIFN-Fc11-Mth is
constructed with SEQ ID NO:15 and SEQ ID NO:1, and pCETP-Fc11-Mth
is constructed with SEQ ID NO:16 and SEQ ID NO:1. For amplified
expression, pSHH-Fc11-Mth-DHFR is constructed with SEQ ID NO:14 and
pSVeCD4DHFR, pIFN-Fc11-Mth-DHFR is constructed with SEQ ID NO:15
and pSVeCD4DHFR, and pCETP-Fc11-Mth-DHFR is constructed with SEQ ID
NO:16 and pSVeCD4DHFR.
[0443] The S-Fc-intein fusion protein is first purified from the
culture supernatant of transfected host cells using chitin affinity
chromatography. Native chemical ligation is carried out on the
chitin column with the
Msc-NH-cys.sup.5-thr.sup.10-.alpha.-thioester peptide (Fc-A) or the
Msc-NH-sec.sup.5-thr.sup.10-.alpha.-thioester peptide (Fc-B) as
described (EXAMPLE 2). The chitin column is then washed thoroughly
to remove unreacted peptide. The intein autocleavage reaction is
then carried out. After cleavage, the X-Fc-thioester intermediate
is directly applied to protein A Sepharose. The bound activated
X-Fc-thioester is used directly for native ligation with cysteine
and/or selenocysteine. The column is then washed to remove excess
amino acids. The Msc-blocked X-Fc-X is eluted from the column,
treated to remove the Msc protecting group, and repurified using
protein A Sepharose to yield the final X-Fc-X product.
EXAMPLE 6
CD4 Symmetroadhesins
[0444] A therapeutic strategy for treating HIV-1 infection is based
upon human CD4, a component of the HIV-1 receptor. CD4
immunoadhesins (Capon et al. (1989) Nature 337, 525-531)
effectively block HIV-1 infectivity by binding to the gp120
envelope protein. The blocking activity resides in the CD4
extracellular domain (residues 1 to 371).
[0445] Accordingly, various CD4 symmetroadhesins are prepared using
CD4-X protein, and analysed for their ability to bind gp120 and
block HIV-1 infectively. The activity of CD4 symmetroadhesins is
compared with CD4 immunoadhesin prepared as described (Capon et
al., ibid).
[0446] CD4-X protein is prepared by the procedures of EXAMPLE 3.
Host cells are transfected with expression vectors that encode the
pre-CD4 chimeric polypeptide containing the CD4 extracellular
domain joined at its C-terminus by a peptide bond to the N-terminus
of a self-splicing intein at the autocleavage site (FIGS.
40A-40B).
[0447] Cleavage of the CD4 signal sequence (part i) by the cellular
signal peptidase provides mature CD4-intein fusion protein (part
ii). Excision of the intein domain provides a CD4-thioester
intermediate (part iii). Finally, native chemical ligation of the
CD4-thioester with free cysteine and/or selenocysteine is employed
to prepare CD4 domains with C-terminal-X-termini (part iv).
[0448] The sequences of these polypeptides are shown in SEQ ID NO:
140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143.
[0449] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pCD4-Mth is constructed
using SEQ ID NO:17 and SEQ ID NO:1. For amplified expression,
plasmid pCD4-Mth-DHFR is constructed using SEQ ID NO:17 and
pSVeCD4DHFR.
[0450] Suitable expression vectors were constructed by ligation of
an insert fragment prepared by digestion with Hind III and EagI and
a vector fragment prepared by digestion with Hind III and PspOM1.
Plasmid pCDNA3-CD4-Mth was constructed using SEQ ID NO:139 and
pCDNA3.1(+).
[0451] FIG. 56 shows expression in human 293 kidney cells of human
CD4-intein fusion proteins. Lanes 1-4 show the CD4-intein fusion
polypeptide of FIG. 40A (ii). Cell supernatants: lanes 1 and 3;
cell lysates: lanes 2 and 4.
[0452] CD4-X is used to prepare various CD4 symmetroadhesins (Table
2). Hemi-symmetroadhesins are prepared using the CD4-X protein
alone, immunosymmetroadhesins are prepared using the CD4-X protein
with S-Fc, X-Fc, or Fc-X, and bi-symmetroadhesins are prepared
using the CD4-X protein with S-Fc-X or X-Fc-X.
[0453] CD4 hemi-symmetroadhesin is prepared using CD4-X protein
exposed to mildly reducing conditions that activate the X-termini,
but do not denature the protein (Fleischman et al. (1962) Arch.
Biochem. Biophys. 1 (Suppl.), 174-180; Edelman et al. (1963) Proc.
Nat. Acad. Sci. (1963) 50, 753-761). CD4-X protein (0.5 to 2.0
mgs/ml) is dissolved in reducing buffer (0.05 M Tris-HCl buffer pH
8.0, made 0.1 M in 2-mercaptoethanol) and incubated for 1 hour at
room temperature. The protein is then exchanged into oxidation
buffer (0.1 M K.sub.2HPO.sub.4) by Sephadex G-100 chromatography,
gently agitated in a round bottom glass test tube stoppered with a
loosely packed cotton plug, and allowed to proceed at room
temperature for 20 hours (Haber and Anfinsen (1961) J. Biol. Chem.
236, 422-424). CD4 immunosymmetroadhesins and CD4
bi-symmetroadhesins are prepared on protein A Sepharose beads using
the S-Fc, X-Fc, Fc-X, S-Fc-X, or X-Fc-X proteins as indicated
(Table 2). The bound proteins are gently agitated in reducing
buffer for 1 hour at room temperature, then washed with oxidation
buffer. CD4-X is treated with reducing buffer, added to the beads,
and the reaction allowed to proceed at room temperature for 20
hours.
[0454] CD4-gp120 saturation binding analysis is carried out as
described (Smith et al. (1987) Science 238, 1704-1707) using
radio-iodinated gp120 prepared as described (Lasky et al. (1987)
Cell 50, 975-985). Reactions (0.2) contain 0.25% NP-40, 0.1% sodium
deoxycholate, 0.06 M NaCl, 0.01 M Tris-HCl, pH 8.0 (1.times. buffer
A) with .sup.125I-gp120 (3 ng to 670 ng at 2.9 nCi/ng). Binding is
carried out for 1 hour at 0.degree. C. in the presence or absence
of 50 micrograms of unlabeled purified gp120. The bound
.sup.125I-gp120 is then determined by immunoprecipitation. Binding
reactions are preabsorbed with 5 microliters of normal rabbit serum
for 1 hour at 0.degree. C., cleared with 40 microliters of 10% w/v
Pansorbin (Calbiochem) for 30 minutes at 0.degree. C., and
incubated overnight at 0.degree. C. with 2 microliters of normal
serum or 5 microliters (0.25 microgram) of OKT4 monoclonal antibody
(Ortho Biotech). Immunoprecipitates are collected with 10
microliters of Pansorbin, washed twice in 2.times. buffer and once
in water, then eluted at 100.degree. C. for 2 minutes in 0.12 M
Tris-HCl pH 6.8, 4% SDS, 0.7 M mercaptoethanol. The fraction of
bound .sup.125I-gp120 is determined in a gamma counter and
Scatchard analysis is used to determine the apparent dissociation
contant.
[0455] HIV-1 blocking studies are carried out as described
(Robert-Guroff et al. (1985) Nature 316, 72-74). Equal volumes of
inhibitor and HIV-1 (60 microliters) are incubated at 4.degree. C.
for 1 hour, then the same volume of H9 cells (Gallo et al. (1984)
Science 224, 500-503) at 5.times.10.sup.6/ml is added and
incubation continued for 1 hour at 37.degree. C. Following
absorption of virus, 2.5.times.10.sup.5 cells in 150 microliters
are transferred to 2 ml of incubation media. After 4 days at
37.degree. C., the cultures are split 1:2 with fresh media and
incubated for an additional 3 days. Cultures are harvested, reverse
transcriptase activity is measured (Groopman et al., AIDS Res. Hum.
Retroviruses (1987) 3, 71-85), and immunofluorescence with HIV-1
positive serum is determined as described (Poiesz et al. (1980)
Proc. Acad. Nat. Sci. 77, 7415-7419). Challenge dose of virus is
100 TCID.sub.50 of HIV-1 strain HTLV-IIIB grown in H9 cells assayed
in the same system. Incubation media is RPMI 1640 media containing
2 mM L-glutamine, 100 units/ml penicillin, 100 micrograms/ml
streptomycin, 2 micrograms/ml polybrene and 20% fetal calf
serum.
EXAMPLE 7
Tumor Necrosis Factor Receptor Symmetroadhesins
[0456] A therapeutic strategy for treating autoimmune disease is
based upon tumor necrosis factor .alpha. (TNF-.alpha.), and its
binding interaction with TNF-.alpha. antibodies and receptors
(TNR). Both are an important therapeutic option in adult rheumatoid
arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis,
psoriatic arthritis, Crohn's disease, and ulcerative colitis.
[0457] Accordingly, various TNR symmetroadhesins are prepared using
TNR-X proteins, and analyzed for their ability to bind TNF-.alpha.
and to block TNF-.alpha. biological activity. The activity of TNR
symmetroadhesins is compared to that of TNR immunoadhesins
(Ashkenazi et al. (1991) Proc. Natl. Acad. Sci. 88,
10535-10539).
[0458] Human TNR include TNR1A (Genbank Acc. No. NM.sub.--001065);
and TNR1B (GenBank Acc. No. NM.sub.--001066). TNF-.alpha. antibody
Di62 includes Di62 heavy chain (Genbank Acc. No. AJ002433); and
Di62 light chain (Genbank Acc. No. AJ002434) (hereafter,
TNR1A=TNR1; TNR1B=TNR2; and Di62-VH-CH+Di62-VkCk=TNR.sub.Fab).
[0459] Di62-Vk is prepared according to the method of EXAMPLE 1.
Di62-VH-X, TNR1A-X, and TNR1B-X, are prepared according to the
method of EXAMPLE 3.
[0460] Host cells are co-transfected with two expression vectors
that encode the Di62-VH-CH-intein chimeric polypeptide and
Di62-VkCk protein result in co-expression of the
Di62-VH-CH-intein:Di62-Vk-Ck protein that is used to prepare the
TNF.sub.Fab-X protein:
1) a pre-Di62-VH-intein chimeric polypeptide containing the
Di62-VH-CH1 domain joined at its C-terminus by a peptide bond to
the N-terminus of a self-splicing intein at the autocleavage site
(FIGS. 41A-41B); and 2) a pre-Di62-Vk polypeptide containing the
Di62-Vk-Ck domain (FIG. 42).
[0461] Cleavage of the homologous Di62-VH-CH signal sequence (part
i) by the cellular signal peptidase provides mature
Di62-VH-CH-intein fusion protein (part ii). Excision of the intein
by protein splicing provides the Di62-VH-CH-thioester intermediate
(part iii). Finally, native chemical ligation of the
Di62-VH-CH-thioester with free cysteine and/or selenocysteine is
employed to prepare Di62-VH-CH-X protein with C-terminal-X-termini
(part iv) (refer to FIGS. 41A-41B).
[0462] Cleavage of the homologous Di62-Vk-Ck signal sequence (part
i) by the cellular signal peptidase provides the mature Di62-Vk-Ck
protein (part ii) (refer to FIG. 42).
[0463] Host cells are transfected with expression vectors that
encode the TNR1A-intein chimeric polypeptide and TNR1B-intein
chimeric polypeptide to prepare the TNF.sub.1-X and TNF.sub.2-X
proteins, respectively:
1) a pre-TNR1A-intein chimeric polypeptide containing the TNR1A
extracellular domain joined at its C-terminus by a peptide bond to
the N-terminus of a self-splicing intein at the autocleavage site
(FIG. 43); and 2) a pre-TNR1B-intein chimeric polypeptide
containing the TNR1B extracellular domain joined at its C-terminus
by a peptide bond to the N-terminus of a self-splicing intein at
the autocleavage site (FIG. 44A).
[0464] Cleavage of the homologous TNR signal sequences (part i) by
the cellular signal peptidase provides mature TNR-intein fusion
proteins (part ii). Excision of the intein by protein splicing
provides TNR-thioester intermediates (part iii). Finally, native
chemical ligation of the TNR-thioesters with free cysteine and/or
selenocysteine is employed to prepare TNR-X proteins with
C-terminal-X-termini (part iv) (refer to FIG. 43, 44A).
[0465] The sequences of these polypeptides are shown in SEQ ID NO:
145, SEQ ID NO:146, SEQ ID NO:147, and SEQ ID NO:148 (Di62-VHCH);
in SEQ ID NO: 150, and SEQ ID NO:151 (Di62-VkCk); in SEQ ID NO:
153, SEQ ID NO:154, SEQ ID NO:155, and SEQ ID NO:156 (TNR1A); in
SEQ ID NO: 158, SEQ ID NO:159, SEQ ID NO:160, and SEQ ID NO:161
(TNR1B); and in SEQ ID NO: 163, and SEQ ID NO:164 (TNR1B
immunoadhesin).
[0466] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pDi62-VHCH-Mth is
constructed using SEQ ID NO:18 and SEQ ID NO:1, plasmid pDi62-VkCk
is constructed using SEQ ID NO:19 and SEQ ID NO:1, plasmid
pTNR1A-Mth is constructed using SEQ ID NO:20 and SEQ ID NO:1, and
plasmid pTNR1A-Mth is constructed using SEQ ID NO:22 and SEQ ID
NO:1. For amplified expression, plasmid pDi62-VHCH-Mth-DHFR is
constructed using SEQ ID NO:18 and pSVeCD4DHFR, plasmid
pDi62-VkCk-DHFR is constructed using SEQ ID NO:19 and pSVeCD4DHFR,
plasmid pTNR1A-Mth-DHFR is constructed using SEQ ID NO:20 and
pSVeCD4DHFR, and plasmid pTNR1A-Mth-DHFR is constructed using SEQ
ID NO:22 and pSVeCD4DHFR.
[0467] Suitable expression vectors are constructed by ligation of
an insert fragment prepared by digestion with Hind III and EagI and
a vector fragment prepared by digestion with Hind III and PspOM1.
Plasmid pCDA3-Di62-VHCH-Mth is constructed using SEQ ID NO:144 and
pCDNA3.1(+). Plasmid pCDA3-Di62-VkCk is constructed using SEQ ID
NO:149 and pCDNA3.1(+). Plasmid pCDNA-3-TNR1A-Mth-DHFR is
constructed using SEQ ID NO:152 and pCDNA3.1(+). Plasmid
pCDNA-3-TNR1B-Mth-DHFR was constructed using SEQ ID NO:157 and
pCDNA3.1(+). Plasmid pCDNA-3-TNR1B-immunoadhesin was constructed
using SEQ ID NO:162 and pCDNA3.1(+).
[0468] FIG. 57 shows the expression in human 293 kidney cells of
human TNR1B fusion proteins. Lanes 2 and 5 show the TNR1B-intein
fusion protein of FIG. 44A (ii). Lanes 1 and 3 show the
TNR1B-immunoadhesin fusion protein of FIG. 44B (ii). Lanes 3 and 6
show proteins from mock-transfected cells. Cell supernatants: lanes
1-3; cell lysates: lanes 4-7. Lane 7: control TNR1B-immunoadhesin
(R&D Systems).
[0469] FIG. 58 shows TNR1B symmetroadhesin subunits with
C-terminal-S-termini. Lanes 1-2 show the TNR1B polypeptide of FIG.
44A (iii) following purification by chitin affinity chromatography
and cleavage/elution with MESNA. Lanes 3 shows the native ligation
product between the TNR1B polypeptide of FIG. 44A (iii) with a
fluorescent-labeled peptide (New England Biolabs). Panel (i):
direct fluorescence; panel (ii): western blot with anti-TNR1B
antibody (R&D Systems); panel (iii): SYPRO Ruby staining
(Sigma-Aldrich).
[0470] FIG. 59 shows TNR1B symmetroadhesin subunits with
C-terminal-S-termini. Lane 5 shows the TNR1B polypeptide of FIG.
44A (iv) following purification by chitin affinity chromatography
and cleavage/elution with cysteine. Lanes 1-4 show
TNR1B-immunoadhesin.
[0471] FIG. 60 shows TNR1B symmetroadhesin. Lanes 1-4 show the
TNR1B symmetroadhesin of FIG. 44A (iv) before oxidation (lanes 1
and 4) and after oxidation in the presence of 10 mM CuSO4. Lanes 3
and 6 show a TNR1B-immunoadhesin control. Lanes 1-3: reducing
conditions; lanes 4-6: non-reducing conditions. The TNR1B
symmetroadhesin monomer (42 kd) and dimer (84 kd) are apparent in
lanes 2 and 5, and lane 5, respectively.
[0472] FIGS. 61A-61C show TNF-alpha saturation binding analysis
with various TNR1B polypeptides on the Biacore T-100. (A) The TNR1B
symmetroadhesin of FIG. 44A (iv) was covalently coupled to a
Biacore CM-5 chip using standard Biacore amine chemistry. (B) TNR1B
immunoadhesin (R&D Systems) was covalently coupled to a Biacore
CM-5 chip using standard Biacore amine chemistry. (C) The TNR1B
symmetroadhesin of FIG. 44A (iv) was covalently coupled to a
Biacore CM-5 chip using standard Biacore thiol chemistry. Following
coupling, saturation binding analysis was carried out using
TNF-alpha (R&D Systems) at the indicated concentations.
[0473] FIGS. 62A-62C shows Scatchard analysis of the TNF-alpha
saturation binding analysis shown in FIG. 61A-61C. (A) TNR1B
symmetroadhesin of FIG. 44A (iv) covalently coupled using amine
chemistry; Kd=Kd=4.697.times.10.sup.-9 M. (B) TNR1B-immunoadhesin
(R&D Systems) covalently coupled using amine chemistry;
Kd=4.089.times.10.sup.-9 M. (C) TNR1B symmetroadhesin of FIG. 44A
(iv) covalently coupled using thiol chemistry;
Kd=0.8476.times.10.sup.-9 M.
[0474] The TNR.sub.1-X, TNR.sub.2-X, and TNR.sub.Fab-X proteins,
individually and in various combinations, are used to prepare
various VGFR symmetroadhesins (Tables 3, 4, and 5). The number of
distinct configurations obtained for each combination, as well as
the general structure for each, is also shown.
[0475] Hemi-symmetroadhesins are prepared using the TNR-X proteins
alone, immunosymmetroadhesins are prepared using the TNR-X proteins
with S-Fc, X-Fc, or Fc-X, and bi-symmetroadhesins are prepared
using the TNR-X proteins with S-Fc-X or X-Fc-X.
[0476] Each TNR-X protein or TNR symmetroadhesin is determined by a
quantitative immunoassay using subtype-specific, affinity-purified
polyclonal antibodies to TNR1A, TNR1B, and Di62 (goat-anti-mouse),
and immunoadhesins (TNR1A-Ig and TNR2A-Ig) as reference standards.
TNR antibodies, TNR-Ig immunoadhesins, and TNF-.alpha. protein are
obtained from R&D Systems (MN).
[0477] Binding of TNGR symmetroadhesins to TNF-.alpha. is studied
as described (Ashkenazi et al. (1991) Proc. Natl. Acad. Sci. 88,
10535-10539). Individual samples (1 microgram/ml) are immobilized
onto microtiter wells coated with goat-anti-human Ig Fc antibody.
Reactions with recombinant human .sup.125I-TNF-.alpha.
(radiodinated by using lactoperoxidase to a specific activity of
19.1 microCi/microgram, 1 microCi=37 kBq) are done in
phosphate-buffered saline (PBS) containing 1% bovine serum albumin
for 1 hour at 24.degree. C. Non-specific binding is determined by
omitting the sample. In competition binding analyses,
.sup.125I-TNF-.alpha. is incubated with immobilized samples in the
presence of increasing concentrations of unlabeled TNF-.alpha.. The
K.sub.d is determined from competition IC.sub.50 values according
to the following equation: K.sub.d=IC.sub.50/(1+[T]/K.sub.dT),
where [T] is the concentration of the tracer (0.1 nm) and K.sub.dT
is the K.sub.d of the tracer determined by saturation binding (80
.mu.M). TNF cytotoxity is studied as described (Kawade and Watanabe
(1984) J. Interferon Res. 4, 571-584). Mouse L-M cells are plated
in microtiter dishes (4.times.10.sup.4 cells per well) and treated
with actinomycin D (3 micrograms/ml) and TNF-.alpha. or TNF-.beta.
(1 nanogram/ml) in the presence or absence of the sample or other
inhibitors. After 20 hours of incubation at 39.degree. C., the cell
survival is determined by a crystal violet dye exclusion test.
[0478] A mouse model for septic shock is studied by endotoxin
injection of 6- to 8-week-old BALB/c mice. Animals are injected
intravenously (i.v.) with an LD.sub.100 dose of Salmonella
abortus-derived endotoxin (175 micrograms per mouse) in
phosphate-buffered saline (PBS), and survival is followed for at
least 78 hour. TNR1-immunoadhesin and CD4-immunoadhesin are used as
the positive and negative controls, respectively. Each is diluted
in PBS and injected i.v. prior to, or after, the administration of
endotoxin.
EXAMPLE 8
Vascular Endothelial Growth Factor Receptor Symmetroadhesins
[0479] A therapeutic strategy for treating angiogenic disease is
based upon vascular endothelial growth factors (VEGF) and their
binding interaction with VEGF receptors (VGFR). VEGF-antibodies and
VGFR-immunoadhesins are promising candidates for treatment in a
number of metastatic carcinomas including colon, rectum, lung, and
breast, and in age-related macular degeneration.
[0480] Accordingly, various VGFR symmetroadhesins are prepared
using VGFR-X proteins, and analyzed for their ability to bind VEGF
and to block VEGF biological activity. The activity of VGFR
symmetroadhesins is compared to that of VGRF immunoadhesins (Park
et al. (1994) J. Biol. Chem. 269, 25646-25654).
[0481] Human VGFR include VGFR1 (Genbank Acc. No. NM.sub.--002019);
VGFR2 (GenBank Acc. No. NM.sub.--002253); and VGFR3 (GenBank Acc.
No. NM.sub.--002020). VGFR1-X, VGFR2-X, and VGFR3-X are prepared
according to the method of EXAMPLE 3. Host cells are transfected
with expression vectors that encode:
1) a pre-VGFR1-intein chimeric polypeptide containing the VGFR1
extracellular domain joined at its C-terminus by a peptide bond to
the N-terminus of a self-splicing intein at the autocleavage site
(FIGS. 45A-45C); 2) a pre-VGFR2-intein chimeric polypeptide
containing the VGFR1 extracellular domain joined at its C-terminus
by a peptide bond to the N-terminus of a self-splicing intein at
the autocleavage site (FIGS. 46A-46C); and 3) a pre-VGFR1-intein
chimeric polypeptide containing the VGFR1 extracellular domain
joined at its C-terminus by a peptide bond to the N-terminus of a
self-splicing intein at the autocleavage site (FIGS. 47A-47C).
[0482] Cleavage of the VGFR signal sequence (part i) by the
cellular signal peptidase provides mature VGFR-intein fusion
proteins (part ii). Excision of the intein domain provides a
VGFR-thioester intermediate (part iii). Finally, native chemical
ligation of the VGFR-thioester with free cysteine and/or
selenocysteine is employed to prepare VGFR domains with
C-terminal-X-termini (part iv).
[0483] The sequences of these polypeptides are shown in SEQ ID NO:
166, SEQ ID NO:167, SEQ ID NO:168, and SEQ ID NO:169 (VGFR1); in
SEQ ID NO: 171, SEQ ID NO:172, SEQ ID NO:173, and SEQ ID NO:174
(VGFR2); and in SEQ ID NO: 176, SEQ ID NO:177, SEQ ID NO:178, and
SEQ ID NO:179 (VGFR3).
[0484] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pVGFR1-Mth is
constructed using SEQ ID NO:22 and SEQ ID NO:1, plasmid pVGR1-Mth
is constructed using SEQ ID NO:23 and SEQ ID NO:1, and plasmid
pVGR1-Mth is constructed using SEQ ID NO:24 and SEQ ID NO:1. For
unamplified expression, plasmid pVGFR1-Mth-DHFR is constructed
using SEQ ID NO:22 and pSVeCD4DHFR, plasmid pVGR1-Mth-DHFR is
constructed using SEQ ID NO:23 and pSVeCD4DHFR, and plasmid
pVGR1-Mth-DHFR is constructed using SEQ ID NO:24 and
pSVeCD4DHFR.
[0485] Suitable expression vectors are constructed by ligation of
an insert fragment prepared by digestion with Hind III and EagI and
a vector fragment prepared by digestion with Hind III and PspOM1.
Plasmid pCDNA3-VGFR1-Mth is constructed using SEQ ID NO:165 and
pCDNA3.1(+). Plasmid pCDNA3-VGFR2-Mth is constructed using SEQ ID
NO:170 and pCDNA3.1(+). Plasmid pCDNA3-VGFR3-Mth is constructed
using SEQ ID NO:175 and pCDNA3.1(+).
[0486] The VGFR1-X, VGFR2-X, and VEGFR3-X proteins, individually
and in various combinations, are used to prepare various VGFR
symmetroadhesins (Tables 6, 7, and 8). The number of distinct
configurations obtained for each combination, as well as the
general structure for each, is also shown.
[0487] Hemi-symmetroadhesins are prepared using the VGFR-X proteins
alone, immunosymmetroadhesins are prepared using the VGFR-X
proteins with S-Fc, X-Fc, or Fc-X, and bi-symmetroadhesins are
prepared using the VGFR-X proteins with S-Fc-X or X-Fc-X.
[0488] Each VGFR-X protein or VGFR symmetroadhesin is determined by
a quantitative immunoassay using subtype-specific,
affinity-purified polyclonal antibodies to VGFR1, VGFR2, and VGFR3,
and immunoadhesins (VGFR1-Ig, VGFR2-Ig, and VGFR3-Ig) as reference
standards. VGFR antibodies, VGFR-Ig immunoadhesins, and
VEGF.sub.165 protein are obtained from R&D Systems (MN).
[0489] Binding of VEGR symmetroadhesins to VEGF is studied using a
VEGF saturation binding assay. Reactions (0.1 ml) contain 10% fetal
bovine serum in PBS (buffer A) with .sup.125I-VEGF.sub.165
(<9000 cpm/well, 5.69.times.10.sup.7 cpm/microgram). Chloramine
T is used to iodinated VEGF.sub.165 as described (Keyt et al.
(1996) J. Biol Chem. 271, 5638-5646). Binding is carried out
overnight at 4.degree. C. in the presence or absence of 50
nanograms of unlabeled purified VEGF.sub.165. The bound
.sup.125I-VEGF.sub.165 is then determined by capture in 96-well
breakaway immunoabsorbent assay plates (Nunc). Plates are coated
overnight at 4.degree. C. with 2 micrograms/ml affinity-purified
goat anti-human Fc IgG (Organon-Teknika) in 50 mM Na.sub.2CO.sub.3,
pH 9.6, and preblocked for 1 hr in buffer A. Binding reactions are
then incubated in the coated wells for 4 hrs at room temperature,
followed by 4 washes with buffer A. The fraction of bound
.sup.125I-VEGF.sub.165 is determined in a gamma counter. Data is
analyzed using a 4-parameter non-linear curve fitting program
(Kalidagraph, Abelbeck Software, Pa.).
[0490] Binding of VEGF to VEGR symmetroadhesins is also studied
using a competition binding assay. ELISA plates are coated with 2
micrograms/ml rabbit F(ab').sub.2 to human IgG Fc (Jackson
ImmunoResearch, Pa.) and blocked with buffer A. KDR-IgG (3
nanograms/ml) in buffer A is added to the plate and incubated for 1
hr. Serially-diluted samples are incubated with 2 nM biotinylated
VEGF for 1 h in tubes. The reactions are then transferred to the
ELISA plates and incubated for 1 h. After washing, the fraction of
biotinylated VEGF bound to KDR-Ig is detected using horseradish
peroxidase-labeled streptavidin (Sigma, Mo.) followed by
3,3',5,5'-tetramethylbenzidine substrate. Data is analyzed using
4-parameter non-linear regression curve fitting analysis.
[0491] Endothelial cell growth inhibition studies are carried out
as described (Leung et al. (1989) Science 246, 1306-1309). Bovine
adrenal cortex capillary endothelial cells are cultured in the
presence of low glucose Dulbecco's modified Eagle's medium (GIBCO)
supplemented with 10% calf serum, and 2 mM glutamine (growth
medium). Cells are seeded at a density of 6.times.10.sup.3
cells/well in 6-well plates. Serially diluted samples are added to
the cells at concentrations between 1 to 5000 nanograms/ml and
incubated for 2 to 3 hr. Purified VEGF.sub.165 is added to a final
concentration of 3 nanograms/ml. Cells are then incubated for 5 to
6 days, removed from plates with trypsin, and cell number
determined in a Coulter counter (Coulter Electronics, FL). Data is
analyzed using 4-parameter non-linear regression curve fitting
analysis.
[0492] In vivo tumor studies are carried out as described (Kim et
al. (1993) Nature 362, 841-844; Borgstrom et al. (1996) Cancer Res.
56, 4032-4039). Human A673 rhabdomyosarcoma cells (ATCC CRL-1598)
are cultured in DMEM/F12 supplemented with 10% fetal bovine serum,
and 2 mM glutamine. Female BALB/c nude mice, 6 to 10 weeks old, are
injected subcutaneously with 2.times.10.sup.6 tumor cells in the
dorsal area in a volume of 200 microliters. Following tumor cell
inoculation (24 hr.), animals (10 per group) are treated with
serially diluted samples at dose of 0.05 mg/kg, 0.5 mg/kg, and 5
mg/kg, administered twice weekly intraperitoneally in a volume of
0.1 ml. Tumor size is determined at weekly intervals. Four weeks
after tumor cell inoculation, animals are euthanized and the tumors
removed and weighed. Statistical analysis is carried out by
ANOVA.
EXAMPLE 9
ErbB Syetroadhesins
[0493] A therapeutic strategy for treating malignant disease is
based upon epidermal growth factor-like receptors (ErbB) and their
ligands, including the neuregulins/heregulins (NRG/HRG), and the
family of EGF-related protein ligands. ErbB-antibodies and
ErbB-immunoadhesins are under clinical investigation, and are
well-proven in treating metastatic breast cancers overexpressing
ErbB2.
[0494] Accordingly, various ErbB symmetroadhesins are prepared
using ErbB-X proteins, and analyzed for their ability to bind
heregulins and block heregulin biological activity. The activity of
ErbB symmetroadhesins is compared to that of ErbB immunoadhesins
(Sliwkowski et al. (1994) J. Biol. Chem. 269, 14661-14665).
[0495] Human ErbB include ErbB1 (Genbank Acc. No. NM.sub.--005228);
ErbB2 (GenBank Acc. No. NM.sub.--004448); ErbB3 (GenBank Acc. No.
NM.sub.--001982); and ErbB4 (GenBank Acc. No. NM.sub.--005235).
ErbB1-X, ErbB2-X, ErbB3-X, and ErbB4-X are prepared according to
the method of EXAMPLE 3. Host cells are transfected with expression
vectors that encode:
1) a pre-ErbB1-intein chimeric polypeptide containing the ErbB1
extracellular domain joined at its C-terminus by a peptide bond to
the N-terminus of a self-splicing intein at the autocleavage site
(FIGS. 48A-48B); 2) a pre-ErbB2-intein chimeric polypeptide
containing the ErbB2 extracellular domain joined at its C-terminus
by a peptide bond to the N-terminus of a self-splicing intein at
the autocleavage site (FIGS. 49A-49B); 3) a pre-ErbB3-intein
chimeric polypeptide containing the ErbB3 extracellular domain
joined at its C-terminus by a peptide bond to the N-terminus of a
self-splicing intein at the autocleavage site (FIGS. 50A-50B); and
4) a pre-ErbB4-intein chimeric polypeptide containing the ErbB4
extracellular domain joined at its C-terminus by a peptide bond to
the N-terminus of a self-splicing intein at the autocleavage site
(FIGS. 51A-50B).
[0496] Cleavage of the homologous ErbB signal sequence (part i) by
the cellular signal peptidase provides mature ErbB-intein fusion
proteins (part ii). Excision of the intein domain by protein
splicing provides a ErbB-thioester intermediate (part iii).
Finally, native chemical ligation of the ErbB-thioester with free
cysteine and/or selenocysteine is employed to prepare VEGR domains
with C-terminal-X-termini (part iv).
[0497] The sequences of these polypeptides are shown in SEQ ID NO:
181, SEQ ID NO:182, SEQ ID NO:183, and SEQ ID NO:184 (ERBB1); in
SEQ ID NO: 186, SEQ ID NO:187, SEQ ID NO:188, and SEQ ID NO:189
(ERBB2); in SEQ ID NO: 191, SEQ ID NO:192, SEQ ID NO:193, and SEQ
ID NO:194 (ERBB3); and in SEQ ID NO: 196, SEQ ID NO:197, SEQ ID
NO:198, and SEQ ID NO:199 (ERBB4).
[0498] Suitable expression vectors are constructed by ligation of
an insert and vector fragment prepared by digestion with EcoRI and
BglII. For unamplified expression, plasmid pErbB1-Mth is
constructed using SEQ ID NO:25 and SEQ ID NO:1, plasmid pErbB2-Mth
is constructed using SEQ ID NO:26 and SEQ ID NO:1, plasmid
pErbB3-Mth is constructed using SEQ ID NO:27 and SEQ ID NO:1, and
plamid pErbB4-Mth is constructed using SEQ ID NO:28 and SEQ ID
NO:1. For amplified expression, plasmid pErbB1-Mth-DHFR is
constructed using SEQ ID NO:25 and pSVeCD4DHFR, plasmid
pErbB2-Mth-DHFR is constructed using SEQ ID NO:26 and pSVeCD4DHFR,
plasmid pErbB3-Mth-DHFR is constructed using SEQ ID NO:27 and
pSVeCD4DHFR, and plamid pErbB4-Mth-DHFR is constructed using SEQ ID
NO:28 and pSVeCD4DHFR.
[0499] Suitable expression vectors are constructed by ligation of
an insert fragment prepared by digestion with Hind III and EagI and
a vector fragment prepared by digestion with Hind III and PspOM1.
Plasmid pCDNA3-ERBB1-Mth is constructed using SEQ ID NO:180 and
pCDNA3.1(+). Plasmid pCDNA3-ERBB2-Mth is constructed using SEQ ID
NO:185 and pCDNA3.1(+). Plasmid pCDNA3-ERBB3-Mth is constructed
using SEQ ID NO:190 and pCDNA3.1(+). Plasmid pCDNA3-ERBB4-Mth is
constructed using SEQ ID NO:195 and pCDNA3.1(+).
[0500] The ErbB1-X, ErbB2-X, ErbB3-X and ErbB4-X proteins,
individually and in various combinations, are used to prepare
various VGFR symmetroadhesins (Tables 9, 10, 11). The number of
distinct configurations obtained for each combination, as well as
the general structure for each, is also shown.
[0501] Hemi-symmetroadhesins are prepared using the ErbB-X proteins
alone, immunosymmetroadhesins are prepared using the ErbB-X
proteins with S-Fc, X-Fc, or Fc-X, and bi-symmetroadhesins are
prepared using the ErbB-X proteins with S-Fc-X or X-Fc-X.
[0502] Each ErbB-X protein or ErbB symmetroadhesin is determined by
a quantitative immunoassay using subtype-specific,
affinity-purified polyclonal antibodies to ErbB1, ErbB2, ErbB3, and
ErbB4, and immunoadhesins (ErbB1-Ig, ErbB2-Ig, ErbB3-Ig, and
ErbB4-Ig) as reference standards. ErbB antibodies, ErbB-Ig
immunoadhesins, NRG1-.alpha..sub.177-241 protein,
NRG1-.beta..sub.176-246 protein, NRG1-.beta..sub.1-246 protein, and
NRG1-SMDF.sub.1-296 protein are obtained from R&D Systems
(MN).
[0503] Binding of ErbB symmetroadhesins to neuregulins is studied
using a HRG saturation binding assay (Sliwkowski et al. (1994) J.
Biol. Chem. 269, 14661-14665). Reactions are performed in Nunc
breakapart immuno-module plates. Plate wells are coated at
4.degree. C. overnight with 100 microliters of 5 micrograms/ml
goat-anti-human antibody (Boehringer Mannheim) in 50 mM carbonate
buffer (pH 9.6). Plates are rinsed twice with 200 microliters wash
buffer (PBS/0.05% Tween-20.TM.) followed by a brief incubation with
100 microliters 1% BSA/PBS for 30 min at room temperature. Buffer
is removed and each well is incubated with 100 microliters of the
sample in 1% BSA/PBS under vigorous side-to-side rotation for 1
hour. Plates are rinsed three times with wash buffer and
competitive binding is carried out by adding various amounts of
cold competitor gamma-HRG and .sup.125I-HRG.beta.1 and incubating
at room temperature for 2 to 3 hours with vigorous side-to-side
rotation. Wells are quickly rinsed three times with wash buffer,
drained and individual wells are counted in a gamma-counter.
Scatchard analysis is performed using a modified Ligand program
(Munson, P. and Robard, D. (1980) Analytical Biochemistry
107:220-239).
[0504] The ability of ErbB symmetroadhesins to inhibit
HRG-dependent proliferation is studied in the MCF7 breast carcinoma
cell line (Lewis et al. (1996) Cancer Res. 56, 1457-1465).
.sup.3H-thymidine incorporation is are carried out in 96-well
format. Serum-starved MCF7-7 cells are plated at 10,000 cells/well
in 50:50 F12/DMEM (high glucose). Varying concentrations of sample
are incubated with 1 nM HRG and added to the cells. After a 15 hour
incubation, cells are labeled with .sup.3H-thymidine to measure DNA
synthesis (20 mL of 1/20 diluted tritiated thymidine stock:
Amersham TRA 120 B363, 1 mCi/ml). Cells are then harvested onto
GF/C unifilters (96 well format) using a Packard Filtermate 196
harvester. Filters are counted using a Packard Topcount
apparatus.
Screening
[0505] This invention is particularly useful for screening
compounds by using consecutive amino acid sequences/or compounds
comprising such in any of a variety of drug screening techniques.
The compound employed in such a test may either be free in
solution, affixed to a solid support, borne on a cell surface, or
located intracellularly. One method of drug screening utilizes
eukaryotic or prokaryotic host cells which are stably transformed
with recombinant nucleic acids expressing the compound. Drugs are
screened against such transformed cells in competitive binding
assays. Such cells, either in viable or fixed form, can be used for
standard binding assays. One may measure, for example, the
formation of complexes between compound or a fragment and the agent
being tested. Alternatively, one can examine the diminution in
complex formation between the compound and its target cell or
target receptors caused by the agent being tested.
[0506] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a disease or
disorder associated with a stretch of consecutive amino acids of
the compound. These methods comprise contacting such an agent with
the compound or fragment thereof and assaying (I) for the presence
of a complex between the agent and the compound or fragment
thereof, or (ii) for the presence of a complex between the compound
or fragment thereof and the cell, by methods well known in the art.
In such competitive binding assays, the compound or fragment
thereof is typically labeled. After suitable incubation, free
compound or fragment thereof is separated from that present in
bound form, and the amount of free or uncomplexed label is a
measure of the ability of the particular agent to bind to the
compound or fragment thereof or to interfere with the compound/cell
complex.
[0507] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on Sep. 13, 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to the instant compound or fragment thereof, the peptide test
compounds are reacted with compound or fragment thereof and washed.
Bound compound or fragment thereof is detected by methods well
known in the art. Purified compound or fragment thereof can also be
coated directly onto plates for use in the aforementioned drug
screening techniques. In addition, non-neutralizing antibodies can
be used to capture the peptide and immobilize it on the solid
support.
[0508] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding compound or fragment thereof specifically compete with a
test compound for binding to compound or fragment thereof. In this
manner, the antibodies can be used to detect the presence of any
peptide which shares one or more antigenic determinants with the
stretch(es) of consecutive amino acids of the compound.
Rational Drug Design
[0509] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a
compound of the invention, or an amino acid sequence of a compound
of the invention) or of small molecules with which they interact,
e.g., agonists, antagonists, or inhibitors. Any of these examples
can be used to fashion drugs which are more active or stable forms
of the a compound of the invention, or an amino acid sequence of a
compound of the invention or which enhance or interfere with the
function of the a compound of the invention, or an amino acid
sequence of a compound of the invention in vivo (cf., Hodgson,
Bio/Technology, 9:19-21 (1991)).
[0510] In one approach, the three-dimensional structure of a
compound of the invention, or an amino acid sequence of a compound
of the invention, or of a compound-inhibitor complex, is determined
by x-ray crystallography, by computer modeling or, most typically,
by a combination of the two approaches. Both the shape and charges
of a compound of the invention, or an amino acid sequence of a
compound of the invention, must be ascertained to elucidate the
structure and to determine active site(s). Relevant structural
information is used to then design analogous molecules or to
identify efficient inhibitors. Useful examples of rational drug
design may include molecules which have improved activity or
stability as shown by Braxton and Wells, Biochemistry, 31:7796-7801
(1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by Athauda et al., J. Biochem.,
113:742-746 (1993).
[0511] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The anti-id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides would then
act as the pharmacore.
[0512] By virtue of the present invention, sufficient amounts of
the compounds of the invention, or an amino acid sequence of a
compound of the invention may be made available to perform such
analytical studies as X-ray crystallography.
Assays of Biological Activity
[0513] The compounds disclosed herein, including components thereof
such as a stretch of consecutive amino acids of a compound, are
readily assayed using one or more standard assays of biological
activity known to those in the art. The following are non-limiting
examples of such assays:
Ability of the Compounds to Inhibit Vascular Endothelial Growth
Factor (VEGF) Stimulated Proliferation of Endothelial Cell
Growth
[0514] The ability of the various compounds of this invention to
inhibit VEGF stimulated proliferation of endothelial cells was
tested. Positive testing in this assay indicates the compound is
useful for inhibiting endothelial cell growth in mammals where such
an effect would be beneficial, e.g., for inhibiting tumor
growth.
[0515] In a specific example of the assay, bovine adrenal cortical
capillary endothelial cells (ACE) (from primary culture, maximum of
12-14 passages) are plated in 96-well plates at 500 cells/well per
100 microliter. Assay media includes low glucose DMEM, 10% calf
serum, 2 mM glutamine, and 1.times.
penicillin/streptomycin/fungizone. Control wells include the
following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE
cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml VEGF; (5) ACE
cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and (6) ACE cells
plus 3 ng/ml VEGF plus 5 ng/ml LIF. The test samples, poly-his
tagged compounds (in 100 microliter volumes), are then added to the
wells (at dilutions of 1%, 0.1% and 0.01%, respectively). The cell
cultures are incubated for 6-7 days at 37.degree. C./5% CO.sub.2
After the incubation, the media in the wells is aspirated, the
cells are washed 1.times. with PBS. An acid phosphatase reaction
mixture (100 microliter; 0.1M sodium acetate, pH 5.5, 0.1% Triton
X-100, 10 mM p-nitrophenyl phosphate) is then added to each well.
After a 2 hour incubation at 37.degree. C., the reaction is stopped
by addition of 10 microliters 1M NaOH. Optical density (OD) is
measured on a microplate reader at 405 nm.
[0516] The activity of the assayed compound is calculated as the
percent inhibition of VEGF (3 ng/ml) stimulated proliferation (as
determined by measuring acid phosphatase activity at OD 405 nm)
relative to the cells without stimulation. TGF-beta can be employed
as an activity reference at 1 ng/ml, since TGF-beta blocks 70-90%
of VEGF-stimulated ACE cell proliferation. The results are
indicative of the utility of the assayed compound in cancer therapy
and specifically in inhibiting tumor angiogenesis. Numerical values
(relative inhibition) are determined by calculating the percent
inhibition of VEGF stimulated proliferation by the assayed compound
relative to cells without stimulation and then dividing that
percentage into the percent inhibition obtained by TGF-beta at 1
ng/ml which is known to block 70-90% of VEGF stimulated cell
proliferation. The results are considered positive if the assayed
compound exhibits 30% or greater inhibition of VEGF stimulation of
endothelial cell growth (relative inhibition 30% or greater).
Retinal Neuron Survival
[0517] This assay can demonstrate if the compound tested has
efficacy in enhancing the survival of retinal neuron cells and,
therefore, is useful for the therapeutic treatment of retinal
disorders or injuries including, for example, treating sight loss
in mammals due to retinitis pigmentosum, AMD, etc.
[0518] Sprague Dawley rat pups at postnatal day 7 (mixed
population: glia and retinal neuronal types) are killed by
decapitation following CO.sub.2 anesthesia and the eyes are removed
under sterile conditions. The neural retina is dissected away from
the pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N.sub.2
and with or without the specific test compound. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere of 5% CO.sub.2. After 2-3 days in culture, cells are
stained with calcein AM then fixed using 4% paraformaldehyde and
stained with DAPI for determination of total cell count. The total
cells (fluorescent) are quantified at 20.times. objective
magnification using CCD camera and NIH image software for
MacIntosh. Fields in the well are chosen at random.
[0519] The effect of various concentration of the tested compound
are reported herein where percent survival is calculated by
dividing the total number of calcein AM positive cells at 2-3 days
in culture by the total number of DAPI-labeled cells at 2-3 days in
culture. Anything above 30% survival is considered positive.
Rod Photoreceptor Cell Survival
[0520] This assay is used to show whether certain compounds of the
invention act to enhance the survival/proliferation of rod
photoreceptor cells and, therefore, are useful for the therapeutic
treatment of retinal disorders or injuries including, for example,
treating sight loss in mammals due to retinitis pigmentosum, AMD,
etc. Sprague Dawley rat pups at 7 day postnatal (mixed population:
glia and retinal neuronal cell types) are killed by decapitation
following CO.sub.2 anesthesis and the eyes are removed under
sterile conditions. The neural retina is dissected away form the
pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N.sub.2.
Cells for all experiments are grown at 37.degree. C. in a water
saturated atmosphere of 5% CO.sub.2. After 2-3 days in culture,
cells are fixed using 4% paraformaldehyde, and then stained using
CellTracker Green CMFDA. Rho 4D2 (ascites or IgG 1:100), a
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated as % survival: total
number of calcein-rhodopsin positive cells at 2-3 days in culture,
divided by the total number of rhodopsin positive cells at time 2-3
days in culture. The total cells (fluorescent) are quantified at
20.times. objective magnification using a CCD camera and NIH image
software for MacIntosh. Fields in the well are chosen at
random.
Induction of Endothelial Cell Apoptosis
[0521] The ability of the compounds disclosed herein to induce
apoptosis in endothelial cells can be tested in human venous
umbilical vein endothelial cells (HUVEC, Cell Systems). A positive
test in the assay is indicative of the usefulness of the compound
in therapeutically treating tumors as well as vascular disorders
where inducing apoptosis of endothelial cells would be
beneficial.
[0522] The cells are plated on 96-well microtiter plates (Amersham
Life Science, cytostar-T scintillating microplate, RPNQ160,
sterile, tissue-culture treated, individually wrapped), in 10%
serum (CSG-medium, Cell Systems), at a density of 2.times.10.sup.4
cells per well in a total volume of 100 .mu.l. On day 2, test
samples containing the tested compound are added in triplicate at
dilutions of 1%, 0.33% and 0.11%. Wells without cells were used as
a blank and wells with cells only are used as a negative control.
As a positive control 1:3 serial dilutions of 50 .mu.l of a
3.times. stock of staurosporine were used. The ability of the
compound to induce apoptosis is determined by processing of the 96
well plates for detection of Annexin V, a member of the calcium and
phospholipid binding proteins, to detect apoptosis.
[0523] 0.2 ml Annexin V-Biotin stock solution (100 .mu.g/ml) was
diluted in 4.6 ml 2.times. Ca.sup.2+ binding buffer and 2.5% BSA
(1:25 dilution). 50 .mu.l of the diluted Annexin V-Biotin solution
was added to each well (except controls) to a final concentration
of 1.0 .mu.g/ml. The samples were incubated for 10-15 minutes with
Annexin-Biotin prior to direct addition of .sup.35S-Streptavidin.
.sup.35S-Streptavidin was diluted in 2.times. Ca.sup.2+ Binding
buffer, 2.5% BSA and was added to all wells at a final
concentration of 3.times.10.sup.4 cpm/well. The plates were then
sealed, centrifuged at 1000 rpm for 15 minutes and placed on
orbital shaker for 2 hours. The analysis was performed on a 1450
Microbeta Trilux (Wallac). Percent above background represents the
percentage amount of counts per minute above the negative controls.
Percents greater than or equal to 30% above background are
considered positive.
PDB12 Cell Inhibition
[0524] This assay will demonstrates if the compounds disclosed
herein have efficacy in inhibiting protein production by PDB12
pancreatic ductal cells and are, therefore, useful in the
therapeutic treatment of disorders which involve protein secretion
by the pancreas, including diabetes, and the like.
[0525] PDB12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
.mu.L/180 .mu.L of growth media. 100 .mu.L of growth media with the
compound test sample or negative control lacking the compound is
then added to well, for a final volume of 200 .mu.L. Controls
contain growth medium containing a protein shown to be inactive in
this assay. Cells are incubated for 4 days at 37.degree. C. 20
.mu.L of Alamar Blue Dye (AB) is then added to each well and the
flourescent reading is measured at 4 hours post addition of AB, on
a microtiter plate reader at 530 nm excitation and 590 nm emission.
The standard employed is cells without Bovine Pituitary Extract
(BPE) and with various concentrations of BPE. Buffer or CM controls
from unknowns are run 2 times on each 96 well plate.
[0526] These assays allow one to calculate a percent decrease in
protein production by comparing the Alamar Blue Dye calculated
protein concentration produced by the compound-treated cells with
the Alamar Blue Dye calculated protein concentration produced by
the negative control cells. A percent decrease in protein
production of greater than or equal to 25% as compared to the
negative control cells is considered positive.
Stimulation of Adult Heart Hypertrophy
[0527] This assay is designed to measure the ability of the various
compounds disclosed herein to stimulate hypertrophy of adult heart.
A positive test in this assay indicates that the compound would be
expected to be useful for the therapeutic treatment of various
cardiac insufficiency disorders.
[0528] Ventricular myocytes freshly isolated from adult (250 g)
Sprague Dawley rats are plated at 2000 cell/well in 180 .mu.l
volume. Cells are isolated and plated on day 1, the
compound-containing test samples or growth medium only (negative
control) (20 .mu.l volume) is added on day 2 and the cells are then
fixed and stained on day 5. After staining, cell size is visualized
wherein cells showing no growth enhancement as compared to control
cells are given a value of 0.0, cells showing small to moderate
growth enhancement as compared to control cells are given a value
of 1.0 and cells showing large growth enhancement as compared to
control cells are given a value of 2.0. Any degree of growth
enhancement as compared to the negative control cells is considered
positive for the assay.
PDB12 Cell Proliferation
[0529] This assay demonstrates whether the various compounds
disclosed herein have efficacy in inducing proliferation of PDB12
pancreatic ductal cells and are, therefore, useful in the
therapeutic treatment of disorders which involve protein secretion
by the pancreas, including diabetes, and the like.
[0530] PDB12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
.mu.L/180 .mu.L of growth media. 100 .mu.L of growth media with the
compound test sample or negative control lacking the compound
tested is then added to well, for a final volume of 200 .mu.L.
Controls contain growth medium containing a protein shown to be
inactive in this assay. Cells are incubated for 4 days at
37.degree. C. 20 .mu.L of Alamar Blue Dye (AB) is then added to
each well and the flourescent reading is measured at 4 hours post
addition of AB, on a microtiter plate reader at 530 nm excitation
and 590 nm emission. The standard employed is cells without Bovine
Pituitary Extract (BPE) and with various concentrations of BPE.
Buffer or growth medium only controls from unknowns are run 2 times
on each 96 well plate.
[0531] Percent increase in protein production is calculated by
comparing the Alamar Blue Dye calculated protein concentration
produced by the test compound-treated cells with the Alamar Blue
Dye calculated protein concentration produced by the negative
control cells. A percent increase in protein production of greater
than or equal to 25% as compared to the negative control cells is
considered positive.
Enhancement of Heart Neonatal Hypertrophy
[0532] This assay is designed to measure the ability of the
compounds disclosed herein to stimulate hypertrophy of neonatal
heart. Testing positive in this assay indicates the compounds to be
useful for the therapeutic treatment of various cardiac
insufficiency disorders.
[0533] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
are obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml, serum
<0.1%, freshly isolated) are added on day 1 to 96-well plates
previously coated with DMEM/F12+4% FCS. Test samples containing the
test compound or growth medium only (negative control) (201/well)
are added directly to the wells on day 1. PGF (20 .mu.l/well) is
then added on day 2 at final concentration of 10.sup.-6 M. The
cells are then stained on day 4 and visually scored on day 5,
wherein cells showing no increase in size as compared to negative
controls are scored 0.0, cells showing a small to moderate increase
in size as compared to negative controls are scored 1.0 and cells
showing a large increase in size as compared to negative controls
are scored 2.0. A positive result in the assay is a score of 1.0 or
greater.
Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay
[0534] This assay is used to determine if the compounds disclosed
herein are active as a stimulator of the proliferation of
stimulated T-lymphocytes. Compounds which stimulate proliferation
of lymphocytes are useful therapeutically where enhancement of an
immune response is beneficial. A therapeutic agent may take the
form of antagonists of the compounds of the invention, for example,
murine-human chimeric, humanized or human antibodies against the
compound.
[0535] The basic protocol for this assay is described in Current
Protocols in Immunology, unit 3.12; edited by J E Coligan, A M
Kruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes
of Health, Published by John Wiley & Sons, Inc.
[0536] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[0537] The assay is prepared by plating in triplicate wells a
mixture of:
[0538] 100:1 of test sample diluted to 1% or to 0.1%, 50:1 of
irradiated stimulator cells, and 50:1 of responder PBMC cells.
[0539] 100 microliters of cell culture media or 100 microliter of
CD4-IgG is used as the control. The wells are then incubated at
37.degree. C., 5% CO.sub.2 for 4 days. On day 5, each well is
pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6
hours the cells are washed 3 times and then the uptake of the label
is evaluated.
[0540] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[0541] Positive increases over control are considered positive with
increases of greater than or equal to 180% being preferred.
However, any value greater than control indicates a stimulatory
effect for the test protein.
Pericyte c-Fos Induction
[0542] This assay shows the ability of the compounds disclosed
herein of the invention act to induce the expression of c-fos in
pericyte cells and, therefore, their use not only as diagnostic
markers for particular types of pericyte-associated tumors but also
for giving rise to antagonists which would be expected to be useful
for the therapeutic treatment of pericyte-associated tumors.
Specifically, on day 1, pericytes are received from VEC
Technologies and all but 5 ml of media is removed from flask. On
day 2, the pericytes are trypsinized, washed, spun and then plated
onto 96 well plates. On day 7, the media is removed and the
pericytes are treated with 100 .mu.l of test compound samples and
controls (positive control=DME+5% serum +/-PDGF at 500 ng/ml;
negative control=protein 32). Replicates are averaged and SD/CV are
determined. Fold increase over Protein 32 (buffer control) value
indicated by chemiluminescence units (RLU) luminometer reading
verses frequency is plotted on a histogram. Two-fold above Protein
32 value is considered positive for the assay. ASY Matrix: Growth
media=low glucose DMEM=20% FBS+1.times. pen/strep+1.times.
fungizone. Assay Media=low glucose DMEM+5% FBS.
Ability of the Compounds of the Invention to Stimulate the Release
of Proteoglycans from Cartilage
[0543] The ability of the compounds disclosed herein to stimulate
the release of proteoglycans from cartilage tissue can be tested as
follows.
[0544] The metacarphophalangeal joint of 4-6 month old pigs is
aseptically dissected, and articular cartilage was removed by free
hand slicing being careful to avoid the underlying bone. The
cartilage was minced and cultured in bulk for 24 hours in a
humidified atmosphere of 95% air, 5% CO.sub.2 in serum free (SF)
media (DME/F12 1:1) with 0.1% BSA and 100 U/ml penicillin and 100
.mu.g/ml streptomycin. After washing three times, approximately 100
mg of articular cartilage is aliquoted into micronics tubes and
incubated for an additional 24 hours in the above SF media. The
compound is then added at 1% either alone or in combination with 18
ng/ml interleukin-1 alpha, a known stimulator of proteoglycan
release from cartilage tissue. The supernatant is then harvested
and assayed for the amount of proteoglycans using the
1,9-dimethyl-methylene blue (DMB) calorimetric assay (Farndale and
Buttle, Biochem. Biophys. Acta 883:173-177 (1985)). A positive
result in this assay indicates that the test compound will find
use, for example, in the treatment of sports-related joint
problems, articular cartilage defects, osteoarthritis or rheumatoid
arthritis.
Skin Vascular Permeability Assay
[0545] This assay is used to test whether compounds of the
invention stimulate an immune response and induce inflammation by
inducing mononuclear cell, eosinophil and PMN infiltration at the
site of injection of the animal. Compounds which stimulate an
immune response are useful therapeutically where stimulation of an
immune response is beneficial. This skin vascular permeability
assay is conducted as follows. Hairless guinea pigs weighing 350
grams or more are anesthetized with ketamine (75-80 mg/Kg) and 5
mg/Kg xylazine intramuscularly (IM). A sample of purified compound
of the invention or a conditioned media test sample is injected
intradermally onto the backs of the test animals with 100 .mu.l per
injection site. It is possible to have about 10-30, preferably
about 16-24, injection sites per animal. One .mu.l of Evans blue
dye (1% in physiologic buffered saline) is injected intracardially.
Blemishes at the injection sites are then measured (mm diameter) at
1 hr and 6 hr post injection. Animals were sacrificed at 6 hrs
after injection. Each skin injection site is biopsied and fixed in
formalin. The skins are then prepared for histopathologic
evaluation. Each site is evaluated for inflammatory cell
infiltration into the skin. Sites with visible inflammatory cell
inflammation are scored as positive. Inflammatory cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic. At least a
minimal perivascular infiltrate at the injection site is scored as
positive, no infiltrate at the site of injection is scored as
negative.
Enhancement of Heart Neonatal Hypertrophy Induced by F2a
[0546] This assay is designed to measure the ability of compounds
disclosed herein to stimulate hypertrophy of neonatal heart, a
positive test indicating usefulness for the therapeutic treatment
of various cardiac insufficiency disorders.
[0547] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
were obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml, serum
<0.1%, freshly isolated) are added on day 1 to 96-well plates
previously coated with DMEM/F12+4% FCS. Test samples containing the
test compound (20 .mu.l/well) are added directly to the wells on
day 1. PGF (20 .mu.l/well) is then added on day 2 at a final
concentration of 10.sup.-6 M. The cells are then stain on day 4 and
visually scored on day .about.5. Visual scores are based on cell
size, wherein cells showing no increase in size as compared to
negative controls are scored 0.0, cells showing a small to moderate
increase in size as compared to negative controls are scored 1.0
and cells showing a large increase in size as compared to negative
controls are scored 2.0. A score of 1.0 or greater is considered
positive.
[0548] No PBS is included, since calcium concentration is critical
for assay response. Plates are coated with DMEM/F12 plus 4% FCS
(200 .mu.l/well). Assay media included: DMEM/F12 (with 2.44 gm
bicarbonate), .mu.g/ml transferrin, 1 .mu.g/ml insulin, 1 .mu.g/ml
aprotinin, 2 mmol/L glutamine, 100 U/ml penicillin G, 100 .mu.g/ml
streptomycin. Protein buffer containing mannitol (4%) gave a
positive signal (score 3.5) at 1/10 (0.4%) and 1/100 (0.04%), but
not at 1/1000 (0.004%). Therefore the test sample buffer containing
mannitol is not run.
Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay
[0549] This example shows that one or more of the compound of the
invention are active as inhibitors of the proliferation of
stimulated T-lymphocytes. Compounds which inhibit proliferation of
lymphocytes are useful therapeutically where suppression of an
immune response is beneficial.
[0550] The basic protocol for this assay is described in Current
Protocols in Immunology, unit 3.12; edited by J E Coligan, A M
Kruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes
of Health, Published by John Wiley & Sons, Inc.
[0551] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[0552] The assay is prepared by plating in triplicate wells a
mixture of:
100:1 of test sample diluted to 1% or to 0.1%, 50:1 of irradiated
stimulator cells, and 50:1 of responder PBMC cells.
[0553] 100 microliters of cell culture media or 100 microliter of
CD4-IgG is used as the control. The wells are then incubated at
37.degree. C., 5% CO.sub.2 for 4 days. On day 5, each well is
pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6
hours the cells are washed 3 times and then the uptake of the label
is evaluated.
[0554] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[0555] Any decreases below control is considered to be a positive
result for an inhibitory compound, with decreases of less than or
equal to 80% being preferred. However, any value less than control
indicates an inhibitory effect for the test protein.
Induction of Endothelial Cell Apoptosis (ELISA)
[0556] The ability of the compounds disclosed herein to induce
apoptosis in endothelial cells can be tested in human venous
umbilical vein endothelial cells (HUVEC, Cell Systems) using a
96-well format, in 0% serum media supplemented with 100 ng/ml VEGF,
0.1% BSA, 1.times. pen/strep.
[0557] A positive result in this assay indicates the usefulness of
the compound for therapeutically treating any of a variety of
conditions associated with undesired endothelial cell growth
including, for example, the inhibition of tumor growth. Coating of
96 well plates can be prepared by allowing gelatinization to occur
for >30 minutes with 100 .mu.l of 0.2% gelatin in PBS solution.
The gelatin mix is aspirated thoroughly before plating HUVEC cells
at a final concentration of 2.times.10.sup.4 cells/ml in 10% serum
containing medium--100 .mu.l volume per well. The cells were grown
for 24 hours before adding test samples containing the compound of
interest.
[0558] To all wells, 100 .mu.l of 0% serum media (Cell Systems)
complemented with 100 ng/ml VEGF, 0.1% BSA, 1.times. pen/strep is
added. Test samples containing the test compound were added in
triplicate at dilutions of 1%, 0.33% and 0.11%. Wells without cells
were used as a blank and wells with cells only are used as a
negative control. As a positive control, 1:3 serial dilutions of 50
.mu.l of a 3.times. stock of staurosporine are used. The cells were
incubated for 24 to 35 hours prior to ELISA.
[0559] ELISA is used to determine levels of apoptosis preparing
solutions according to the Boehringer Manual [Boehringer, Cell
Death Detection ELISA plus, Cat No. 1 920 685]. Sample
preparations: 96 well plates are spun down at 1 krpm for 10 minutes
(200 g); the supernatant is removed by fast inversion, placing the
plate upside down on a paper towel to remove residual liquid. To
each well, 200 .mu.l of 1.times. Lysis buffer is added and
incubation allowed at room temperature for 30 minutes without
shaking. The plates were spun down for 10 minutes at 1 krpm, and 20
.mu.l of the lysate (cytoplasmic fraction) is transferred into
streptavidin coated MTP. 80 .mu.l of immunoreagent mix was added to
the 20 .mu.l lystate in each well. The MTP was covered with
adhesive foil and incubated at room temperature for 2 hours by
placing it on an orbital shaker (200 rpm). After two hours, the
supernatant was removed by suction and the wells rinsed three times
with 250 .mu.l of 1.times. incubation buffer per well (removed by
suction). Substrate solution was added (100 .mu.l) into each well
and incubated on an orbital shaker at room temperature at 250 rpm
until color development was sufficient for a photometric analysis
(approx. after 10-20 minutes). A 96 well reader was used to read
the plates at 405 nm, reference wavelength, 492 nm. The levels
obtained for PIN 32 (control buffer) was set to 100%. Samples with
levels >130% were considered positive for induction of
apoptosis.
Human Venous Endothelial Cell Calcium Flux Assay
[0560] This assay is designed to determine whether compounds of the
present invention show the ability to stimulate calcium flux in
human umbilical vein endothelial cells (HUVEC, Cell Systems).
Calcium influx is a well documented response upon binding of
certain ligands to their receptors. A test compound that results in
a positive response in the present calcium influx assay can be said
to bind to a specific receptor and activate a biological signaling
pathway in human endothelial cells. This could ultimately lead, for
example, to endothelial cell division, inhibition of endothelial
cell proliferation, endothelial tube formation, cell migration,
apoptosis, etc.
[0561] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50:50 without glycine, 1% glutamine, 10
mM Hepes, 10% FBS, 10 ng/ml bFGF), are plated on 96-well microtiter
ViewPlates-96 (Packard Instrument Company Part #6005182) microtiter
plates at a cell density of 2.times.10.sup.4 cells/well. The day
after plating, the cells are washed three times with buffer (HBSS
plus 10 mM Hepes), leaving 100 .mu.l/well. Then 100 .mu.l/well of 8
.mu.M Fluo-3 (2.times. is added. The cells are incubated for 1.5
hours at 37.degree. C./5% CO.sub.2. After incubation, the cells are
then washed 3.times. with buffer (described above) leaving 100
.mu.l/well. Test samples of the compound are prepared on different
96-well plates at 5.times. concentration in buffer. The positive
control corresponded to 50 .mu.M ionomycin (5.times.); the negative
control corresponded to Protein 32. Cell plate and sample plates
are run on a FLIPR (Molecular Devices) machine. The FLIPR machine
added 25 .mu.l of test sample to the cells, and readings are taken
every second for one minute, then every 3 seconds for the next
three minutes.
[0562] The fluorescence change from baseline to the maximum rise of
the curve (.DELTA. change) is calculated, and replicates averaged.
The rate of fluorescence increase is monitored, and only those
samples which had a .DELTA. change greater than 1000 and a rise
within 60 seconds, are considered positive.
Fibroblast (BHK-21) Proliferation
[0563] This assay will show if the compounds of the invention act
to induce proliferation of mammalian fibroblast cells in culture
and, therefore, function is useful growth factors in mammalian
systems.
[0564] The assay is performed is follows. BHK-21 fibroblast cells
plated in standard growth medium at 2500 cells/well in a total
volume of 100 .mu.l. The compound, beta-FGF (positive control) or
nothing (negative control) are then added to the wells in the
presence of 1 .mu.g/ml of heparin for a total final volume of 200
.mu.l. The cells are then incubated at 37.degree. C. for 6 to 7
days. After incubation, the media is removed, the cells are washed
with PBS and then an acid phosphatase substrate reaction mixture
(100 .mu.l/well) is added. The cells are then incubated at
37.degree. C. for 2 hours. 10 .mu.L per well of 1N NaOH is then
added to stop the acid phosphatase reaction. The plates are then
read at OD 405 nm. A positive in the assay is acid phosphatase
activity which is at least 50% above the negative control.
Inhibition of Heart Adult Hypertrophy
[0565] This assay is designed to measure the inhibition of heart
adult hypertrophy. Compounds testing positive in this assay may
find use in the therapeutic treatment of cardiac disorders
associated with cardiac hypertrophy.
[0566] Ventricular myocytes are freshly isolated from adult (250 g)
Harlan Sprague Dawley rats and the cells are plated at 2000/well in
180 .mu.l volume. On day two, test samples (20 .mu.L) containing
the test compound are added. On day five, the cells are fixed and
then stained. An increase in ANP message can also be measured by
PCR from cells after a few hours. Results are based on a visual
score of cell size: 0=no inhibition, -1=small inhibition, -2=large
inhibition. A score of less than 0 is considered positive. Activity
reference corresponds to phenylephrine (PE) at 0.1 mM, is a
positive control. Assay media included: M199 (modified)-glutamine
free, NaHCO.sub.3, phenol red, supplemented with 100 nM insulin,
0.2% BSA, 5 mM cretine, 2 mM L-carnitine, 5 mM taurine, 100 U/ml
penicillin G, 100 .mu.g/ml streptomycin (CCT medium). Only inner 60
wells are used in 96 well plates. Of these, 6 wells are reserved
for negative and positive (PE) controls.
Induction of c-Fos in Endothelial Cells
[0567] This assay is designed to determine whether compounds of the
invention show the ability to induce c-fos in endothelial cells.
Compounds testing positive in this assay would be expected to be
useful for the therapeutic treatment of conditions or disorders
where angiogenesis would be beneficial including, for example,
wound healing, and the like (is would agonists of these compounds).
Antagonists of the compounds testing positive in this assay would
be expected to be useful for the therapeutic treatment of cancerous
tumors.
[0568] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50% Ham's F12 w/o GHT: low glucose, and
50% DMEM without glycine: with NaHCO.sub.3, 1% glutamine, 10 mM
HEPES, 10% FBS, 10 ng/ml bFGF) are plated on 96-well microtiter
plates at a cell density of 1.times.10.sup.4 cells/well. The day
after plating, the cells are starved by removing the growth media
and treating the cells with 100 .mu.l/well test samples and
controls (positive control=growth media; negative control=Protein
32 buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The
cells are incubated for 30 minutes at 37.degree. C., in 5%
CO.sub.2. The samples are removed, and the first part of the bDNA
kit protocol (Chiron Diagnostics, cat. #6005-037) is followed,
where each capitalized reagent/buffer listed below is available
from the kit.
[0569] Briefly, the amounts of the TM Lysis Buffer and Probes
needed for the tests are calculated based on information provided
by the manufacturer. The appropriate amounts of thawed Probes are
added to the TM Lysis Buffer. The Capture Hybridization Buffer is
warmed to room temperature. The bDNA strips are set up in the metal
strip holders, and 100 .mu.l of Capture Hybridization Buffer is
added to each b-DNA well needed, followed by incubation for at
least 30 minutes. The test plates with the cells are removed from
the incubator, and the media is gently removed using the vacuum
manifold. 100 .mu.l of Lysis Hybridization Buffer with Probes are
quickly pipetted into each well of the microtiter plates. The
plates are then incubated at 55.degree. C. for 15 minutes. Upon
removal from the incubator, the plates are placed on the vortex
mixer with the microtiter adapter head and vortexed on the #2
setting for one minute. 80 .mu.l of the lysate is removed and added
to the bDNA wells containing the Capture Hybridization Buffer, and
pipetted up and down to mix. The plates are incubated at 53.degree.
C. for at least 16 hours.
[0570] On the next day, the second part of the bDNA kit protocol is
followed. Specifically, the plates are removed from the incubator
and placed on the bench to cool for 10 minutes. The volumes of
additions needed are calculated based upon information provided by
the manufacturer. An Amplifier Working Solution is prepared by
making a 1:100 dilution of the Amplifier Concentrate (20 fm/.mu.l)
in AL Hybridization Buffer. The hybridization mixture is removed
from the plates and washed twice with Wash A. 50 .mu.l of Amplifier
Working Solution is added to each well and the wells are incubated
at 53.degree. C. for 30 minutes. The plates are then removed from
the incubator and allowed to cool for 10 minutes. The Label Probe
Working Solution is prepared by making a 1:100 dilution of Label
Concentrate (40 pmoles/.mu.l) in AL Hybridization Buffer. After the
10-minute cool-down period, the amplifier hybridization mixture is
removed and the plates are washed twice with Wash A. 50 .mu.l of
Label Probe Working Solution is added to each well and the wells
are incubated at 53.degree. C. for 15 minutes. After cooling for 10
minutes, the Substrate is warmed to room temperature. Upon addition
of 3 .mu.l of Substrate Enhancer to each ml of Substrate needed for
the assay, the plates are allowed to cool for 10 minutes, the label
hybridization mixture is removed, and the plates are washed twice
with Wash A and three times with Wash D. 50 .mu.l of the Substrate
Solution with Enhancer is added to each well. The plates are
incubated for 30 minutes at 37.degree. C. and RLU is read in an
appropriate luminometer.
[0571] The replicates are averaged and the coefficient of variation
is determined. The measure of activity of the fold increase over
the negative control (Protein 32/HEPES buffer described above)
value is indicated by chemiluminescence units (RLU). The results
are considered positive if the compound exhibits at least a
two-fold value over the negative buffer control. Negative
control=1.00 RLU at 1.00% dilution. Positive control=8.39 RLU at
1.00% dilution.
Guinea Pig Vascular Leak
[0572] This assay is designed to determine whether the compounds of
the present invention show the ability to induce vascular
permeability. Compounds testing positive in this assay are expected
to be useful for the therapeutic treatment of conditions which
would benefit from enhanced vascular permeability including, for
example, conditions which may benefit from enhanced local immune
system cell infiltration.
[0573] Hairless guinea pigs weighing 350 grams or more are
anesthetized with Ketamine (75-80 mg/kg) and 5 mg/kg Xylazine
intramuscularly. Test samples containing the tested compound or a
physiological buffer without the test compound are injected into
skin on the back of the test animals with 100 .mu.l per injection
site intradermally. There are approximately 16-24 injection sites
per animal. One ml of Evans blue dye (1% in PBS) is then injected
intracardially. Skin vascular permeability responses to the
compounds (i.e., blemishes at the injection sites of injection) are
visually scored by measuring the diameter (in mm) of blue-colored
leaks from the site of injection at 1 and 6 hours post
administration of the test materials. The mm diameter of blueness
at the site of injection is observed and recorded is well is the
severity of the vascular leakage. Blemishes of at least 5 mm in
diameter are considered positive for the assay when testing
purified proteins, being indicative of the ability to induce
vascular leakage or permeability. A response greater than 7 mm
diameter is considered positive for conditioned media samples.
Human VEGF at 0.1 .mu.g/100 .mu.l is used is a positive control,
inducing a response of 15-23 mm diameter.
Detection of Endothelial Cell Apoptosis (FACS)
[0574] The ability of the compounds disclosed herein to induce
apoptosis in endothelial cells is tested in human venous umbilical
vein endothelial cells (HUVEC, Cell Systems) in gelatinized T175
flasks using HUVEC cells below passage 10. Compounds testing
positive in this assay are expected to be useful for
therapeutically treating conditions where apoptosis of endothelial
cells would be beneficial including, for example, the therapeutic
treatment of tumors.
[0575] On day one, the cells are split [420,000 cells per
gelatinized 6 cm dishes (11.times.10.sup.3 cells/cm.sup.2 Falcon,
Primaria)] and grown in media containing serum (CS-C, Cell System)
overnight or for 16 hours to 24 hours.
[0576] On day 2, the cells are washed 1.times. with 5 ml PBS; 3 ml
of 0% serum medium is added with VEGF (100 ng/ml); and 30 .mu.l of
the PRO test compound (final dilution 1%) or 0% serum medium
(negative control) is added. The mixtures are incubated for 48
hours before harvesting.
[0577] The cells are then harvested for FACS analysis. The medium
is aspirated and the cells washed once with PBS. 5 ml of 1.times.
trypsin is added to the cells in a T-175 flask, and the cells are
allowed to stand until they are released from the plate (about 5-10
minutes). Trypsinization is stopped by adding 5 ml of growth media.
The cells are spun at 1000 rpm for 5 minutes at 4.degree. C. The
media is aspirated and the cells are resuspended in 10 ml of 10%
serum complemented medium (Cell Systems), 5 .mu.l of Annexin-FITC
(BioVison) added and chilled tubes are submitted for FACS. A
positive result is determined to be enhanced apoptosis in the
compound treated samples is compared to the negative control.
Induction of c-Fos in Cortical Neurons
[0578] This assay is designed to determine whether the compounds
tested show the ability to induce c-fos in cortical neurons.
Compounds testing positive in this assay would be expected to be
useful for the therapeutic treatment of nervous system disorders
and injuries where neuronal proliferation would be beneficial.
[0579] Cortical neurons are dissociated and plated in growth medium
at 10,000 cells per well in 96 well plates. After approximately 2
cellular divisions, the cells are treated for 30 minutes with the
compound or nothing (negative control). The cells are then fixed
for 5 minutes with cold methanol and stained with an antibody
directed against phosphorylated CREB. mRNA levels are then
calculated using chemiluminescence. A positive in the assay is any
factor that results in at least a 2-fold increase in c-fos message
is compared to the negative controls.
Stimulation of Endothelial Tube Formation
[0580] This assay is designed to determine whether the compounds of
the invention show the ability to promote endothelial vacuole and
lumen formation in the absence of exogenous growth factors.
Compounds testing positive in this assay would be expected to be
useful for the therapeutic treatment of disorders where endothelial
vacuole and/or lumen formation would be beneficial including, for
example, where the stimulation of pinocytosis, ion pumping,
vascular permeability and/or junctional formation would be
beneficial.
[0581] HUVEC cells (passage <8 from primary) are mixed with type
I rat tail collagen (final concentration 2.6 mg/ml) at a density of
6.times.10.sup.5 cells per ml and plated at 50 .mu.l per well of
M199 culture media supplement with 1% FBS and 1 .mu.M 6-FAM-FITC
dye to stain the vacuoles while they are forming and in the
presence of the test compound. The cells are then incubated at
37.degree. C./5% CO.sub.2 for 48 hours, fixed with 3.7% formalin
room temperature for 10 minutes, washed 5 times with M199 medium
and then stained with Rh-Phalloidin at 4.degree. C. overnight
followed by nuclear staining with 4 .mu.M DAPI. A positive result
in the assay is when vacuoles are present in greater than 50% of
the cells.
Detection of Compounds of the Invention that Affect Glucose and/or
FFA Uptake in Skeletal Muscle
[0582] This assay is designed to determine whether compounds of the
invention show the ability to affect glucose or FFA uptake by
skeletal muscle cells. Compounds testing positive in this assay
would be expected to be useful for the therapeutic treatment of
disorders where either the stimulation or inhibition of glucose
uptake by skeletal muscle would be beneficial including, for
example, diabetes or hyper- or hypo-insulinemia.
[0583] In a 96 well format, a compound to be assayed is added to
primary rat differentiated skeletal muscle, and allowed to incubate
overnight. Then fresh media with the compound and +/- insulin are
added to the wells. The sample media is then monitored to determine
glucose and FFA uptake by the skeletal muscle cells. The insulin
will stimulate glucose and FFA uptake by the skeletal muscle, and
insulin in media without the compound is used is a positive
control, and a limit for scoring. As the compound being tested may
either stimulate or inhibit glucose and FFA uptake, results are
scored is positive in the assay if greater than 1.5 times or less
than 0.5 times the insulin control.
Rod Photoreceptor Cell Survival Assay
[0584] This assay tests the ability of the compounds of this
invention act to enhance the survival/proliferation of rod
photoreceptor cells and, therefore, are useful for the therapeutic
treatment of retinal disorders or injuries including, for example,
treating sight loss in mammals due to retinitis pigmentosum, AMD,
etc.
[0585] Sprague Dawley rat pups (postnatal day 7, mixed population:
glia and netinal neural cell types) are killed by decapitation
following CO.sub.2 anesthesia and the eyes removed under sterile
conditions. The neural retina is dissected away from the pigment
epithelium and other ocular tissue and then dissociated into a
single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. in
this solution for 7-10 minutes after which the trypsin is
inactivated by adding 1 ml soybean trypsin inhibitor. The cells are
plated at a density of approximately 10,000 cells/ml into 96 well
plates in DMEM/F12 supplemented with N.sub.2. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere of 5% CO.sub.2. After 7-10 days in culture, the cells
are stained using calcein AM or CellTracker Green CMFDA and then
fixed using 4% paraformaldehyde. Rho 4D2 (ascities or IgG 1:100)
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated is % survival: total
number of calcein--rhodopsin positive cells at 7-10 days in
culture, divided by the total number of rhodopsin positive cells at
time 7-10 days in culture. The total cells (fluorescent) are
quantified at 20.times. objective magnification using a CCD camera
and NIH image software for MacIntosh. Fields in the well are chosen
at random.
In Vitro Antitumor Assay
[0586] The antiproliferative activity of the compounds disclosed
herein can be determined in the investigational, disease-oriented
in vitro anti-cancer drug discovery assay of the National Cancer
Institute (NCl), using a sulforhodamine B (SRB) dye binding assay
essentially is described by Skehan et al., J. Natl. Cancer Inst.
82:1107-1112 (1990). The 60 tumor cell lines employed in this study
("the NCl panel"), as well as conditions for their maintenance and
culture in vitro, have been described by Monks et al., J. Natl.
Cancer Inst. 83:757-766 (1991). The purpose of this screen is to
initially evaluate the cytotoxic and/or cytostatic activity of the
test compounds against different types of tumors (Monks et al.,
supra; Boyd, Cancer: Princ. Pract. Oncol. Update 3(10):1-12
[1989]).
[0587] Cells from approximately 60 human tumor cell lines are
harvested with trypsin/EDTA (Gibco), washed once, resuspended in
IMEM and their viability is determined. The cell suspensions are
added by pipet (100 .mu.L volume) into separate 96-well microtiter
plates. The cell density for the 6-day incubation is less than for
the 2-day incubation to prevent overgrowth. Inoculates are allowed
a preincubation period of 24 hours at 37.degree. C. for
stabilization. Dilutions at twice the intended test concentration
are added at time zero in 100 .mu.L aliquots to the microtiter
plate wells (1:2 dilution). Test compounds are evaluated at five
half-log dilutions (1000 to 100.000-fold). Incubations took place
for two days and six days in a 5% CO.sub.2 atmosphere and 100%
humidity.
[0588] After incubation, the medium is removed and the cells are
fixed in 0.1 ml of 10% trichloroacetic acid at 40.degree. C. The
plates are rinsed five times with deionized water, dried, stained
for 30 minutes with 0.1 ml of 0.4% sulforhodamine B dye (Sigma)
dissolved in 1% acetic acid, rinsed four times with 1% acetic acid
to remove unbound dye, dried, and the stain is extracted for five
minutes with 0.1 ml of 10 mM Tris base
[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) of
sulforhodamine B at 492 nm is measured using a computer-interfaced,
96-well microtiter plate reader.
[0589] A test sample is considered positive if it shows at least
50% growth inhibitory effect at one or more concentrations.
Determination of Compounds of this Invention that Affect Glucose or
FFA Uptake by Primary Rat Adipocytes
[0590] This assay is designed to determine whether the compounds of
this invention show the ability to affect glucose or FFA uptake by
adipocyte cells. Compounds testing positive in this assay would be
expected to be useful for the therapeutic treatment of disorders
where either the stimulation or inhibition of glucose uptake by
adipocytes would be beneficial including, for example, obesity,
diabetes or hyper- or hypo-insulinemia.
[0591] In a 96 well format, compounds to be assayed are added to
primary rat adipocytes, and allowed to incubate overnight. Samples
are taken at 4 and 16 hours and assayed for glycerol, glucose and
FFA uptake. After the 16 hour incubation, insulin is added to the
media and allowed to incubate for 4 hours. At this time, a sample
is taken and glycerol, glucose and FFA uptake is measured. Media
containing insulin without the compound is used as a positive
reference control. As the compound being tested may either
stimulate or inhibit glucose and FFA uptake, results are scored as
positive in the assay if greater than 1.5 times or less than 0.5
times the insulin control.
Chondrocyte Re-Differentiation Assay
[0592] This assay shows whether the compounds of the invention act
to induce redifferentiation of chondrocytes, and therefore are
expected to be useful for the treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and
arthritis. The assay is performed as follows. Porcine chondrocytes
are isolated by overnight collagenase digestion of articulary
cartilage of metacarpophalangeal joints of 4-6 month old female
pigs. The isolated cells are then seeded at 25,000 cells/cm.sup.2
in Ham F-12 containing 10% FBS and 4 .mu.g/ml gentamycin. The
culture media is changed every third day and the cells are then
seeded in 96 well plates at 5,000 cells/well in 100 .mu.l of the
same media without serum and 100 .mu.l of the test compound, 5 nM
staurosporin (positive control) or medium alone (negative control)
is added to give a final volume of 200 .mu.l/well. After 5 days of
incubation at 37.degree. C., a picture of each well is taken and
the differentiation state of the chondrocytes is determined. A
positive result in the assay occurs when the redifferentiation of
the chondrocytes is determined to be more similar to the positive
control than the negative control.
Fetal Hemoglobin Induction in an Erythroblastic Cell Line
[0593] This assay is useful for screening compounds for the ability
to induce the switch from adult hemoglobin to fetal hemoglobin in
an erythroblastic cell line. Compounds testing positive in this
assay are expected to be useful for therapeutically treating
various mammalian hemoglobin-associated disorders such as the
various thalassemias. The assay is performed as follows.
Erythroblastic cells are plated in standard growth medium at 1000
cells/well in a 96 well format. The tested compound is added to the
growth medium at a concentration of 0.2% or 2% and the cells are
incubated for 5 days at 37.degree. C. As a positive control, cells
are treated with 100 .mu.M hemin and as a negative control, the
cells are untreated. After 5 days, cell lysates are prepared and
analyzed for the expression of gamma globin (a fetal marker). A
positive in the assay is a gamma globin level at least 2-fold above
the negative control.
Mouse Kidney Mesangial Cell Proliferation Assay
[0594] This assay shows whether compounds of the invention act to
induce proliferation of mammalian kidney mesangial cells and
therefore are useful for treating kidney disorders associated with
decreased mesangial cell function such as Berger disease or other
nephropathies associated with Schonlein-Henoch purpura, celiac
disease, dermatitis herpetiformis or Crohn disease. The assay is
performed as follows. On day one, mouse kidney mesangial cells are
plated on a 96 well plate in growth media (3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95% fetal
bovine serum, 5% supplemented with 14 mM HEPES) and grown
overnight. On day 2, the test compound is diluted at 2
concentrations (1% and 0.1%) in serum-free medium and added to the
cells. Control samples are serum-free medium alone. On day 4, 20
.mu.l of the Cell Titer 96 Aqueous one solution reagent (Progema)
was added to each well and the colormetric reaction was allowed to
proceed for 2 hours. The absorbance (OD) is then measured at 490
nm. A positive in the assay is an absorbance reading which is at
least 15% above the control reading.
Proliferation of Rat Utricular Supporting Cells
[0595] This assay is used to determine of compounds of the
invention act as potent mitogens for inner ear supporting cells
which are auditory hair cell progenitors and, therefore, are useful
for inducing the regeneration of auditory hair cells and treating
hearing loss in mammals. The assay is performed as follows. Rat
UEC-4 utricular epithelial cells are aliquoted into 96 well plates
with a density of 3000 cells/well in 200 .mu.l of serum-containing
medium at 33.degree. C. The cells are cultured overnight and are
then switched to serum-free medium at 37.degree. C. Various
dilutions of the compounds (or nothing for a control) are then
added to the cultures and the cells are incubated for 24 hours.
After the 24 hour incubation, .sup.3H-thymidine (1 .mu.Ci/well) is
added and the cells are then cultured for an additional 24 hours.
The cultures are then washed to remove unincorporated radiolabel,
the cells harvested and Cpm per well determined. Cpm of at least
30% or greater in the test compound treated cultures as compared to
the control cultures is considered a positive in the assay.
Chondrocyte Proliferation Assay
[0596] This assay is designed to determine whether compounds of the
present invention show the ability to induce the proliferation
and/or redifferentiation of chondrocytes in culture. Compounds
testing positive in this assay would be expected to be useful for
the therapeutic treatment of various bone and/or cartilage
disorders such as, for example, sports injuries and arthritis.
[0597] Porcine chondrocytes are isolated by overnight collagenase
digestion of articular cartilage of the metacarpophalangeal joint
of 4-6 month old female pigs. The isolated cells are then seeded at
25,000 cells/cm.sup.2 in Ham F-12 containing 10% FBS and 4 .mu.g/ml
gentamycin. The culture media is changed every third day and the
cells are reseeded to 25,000 cells/cm.sup.2 every five days. On day
12, the cells are seeded in 96 well plates at 5,000 cells/well in
100 .mu.l of the same media without serum and 100 .mu.l of either
serum-free medium (negative control), staurosporin (final
concentration of 5 nM; positive control) or the test compound are
added to give a final volume of 200 .mu.l/well. After 5 days at
37.degree. C., 20 .mu.l of Alamar blue is added to each well and
the plates are incubated for an additional 3 hours at 37.degree. C.
The fluorescence is then measured in each well (Ex: 530 nm; Em: 590
nm). The fluorescence of a plate containing 200 .mu.l of the
serum-free medium is measured to obtain the background. A positive
result in the assay is obtained when the fluorescence of the
compound treated sample is more like that of the positive control
than the negative control.
Inhibition of Heart Neonatal Hypertrophy Induced by LIF+ET-1
[0598] This assay is designed to determine whether the compounds of
the present invention show the ability to inhibit neonatal heart
hypertrophy induced by LIF and endothelin-1 (ET-1). A test compound
that provides a positive response in the present assay would be
useful for the therapeutic treatment of cardiac insufficiency
diseases or disorders characterized or associated with an undesired
hypertrophy of the cardiac muscle.
[0599] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
(180 .mu.l at 7.5.times.10.sup.4/ml, serum <0.1, freshly
isolated) are introduced on day 1 to 96-well plates previously
coated with DMEM/F12+4% FCS. Test compound samples or growth medium
alone (negative control) are then added directly to the wells on
day 2 in 20 .mu.l volume. LIF+ET-1 are then added to the wells on
day 3. The cells are stained after an additional 2 days in culture
and are then scored visually the next day. A positive in the assay
occurs when the compound treated myocytes are visually smaller on
the average or less numerous than the untreated myocytes.
Sequence CWU 1
1
19914195DNAEscherichia coli 1ctaaattgta agcgttaata ttttgttaaa
attcgcgtta aatttttgtt aaatcagctc 60attttttaac caataggccg aaatcggcaa
aatcccttat aaatcaaaag aatagaccga 120gatagggttg agtgttgttc
cagtttggaa caagagtcca ctattaaaga acgtggactc 180caacgtcaaa
gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
240ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
ctaaagggag 300cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
gcgagaaagg aagggaagaa 360agcgaaagga gcgggcgcta gggcgctggc
aagtgtagcg gtcacgctgc gcgtaaccac 420cacacccgcc gcgcttaatg
cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480caactgttgg
gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
540gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
cacgacgttg 600taaaacgacg gccagtgagc gcgcaagcgg ccgcaacccg
ggaaaagctt ggccattgca 660tacgttgtat ccatatcata atatgtacat
ttatattggc tcatgtccaa cattaccgcc 720atgttgacat tgattattga
ctagttatta atagtaatca attacggggt cattagttca 780tagcccatat
atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
840gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
taacgccaat 900agggactttc cattgacgtc aatgggtgga gtatttacgg
taaactgccc acttggcagt 960acatcaagtg tatcatatgc caagtacgcc
ccctattgac gtcaatgacg gtaaatggcc 1020cgcctggcat tatgcccagt
acatgacctt atgggacttt cctacttggc agtacatcta 1080cgtattagtc
atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
1140atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
atgggagttt 1200gttttggcac caaaatcaac gggactttcc aaaatgtcgt
aacaactccg ccccattgac 1260gcaaatgggc ggtaggcgtg tacggtggga
ggtctatata agcagagctc gtttagtgaa 1320ccgtcagatc gcctggagac
gccatccacg ctgttttgac ctccatagaa gacaccggga 1380ccgatccagc
ctccgcggcc gggaacggtg cattggaacg cggattcccc gtgccaagag
1440tgacgtaagt accgcctata gagtctatag gcccaccccc ttggcttctt
atgcatgctc 1500ccctgctccg acccgggctc ctcgcccgcc cggacccaca
ggccaccctc aaccgtcctg 1560gccccggacc caaaccccac ccctcactct
gcttctcccc gcaggagaat tcaatcgcga 1620aagggcccaa agatctgcca
taccacattt gtagaggttt tacttgcttt aaaaaacctc 1680ccacacctcc
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
1740attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
aaataaagca 1800tttttttcac tgcattctag ttgtggtttg tccaaactca
tcaatgtatc ttatcatgtc 1860tggagctagc atcccgcccc taactccgcc
ctgttccgcc cattctccgc cccatggctg 1920actaattttt tttatttatg
cagaggccga ggccgcctcg gcctctgagc tattccagaa 1980gtagtgagga
ggcttttttg gaggcctagg cttttgcgtc gagaagcgcg cttggcgtaa
2040tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
acacaacata 2100cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
gagtgagcta actcacatta 2160attgcgttgc gctcactgcc cgctttccag
tcgggaaacc tgtcgtgcca gctgcattaa 2220tgaatcggcc aacgcgcggg
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 2280ctcactgact
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
2340gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
gtgagcaaaa 2400ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
tggcgttttt ccataggctc 2460cgcccccctg acgagcatca caaaaatcga
cgctcaagtc agaggtggcg aaacccgaca 2520ggactataaa gataccaggc
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2580accctgccgc
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
2640catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
gctgggctgt 2700gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
ccggtaacta tcgtcttgag 2760tccaacccgg taagacacga cttatcgcca
ctggcagcag ccactggtaa caggattagc 2820agagcgaggt atgtaggcgg
tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2880actagaagga
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
2940gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
ttttgtttgc 3000aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
atcctttgat cttttctacg 3060gggtctgacg ctcagtggaa cgaaaactca
cgttaaggga ttttggtcat gagattatca 3120aaaaggatct tcacctagat
ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3180atatatgagt
aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
3240gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
gataactacg 3300atacgggagg gcttaccatc tggccccagt gctgcaatga
taccgcgaga cccacgctca 3360ccggctccag atttatcagc aataaaccag
ccagccggaa gggccgagcg cagaagtggt 3420cctgcaactt tatccgcctc
catccagtct attaattgtt gccgggaagc tagagtaagt 3480agttcgccag
ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
3540cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
gcgagttaca 3600tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
gtcctccgat cgttgtcaga 3660agtaagttgg ccgcagtgtt atcactcatg
gttatggcag cactgcataa ttctcttact 3720gtcatgccat ccgtaagatg
cttttctgtg actggtgagt actcaaccaa gtcattctga 3780gaatagtgta
tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
3840ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
gcgaaaactc 3900tcaaggatct taccgctgtt gagatccagt tcgatgtaac
ccactcgtgc acccaactga 3960tcttcagcat cttttacttt caccagcgtt
tctgggtgag caaaaacagg aaggcaaaat 4020gccgcaaaaa agggaataag
ggcgacacgg aaatgttgaa tactcatact cttccttttt 4080caatattatt
gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
4140atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccac
41952824DNAEscherichia coli 2gttaacgaat tcccaccatg attgaacaag
atggattgca cgcaggttct ccggccgctt 60gggtggagag gctattcggc tatgactggg
cacaacagac aatcggctgc tctgatgccg 120ccgtgttccg gctgtcagcg
caggggcgcc cggttctttt tgtcaagacc gacctgtccg 180gtgccctgaa
tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
240ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
ctgctattgg 300gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
tcctgccgag aaagtatcca 360tcatggctga tgcaatgcgg cggctgcata
cgcttgatcc ggctacctgc ccattcgacc 420accaagcgaa acatcgcatc
gagcgagcac gtactcggat ggaagccggt cttgtcgatc 480aggatgatct
ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
540aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
tgcttgccga 600atatcatggt ggaaaatggc cgcttttctg gattcatcga
ctgtggccgg ctgggtgtgg 660cggaccgcta tcaggacata gcgttggcta
cccgtgatat tgctgaagag cttggcggcg 720aatgggctga ccgcttcctc
gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 780ccttctatcg
ccttcttgac gagttcttct gaagatctgt taac 8243785DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 3gttaacgaat tcccaccatg ctgctgctgg cgagatgtct
gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgtg acaaaactca
cacatgccca ccgtgcccag 120cacctgaact cctgggggga ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc ccggacccct
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 240ctgaggtcaa
gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc 360aggactggct gaatggcaag gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc aaagccaaag
ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc ccgggatgag
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 540gcttctatcc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact
600acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga acgtcttctc
atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg cagaagagcc
tctccctgtc tccgggtaaa tgaagatctg 780ttaac 7854785DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 4gttaacgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgtg acaaaactca
cacatgccca ccgtgcccag 120cacctgaact cctgggggga ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc ccggacccct
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 240ctgaggtcaa
gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc 360aggactggct gaatggcaag gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc aaagccaaag
ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc ccgggatgag
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 540gcttctatcc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact
600acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga acgtcttctc
atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg cagaagagcc
tctccctgtc tccgggtaaa tgaagatctg 780ttaac 7855767DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 5gttaacgaat tcccaccatg ctggctgcca cagtcctgac
cctggccctg ctgggcaatg 60cccatgcctg tgacaaaact cacacatgcc caccgtgccc
agcacctgaa ctcctggggg 120gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc aagttcaact 240ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca tcccgggatg 480agctgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cgcagaagag cctctccctg tctccgggta
aatgaagatc tgttaac 7676767DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 6gttaacgaat
tcccaccatg ctgctgctgg cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt
atgctcggga ctggcgtgcc caccgtgccc agcacctgaa ctcctggggg
120gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc
tcccggaccc 180ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga
ccctgaggtc aagttcaact 240ggtacgtgga cggcgtggag gtgcataatg
ccaagacaaa gccgcgggag gagcagtaca 300acagcacgta ccgtgtggtc
agcgtcctca ccgtcctgca ccaggactgg ctgaatggca 360aggagtacaa
gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag aaaaccatct
420ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca
tcccgggatg 480agctgaccaa gaaccaggtc agcctgacct gcctggtcaa
aggcttctat cccagcgaca 540tcgccgtgga gtgggagagc aatgggcagc
cggagaacaa ctacaagacc acgcctcccg 600tgctggactc cgacggctcc
ttcttcctct acagcaagct caccgtggac aagagcaggt 660ggcagcaggg
gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca
720cgcagaagag cctctccctg tctccgggta aatgaagatc tgttaac
7677767DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 7gttaacgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc caccgtgccc
agcacctgaa ctcctggggg 120gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc aagttcaact 240ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca tcccgggatg 480agctgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cgcagaagag cctctccctg tctccgggta
aatgaagatc tgttaac 7678749DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 8gttaacgaat
tcccaccatg ctggctgcca cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg
cccaccgtgc ccagcacctg aactcctggg gggaccgtca gtcttcctct
120tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc
acatgcgtgg 180tggtggacgt gagccacgaa gaccctgagg tcaagttcaa
ctggtacgtg gacggcgtgg 240aggtgcataa tgccaagaca aagccgcggg
aggagcagta caacagcacg taccgtgtgg 300tcagcgtcct caccgtcctg
caccaggact ggctgaatgg caaggagtac aagtgcaagg 360tctccaacaa
agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
420cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc
aagaaccagg 480tcagcctgac ctgcctggtc aaaggcttct atcccagcga
catcgccgtg gagtgggaga 540gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac tccgacggct 600ccttcttcct ctacagcaag
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct 660tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc
720tgtctccggg taaatgaaga tctgttaac 74991427DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 9gttaacgaat tcccaccatg agctttccat gtaaatttgt
agccagcttc cttctgattt 60tcaatgtttc ttccaaaggt gcagtctcca aaactcacac
atgcccaccg tgcccagcac 120ctgaactcct ggggggaccg tcagtcttcc
tcttcccccc aaaacccaag gacaccctca 180tgatctcccg gacccctgag
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg 240aggtcaagtt
caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc
300gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc
ctgcaccagg 360actggctgaa tggcaaggag tacaagtgca aggtctccaa
caaagccctc ccagccccca 420tcgagaaaac catctccaaa gccaaagggc
agccccgaga accacaggtg tacaccctgc 480ccccatcccg ggatgagctg
accaagaacc aggtcagcct gacctgcctg gtcaaaggct 540tctatcccag
cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca
600agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc
aagctcaccg 660tggacaagag caggtggcag caggggaacg tcttctcatg
ctccgtgatg catgaggctc 720tgcacaacca ctacacgcag aagagcctct
ccctgtctcc ggagctgcaa ctggaggaga 780gctgtgcgga ggcgcaggac
ggggagctgg acgggtgcgt atccggtgac accattgtaa 840tgactagtgg
cggtccgcgc actgtggctg aactggaggg caaaccgttc accgcactga
900ttcgcggctc tggctaccca tgcccctcag gtttcttccg cacctgtgaa
cgtgacgtat 960atgatctgcg tacacgtgag ggtcattgct tacgtttgac
ccatgatcac cgtgttctgg 1020tgatggatgg tggcctggaa tggcgtgccg
cgggtgaact ggaacgcggc gaccgcctgg 1080tgatggatga tgcagctggc
gagtttccgg cactggcaac cttccgtggc ctgcgtggcg 1140ctggccgcca
ggatgtttat gacgctactg tttacggtgc tagcgcattc actgctaatg
1200gcttcattgt acacgcatgt ggcgagcagc ccgggaccgg tctgaactca
ggcctcacga 1260caaatcctgg tgtatccgct tggcaggtca acacagctta
tactgcggga caattggtca 1320catataacgg caagacgtat aaatgtttgc
agccccacac ctccttggca ggatgggaac 1380catccaacgt tcctgccttg
tggcagcttc aatgaagatc tgttaac 1427101430DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 10gttaacgaat tcccaccatg aaccggggag tcccttttag
gcacttgctt ctggtgctgc 60aactggcgct cctcccagca gccactcagg gaaaaactca
cacatgccca ccgtgcccag 120cacctgaact cctgggggga ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc ccggacccct
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 240ctgaggtcaa
gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc 360aggactggct gaatggcaag gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc aaagccaaag
ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc ccgggatgag
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 540gcttctatcc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact
600acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga acgtcttctc
atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg cagaagagcc
tctccctgtc tccggagctg caactggagg 780agagctgtgc ggaggcgcag
gacggggagc tggacgggtg cgtatccggt gacaccattg 840taatgactag
tggcggtccg cgcactgtgg ctgaactgga gggcaaaccg ttcaccgcac
900tgattcgcgg ctctggctac ccatgcccct caggtttctt ccgcacctgt
gaacgtgacg 960tatatgatct gcgtacacgt gagggtcatt gcttacgttt
gacccatgat caccgtgttc 1020tggtgatgga tggtggcctg gaatggcgtg
ccgcgggtga actggaacgc ggcgaccgcc 1080tggtgatgga tgatgcagct
ggcgagtttc cggcactggc aaccttccgt ggcctgcgtg 1140gcgctggccg
ccaggatgtt tatgacgcta ctgtttacgg tgctagcgca ttcactgcta
1200atggcttcat tgtacacgca tgtggcgagc agcccgggac cggtctgaac
tcaggcctca 1260cgacaaatcc tggtgtatcc gcttggcagg tcaacacagc
ttatactgcg ggacaattgg 1320tcacatataa cggcaagacg tataaatgtt
tgcagcccca cacctccttg gcaggatggg 1380aaccatccaa cgttcctgcc
ttgtggcagc ttcaatgaag atctgttaac 1430111430DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 11gttaacgaat tcccaccatg ctgctgctgg cgagatgtct
gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgtg acaaaactca
cacatgccca ccgtgcccag 120cacctgaact cctgggggga ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc ccggacccct
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc 240ctgaggtcaa
gttcaactgg tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc 360aggactggct gaatggcaag gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc aaagccaaag
ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc ccgggatgag
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag 540gcttctatcc
cagcgacatc gccgtggagt gggagagcaa tgggcagccg gagaacaact
600acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac
agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga acgtcttctc
atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg cagaagagcc
tctccctgtc tccggagctg caactggagg 780agagctgtgc ggaggcgcag
gacggggagc tggacgggtg cgtatccggt gacaccattg 840taatgactag
tggcggtccg cgcactgtgg ctgaactgga gggcaaaccg ttcaccgcac
900tgattcgcgg ctctggctac ccatgcccct caggtttctt ccgcacctgt
gaacgtgacg 960tatatgatct gcgtacacgt gagggtcatt gcttacgttt
gacccatgat caccgtgttc 1020tggtgatgga tggtggcctg gaatggcgtg
ccgcgggtga actggaacgc ggcgaccgcc 1080tggtgatgga tgatgcagct
ggcgagtttc cggcactggc aaccttccgt ggcctgcgtg 1140gcgctggccg
ccaggatgtt tatgacgcta ctgtttacgg tgctagcgca ttcactgcta
1200atggcttcat tgtacacgca tgtggcgagc agcccgggac cggtctgaac
tcaggcctca 1260cgacaaatcc tggtgtatcc gcttggcagg tcaacacagc
ttatactgcg ggacaattgg 1320tcacatataa cggcaagacg tataaatgtt
tgcagcccca cacctccttg gcaggatggg 1380aaccatccaa cgttcctgcc
ttgtggcagc ttcaatgaag atctgttaac 1430121430DNAArtificial
Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 12gttaacgaat tcccaccatg gccttgacct
ttgctttact ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgtg
acaaaactca cacatgccca ccgtgcccag 120cacctgaact cctgggggga
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc
ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
240ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc 300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc 360aggactggct gaatggcaag gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc
aaagccaaag ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc
ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
540gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
gagaacaact 600acaagaccac gcctcccgtg ctggactccg acggctcctt
cttcctctac agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg
cagaagagcc tctccctgtc tccggagctg caactggagg 780agagctgtgc
ggaggcgcag gacggggagc tggacgggtg cgtatccggt gacaccattg
840taatgactag tggcggtccg cgcactgtgg ctgaactgga gggcaaaccg
ttcaccgcac 900tgattcgcgg ctctggctac ccatgcccct caggtttctt
ccgcacctgt gaacgtgacg 960tatatgatct gcgtacacgt gagggtcatt
gcttacgttt gacccatgat caccgtgttc 1020tggtgatgga tggtggcctg
gaatggcgtg ccgcgggtga actggaacgc ggcgaccgcc 1080tggtgatgga
tgatgcagct ggcgagtttc cggcactggc aaccttccgt ggcctgcgtg
1140gcgctggccg ccaggatgtt tatgacgcta ctgtttacgg tgctagcgca
ttcactgcta 1200atggcttcat tgtacacgca tgtggcgagc agcccgggac
cggtctgaac tcaggcctca 1260cgacaaatcc tggtgtatcc gcttggcagg
tcaacacagc ttatactgcg ggacaattgg 1320tcacatataa cggcaagacg
tataaatgtt tgcagcccca cacctccttg gcaggatggg 1380aaccatccaa
cgttcctgcc ttgtggcagc ttcaatgaag atctgttaac 1430131412DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 13gttaacgaat tcccaccatg ctggctgcca cagtcctgac
cctggccctg ctgggcaatg 60cccatgcctg tgacaaaact cacacatgcc caccgtgccc
agcacctgaa ctcctggggg 120gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc aagttcaact 240ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca tcccgggatg 480agctgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cgcagaagag cctctccctg tctccggagc
tgcaactgga ggagagctgt gcggaggcgc 780aggacgggga gctggacggg
tgcgtatccg gtgacaccat tgtaatgact agtggcggtc 840cgcgcactgt
ggctgaactg gagggcaaac cgttcaccgc actgattcgc ggctctggct
900acccatgccc ctcaggtttc ttccgcacct gtgaacgtga cgtatatgat
ctgcgtacac 960gtgagggtca ttgcttacgt ttgacccatg atcaccgtgt
tctggtgatg gatggtggcc 1020tggaatggcg tgccgcgggt gaactggaac
gcggcgaccg cctggtgatg gatgatgcag 1080ctggcgagtt tccggcactg
gcaaccttcc gtggcctgcg tggcgctggc cgccaggatg 1140tttatgacgc
tactgtttac ggtgctagcg cattcactgc taatggcttc attgtacacg
1200catgtggcga gcagcccggg accggtctga actcaggcct cacgacaaat
cctggtgtat 1260ccgcttggca ggtcaacaca gcttatactg cgggacaatt
ggtcacatat aacggcaaga 1320cgtataaatg tttgcagccc cacacctcct
tggcaggatg ggaaccatcc aacgttcctg 1380ccttgtggca gcttcaatga
agatctgtta ac 1412141412DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 14gttaacgaat tcccaccatg
ctgctgctgg cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga
ctggcgtgcc caccgtgccc agcacctgaa ctcctggggg 120gaccgtcagt
cttcctcttc cccccaaaac ccaaggacac cctcatgatc tcccggaccc
180ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc
aagttcaact 240ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa
gccgcgggag gagcagtaca 300acagcacgta ccgtgtggtc agcgtcctca
ccgtcctgca ccaggactgg ctgaatggca 360aggagtacaa gtgcaaggtc
tccaacaaag ccctcccagc ccccatcgag aaaaccatct 420ccaaagccaa
agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggatg
480agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat
cccagcgaca 540tcgccgtgga gtgggagagc aatgggcagc cggagaacaa
ctacaagacc acgcctcccg 600tgctggactc cgacggctcc ttcttcctct
acagcaagct caccgtggac aagagcaggt 660ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga ggctctgcac aaccactaca 720cgcagaagag
cctctccctg tctccggagc tgcaactgga ggagagctgt gcggaggcgc
780aggacgggga gctggacggg tgcgtatccg gtgacaccat tgtaatgact
agtggcggtc 840cgcgcactgt ggctgaactg gagggcaaac cgttcaccgc
actgattcgc ggctctggct 900acccatgccc ctcaggtttc ttccgcacct
gtgaacgtga cgtatatgat ctgcgtacac 960gtgagggtca ttgcttacgt
ttgacccatg atcaccgtgt tctggtgatg gatggtggcc 1020tggaatggcg
tgccgcgggt gaactggaac gcggcgaccg cctggtgatg gatgatgcag
1080ctggcgagtt tccggcactg gcaaccttcc gtggcctgcg tggcgctggc
cgccaggatg 1140tttatgacgc tactgtttac ggtgctagcg cattcactgc
taatggcttc attgtacacg 1200catgtggcga gcagcccggg accggtctga
actcaggcct cacgacaaat cctggtgtat 1260ccgcttggca ggtcaacaca
gcttatactg cgggacaatt ggtcacatat aacggcaaga 1320cgtataaatg
tttgcagccc cacacctcct tggcaggatg ggaaccatcc aacgttcctg
1380ccttgtggca gcttcaatga agatctgtta ac 1412151412DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 15gttaacgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc caccgtgccc
agcacctgaa ctcctggggg 120gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc aagttcaact 240ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca tcccgggatg 480agctgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cgcagaagag cctctccctg tctccggagc
tgcaactgga ggagagctgt gcggaggcgc 780aggacgggga gctggacggg
tgcgtatccg gtgacaccat tgtaatgact agtggcggtc 840cgcgcactgt
ggctgaactg gagggcaaac cgttcaccgc actgattcgc ggctctggct
900acccatgccc ctcaggtttc ttccgcacct gtgaacgtga cgtatatgat
ctgcgtacac 960gtgagggtca ttgcttacgt ttgacccatg atcaccgtgt
tctggtgatg gatggtggcc 1020tggaatggcg tgccgcgggt gaactggaac
gcggcgaccg cctggtgatg gatgatgcag 1080ctggcgagtt tccggcactg
gcaaccttcc gtggcctgcg tggcgctggc cgccaggatg 1140tttatgacgc
tactgtttac ggtgctagcg cattcactgc taatggcttc attgtacacg
1200catgtggcga gcagcccggg accggtctga actcaggcct cacgacaaat
cctggtgtat 1260ccgcttggca ggtcaacaca gcttatactg cgggacaatt
ggtcacatat aacggcaaga 1320cgtataaatg tttgcagccc cacacctcct
tggcaggatg ggaaccatcc aacgttcctg 1380ccttgtggca gcttcaatga
agatctgtta ac 1412161394DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 16gttaacgaat tcccaccatg
ctggctgcca cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg cccaccgtgc
ccagcacctg aactcctggg gggaccgtca gtcttcctct 120tccccccaaa
acccaaggac accctcatga tctcccggac ccctgaggtc acatgcgtgg
180tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg
gacggcgtgg 240aggtgcataa tgccaagaca aagccgcggg aggagcagta
caacagcacg taccgtgtgg 300tcagcgtcct caccgtcctg caccaggact
ggctgaatgg caaggagtac aagtgcaagg 360tctccaacaa agccctccca
gcccccatcg agaaaaccat ctccaaagcc aaagggcagc 420cccgagaacc
acaggtgtac accctgcccc catcccggga tgagctgacc aagaaccagg
480tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg
gagtgggaga 540gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac tccgacggct 600ccttcttcct ctacagcaag ctcaccgtgg
acaagagcag gtggcagcag gggaacgtct 660tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacgcagaag agcctctccc 720tgtctccgga
gctgcaactg gaggagagct gtgcggaggc gcaggacggg gagctggacg
780ggtgcgtatc cggtgacacc attgtaatga ctagtggcgg tccgcgcact
gtggctgaac 840tggagggcaa accgttcacc gcactgattc gcggctctgg
ctacccatgc ccctcaggtt 900tcttccgcac ctgtgaacgt gacgtatatg
atctgcgtac acgtgagggt cattgcttac 960gtttgaccca tgatcaccgt
gttctggtga tggatggtgg cctggaatgg cgtgccgcgg 1020gtgaactgga
acgcggcgac cgcctggtga tggatgatgc agctggcgag tttccggcac
1080tggcaacctt ccgtggcctg cgtggcgctg gccgccagga tgtttatgac
gctactgttt 1140acggtgctag cgcattcact gctaatggct tcattgtaca
cgcatgtggc gagcagcccg 1200ggaccggtct gaactcaggc ctcacgacaa
atcctggtgt atccgcttgg caggtcaaca 1260cagcttatac tgcgggacaa
ttggtcacat ataacggcaa gacgtataaa tgtttgcagc 1320cccacacctc
cttggcagga tgggaaccat ccaacgttcc tgccttgtgg cagcttcaat
1380gaagatctgt taac 1394171820DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 17gttaacgaat
tcccaccatg aaccggggag tcccttttag gcacttgctt ctggtgctgc 60aactggcgct
cctcccagca gccactcagg gaaagaaagt ggtgctgggc aaaaaagggg
120atacagtgga actgacctgt acagcttccc agaagaagag catacaattc
cactggaaaa 180actccaacca gataaagatt ctgggaaatc agggctcctt
cttaactaaa ggtccatcca 240agctgaatga tcgcgctgac tcaagaagaa
gcctttggga ccaaggaaac ttccccctga 300tcatcaagaa tcttaagata
gaagactcag atacttacat ctgtgaagtg gaggaccaga 360aggaggaggt
gcaattgcta gtgttcggat tgactgccaa ctctgacacc cacctgcttc
420aggggcagag cctgaccctg accttggaga gcccccctgg tagtagcccc
tcagtgcaat 480gtaggagtcc aaggggtaaa aacatacagg gggggaagac
cctctccgtg tctcagctgg 540agctccagga tagtggcacc tggacatgca
ctgtcttgca gaaccagaag aaggtggagt 600tcaaaataga catcgtggtg
ctagctttcc agaaggcctc cagcatagtc tataagaaag 660agggggaaca
ggtggagttc tccttcccac tcgcctttac agttgaaaag ctgacgggca
720gtggcgagct gtggtggcag gcggagaggg cttcctcctc caagtcttgg
atcacctttg 780acctgaagaa caaggaagtg tctgtaaaac gggttaccca
ggaccctaag ctccagatgg 840gcaagaagct cccgctccac ctcaccctgc
cccaggcctt gcctcagtat gctggctctg 900gaaacctcac cctggccctt
gaagcgaaaa caggaaagtt gcatcaggaa gtgaacctgg 960tggtgatgag
agccactcag ctccagaaaa atttgacctg tgaggtgtgg ggacccacct
1020cccctaagct gatgctgagc ttgaaactgg agaacaagga ggcaaaggtc
tcgaagcggg 1080agaaggcggt gtgggtgctg aaccctgagg cggggatgtg
gcagtgtctg ctgagtgact 1140cgggacaggt cctgctggaa tccaacatca
aggttctgcc cacatggtcc accccggtgc 1200agccagggtg cgtatccggt
gacaccattg taatgactag tggcggtccg cgcactgtgg 1260ctgaactgga
gggcaaaccg ttcaccgcac tgattcgcgg ctctggctac ccatgcccct
1320caggtttctt ccgcacctgt gaacgtgacg tatatgatct gcgtacacgt
gagggtcatt 1380gcttacgttt gacccatgat caccgtgttc tggtgatgga
tggtggcctg gaatggcgtg 1440ccgcgggtga actggaacgc ggcgaccgcc
tggtgatgga tgatgcagct ggcgagtttc 1500cggcactggc aaccttccgt
ggcctgcgtg gcgctggccg ccaggatgtt tatgacgcta 1560ctgtttacgg
tgctagcgca ttcactgcta atggcttcat tgtacacgca tgtggcgagc
1620agcccgggac cggtctgaac tcaggcctca cgacaaatcc tggtgtatcc
gcttggcagg 1680tcaacacagc ttatactgcg ggacaattgg tcacatataa
cggcaagacg tataaatgtt 1740tgcagcccca cacctccttg gcaggatggg
aaccatccaa cgttcctgcc ttgtggcagc 1800ttcaatgaag atctgttaac
1820181364DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 18gttaacgaat tcccaccatg gcttgggtgt
ggaccttgct tttcctgatg gcagctgccc 60aaagtatcca agcacagatc cagttggtcc
agtctggacc tgagctgaag aagcctggag 120agacagtcaa gatctcctgc
aaggcttcag gatatacctt cacacactat ggaatgaact 180gggtgaagca
ggctccagga aagggtttaa agtggatggg ctggataaac acctacactg
240gagagccaac atatgctgat gacttcaagg aacactttgc cttctctttg
gaaacctctg 300ccagcactgt ctttttgcag atcaacaacc tcaaaaatga
ggacacggcc acatatttct 360gtgcaagaga acggggggat gctatggact
actggggtca gggaacctcc gtcaccgtct 420cctcagcctc caccaagggc
ccatcggtct tccccctggc accctcctcc aagagcacct 480ctgggggcac
agcggccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg
540tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct
gtcctacagt 600cctcaggact ctactccctc agcagcgtgg tgaccgtgcc
ctccagcagc ttgggcaccc 660agacctacat ctgcaacgtg aatcacaagc
ccagcaacac caaggtggac aagaaagttg 720agcccaaatc ttgtgacaaa
actcacacag ggtgcgtatc cggtgacacc attgtaatga 780ctagtggcgg
tccgcgcact gtggctgaac tggagggcaa accgttcacc gcactgattc
840gcggctctgg ctacccatgc ccctcaggtt tcttccgcac ctgtgaacgt
gacgtatatg 900atctgcgtac acgtgagggt cattgcttac gtttgaccca
tgatcaccgt gttctggtga 960tggatggtgg cctggaatgg cgtgccgcgg
gtgaactgga acgcggcgac cgcctggtga 1020tggatgatgc agctggcgag
tttccggcac tggcaacctt ccgtggcctg cgtggcgctg 1080gccgccagga
tgtttatgac gctactgttt acggtgctag cgcattcact gctaatggct
1140tcattgtaca cgcatgtggc gagcagcccg ggaccggtct gaactcaggc
ctcacgacaa 1200atcctggtgt atccgcttgg caggtcaaca cagcttatac
tgcgggacaa ttggtcacat 1260ataacggcaa gacgtataaa tgtttgcagc
cccacacctc cttggcagga tgggaaccat 1320ccaacgttcc tgccttgtgg
cagcttcaat gaagatctgt taac 136419737DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 19gttaacgaat tcccaccatg aagtcacaga cccaggtctt
cgtatttcta ctgctctgtg 60tgtctggtgc tcatgggagt attgtgatga cccagactcc
caaattcctg cttgtatcag 120caggagacag ggttaccata acctgcacgg
ccagtcagag tgtgagtaat gatgtagttt 180ggtaccaaca gaagccaggg
cagtctccta aaatgctgat gtattctgca ttcaatcgct 240acactggagt
ccctgatcgt ttcactggca gaggatacgg gacggatttc actttcacca
300tcagctctgt gcaggctgaa gacctggcag tttatttctg tcagcaggat
tataactctc 360ctcggacgtt cggtggaggc accaagctgg agatcaaacg
aactgtggct gcaccatctg 420tcttcatctt cccgccatct gatgagcagt
tgaaatctgg aactgcctct gttgtgtgcc 480tgctgaataa cttctatccc
agagaggcca aagtacagtg gaaggtggat aacgccctcc 540aatcgggtaa
ctcccaggag agtgtcacag agcaggacag caaggacagc acctacagcc
600tcagcagcac cctgacgctg agcaaagcag actacgagaa acacaaagtc
tacgcctgcg 660aagtcaccca tcagggcctg agctcgcccg tcacaaagag
cttcaacagg ggagagtgtt 720agtgaagatc tgttaac 737201268DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 20gttaacgaat tcccaccatg ggcctctcca ccgtgcctga
cctgctgctg ccgctggtgc 60tcctggagct gttggtggga atatacccct caggggttat
tggactggtc cctcacctag 120gggacaggga gaagagagat agtgtgtgtc
cccaaggaaa atatatccac cctcaaaata 180attcgatttg ctgtaccaag
tgccacaaag gaacctactt gtacaatgac tgtccaggcc 240cggggcagga
tacggactgc agggagtgtg agagcggctc cttcaccgct tcagaaaacc
300acctcagaca ctgcctcagc tgctccaaat gccgaaagga aatgggtcag
gtggagatct 360cttcttgcac agtggaccgg gacaccgtgt gtggctgcag
gaagaaccag taccggcatt 420attggagtga aaaccttttc cagtgcttca
attgcagcct ctgcctcaat gggaccgtgc 480acctctcctg ccaggagaaa
cagaacaccg tgtgcacctg ccatgcaggt ttctttctaa 540gagaaaacga
gtgtgtctcc tgtagtaact gtaagaaaag cctggagtgc acgaagttgt
600gcctacccca gattgagaat gttaagggca ctgaggactc aggcaccaca
gtggggtgcg 660tatccggtga caccattgta atgactagtg gcggtccgcg
cactgtggct gaactggagg 720gcaaaccgtt caccgcactg attcgcggct
ctggctaccc atgcccctca ggtttcttcc 780gcacctgtga acgtgacgta
tatgatctgc gtacacgtga gggtcattgc ttacgtttga 840cccatgatca
ccgtgttctg gtgatggatg gtggcctgga atggcgtgcc gcgggtgaac
900tggaacgcgg cgaccgcctg gtgatggatg atgcagctgg cgagtttccg
gcactggcaa 960ccttccgtgg cctgcgtggc gctggccgcc aggatgttta
tgacgctact gtttacggtg 1020ctagcgcatt cactgctaat ggcttcattg
tacacgcatg tggcgagcag cccgggaccg 1080gtctgaactc aggcctcacg
acaaatcctg gtgtatccgc ttggcaggtc aacacagctt 1140atactgcggg
acaattggtc acatataacg gcaagacgta taaatgtttg cagccccaca
1200cctccttggc aggatgggaa ccatccaacg ttcctgcctt gtggcagctt
caatgaagat 1260ctgttaac 1268211403DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 21gttaacgaat
tcccaccatg gcgcccgtcg ccgtctgggc cgcgctggcc gtcggactgg 60agctctgggc
tgcggcgcac gccttgcccg cccaggtggc atttacaccc tacgccccgg
120agcccgggag cacatgccgg ctcagagaat actatgacca gacagctcag
atgtgctgca 180gcaaatgctc gccgggccaa catgcaaaag tcttctgtac
caagacctcg gacaccgtgt 240gtgactcctg tgaggacagc acatacaccc
agctctggaa ctgggttccc gagtgcttga 300gctgtggctc ccgctgtagc
tctgaccagg tggaaactca agcctgcact cgggaacaga 360accgcatctg
cacctgcagg cccggctggt actgcgcgct gagcaagcag gaggggtgcc
420ggctgtgcgc gccgctgcgc aagtgccgcc cgggcttcgg cgtggccaga
ccaggaactg 480aaacatcaga cgtggtgtgc aagccctgtg ccccggggac
gttctccaac acgacttcat 540ccacggatat ttgcaggccc caccagatct
gtaacgtggt ggccatccct gggaatgcaa 600gcatggatgc agtctgcacg
tccacgtccc ccacccggag tatggcccca ggggcagtac 660acttacccca
gccagtgtcc acacgatccc aacacacgca gccaactcca gaacccagca
720ctgctccaag cacctccttc ctgctcccaa tgggccccag ccccccagct
gaagggagca 780ctggcgacgg gtgcgtatcc ggtgacacca ttgtaatgac
tagtggcggt ccgcgcactg 840tggctgaact ggagggcaaa ccgttcaccg
cactgattcg cggctctggc tacccatgcc 900cctcaggttt cttccgcacc
tgtgaacgtg acgtatatga tctgcgtaca cgtgagggtc 960attgcttacg
tttgacccat gatcaccgtg ttctggtgat ggatggtggc ctggaatggc
1020gtgccgcggg tgaactggaa cgcggcgacc gcctggtgat ggatgatgca
gctggcgagt 1080ttccggcact ggcaaccttc cgtggcctgc gtggcgctgg
ccgccaggat gtttatgacg 1140ctactgttta cggtgctagc gcattcactg
ctaatggctt cattgtacac gcatgtggcg 1200agcagcccgg gaccggtctg
aactcaggcc tcacgacaaa tcctggtgta tccgcttggc 1260aggtcaacac
agcttatact gcgggacaat tggtcacata taacggcaag acgtataaat
1320gtttgcagcc ccacacctcc ttggcaggat gggaaccatc caacgttcct
gccttgtggc 1380agcttcaatg aagatctgtt aac 1403222906DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 22gttaacgaat tcccaccatg gtcagctact gggacaccgg
ggtcctgctg tgcgcgctgc 60tcagctgtct gcttctcaca ggatctagtt caggttcaaa
attaaaagat cctgaactga 120gtttaaaagg cacccagcac atcatgcaag
caggccagac actgcatctc caatgcaggg 180gggaagcagc ccataaatgg
tctttgcctg aaatggtgag taaggaaagc gaaaggctga 240gcataactaa
atctgcctgt ggaagaaatg gcaaacaatt ctgcagtact ttaaccttga
300acacagctca agcaaaccac actggcttct acagctgcaa atatctagct
gtacctactt 360caaagaagaa ggaaacagaa tctgcaatct atatatttat
tagtgataca ggtagacctt 420tcgtagagat gtacagtgaa atccccgaaa
ttatacacat gactgaagga agggagctcg 480tcattccctg ccgggttacg
tcacctaaca tcactgttac tttaaaaaag tttccacttg 540acactttgat
ccctgatgga aaacgcataa tctgggacag tagaaagggc ttcatcatat
600caaatgcaac gtacaaagaa atagggcttc tgacctgtga agcaacagtc
aatgggcatt 660tgtataagac aaactatctc acacatcgac aaaccaatac
aatcatagat gtccaaataa 720gcacaccacg cccagtcaaa ttacttagag
gccatactct tgtcctcaat tgtactgcta 780ccactccctt gaacacgaga
gttcaaatga cctggagtta ccctgatgaa aaaaataaga 840gagcttccgt
aaggcgacga attgaccaaa gcaattccca tgccaacata ttctacagtg
900ttcttactat tgacaaaatg cagaacaaag acaaaggact ttatacttgt
cgtgtaagga 960gtggaccatc attcaaatct gttaacacct cagtgcatat
atatgataaa gcattcatca 1020ctgtgaaaca tcgaaaacag caggtgcttg
aaaccgtagc tggcaagcgg tcttaccggc 1080tctctatgaa agtgaaggca
tttccctcgc cggaagttgt atggttaaaa gatgggttac 1140ctgcgactga
gaaatctgct cgctatttga ctcgtggcta ctcgttaatt atcaaggacg
1200taactgaaga ggatgcaggg aattatacaa tcttgctgag cataaaacag
tcaaatgtgt 1260ttaaaaacct cactgccact ctaattgtca atgtgaaacc
ccagatttac gaaaaggccg 1320tgtcatcgtt tccagacccg gctctctacc
cactgggcag cagacaaatc ctgacttgta 1380ccgcatatgg tatccctcaa
cctacaatca agtggttctg gcacccctgt aaccataatc 1440attccgaagc
aaggtgtgac ttttgttcca ataatgaaga gtcctttatc ctggatgctg
1500acagcaacat gggaaacaga attgagagca tcactcagcg catggcaata
atagaaggaa 1560agaataagat ggctagcacc ttggttgtgg ctgactctag
aatttctgga atctacattt 1620gcatagcttc caataaagtt gggactgtgg
gaagaaacat aagcttttat atcacagatg 1680tgccaaatgg gtttcatgtt
aacttggaaa aaatgccgac ggaaggagag gacctgaaac 1740tgtcttgcac
agttaacaag ttcttataca gagacgttac ttggatttta ctgcggacag
1800ttaataacag aacaatgcac tacagtatta gcaagcaaaa aatggccatc
actaaggagc 1860actccatcac tcttaatctt accatcatga atgtttccct
gcaagattca ggcacctatg 1920cctgcagagc caggaatgta tacacagggg
aagaaatcct ccagaagaaa gaaattacaa 1980tcagagatca ggaagcacca
tacctcctgc gaaacctcag tgatcacaca gtggccatca 2040gcagttccac
cactttagac tgtcatgcta atggtgtccc cgagcctcag atcacttggt
2100ttaaaaacaa ccacaaaata caacaagagc ctggaattat tttaggacca
ggaagcagca 2160cgctgtttat tgaaagagtc acagaagagg atgaaggtgt
ctatcactgc aaagccacca 2220accagaaggg ctctgtggaa agttcagcat
acctcactgt tcaaggaacc tcggacaagt 2280ctaatctgga ggggtgcgta
tccggtgaca ccattgtaat gactagtggc ggtccgcgca 2340ctgtggctga
actggagggc aaaccgttca ccgcactgat tcgcggctct ggctacccat
2400gcccctcagg tttcttccgc acctgtgaac gtgacgtata tgatctgcgt
acacgtgagg 2460gtcattgctt acgtttgacc catgatcacc gtgttctggt
gatggatggt ggcctggaat 2520ggcgtgccgc gggtgaactg gaacgcggcg
accgcctggt gatggatgat gcagctggcg 2580agtttccggc actggcaacc
ttccgtggcc tgcgtggcgc tggccgccag gatgtttatg 2640acgctactgt
ttacggtgct agcgcattca ctgctaatgg cttcattgta cacgcatgtg
2700gcgagcagcc cgggaccggt ctgaactcag gcctcacgac aaatcctggt
gtatccgctt 2760ggcaggtcaa cacagcttat actgcgggac aattggtcac
atataacggc aagacgtata 2820aatgtttgca gccccacacc tccttggcag
gatgggaacc atccaacgtt cctgccttgt 2880ggcagcttca atgaagatct gttaac
2906232924DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 23gttaacgaat tcccaccatg cagagcaagg
tgctgctggc cgtcgccctg tggctctgcg 60tggagacccg ggccgcctct gtgggtttgc
ctagtgtttc tcttgatctg cccaggctca 120gcatacaaaa agacatactt
acaattaagg ctaatacaac tcttcaaatt acttgcaggg 180gacagaggga
cttggactgg ctttggccca ataatcagag tggcagtgag caaagggtgg
240aggtgactga gtgcagcgat ggcctcttct gtaagacact cacaattcca
aaagtgatcg 300gaaatgacac tggagcctac aagtgcttct accgggaaac
tgacttggcc tcggtcattt 360atgtctatgt tcaagattac agatctccat
ttattgcttc tgttagtgac caacatggag 420tcgtgtacat tactgagaac
aaaaacaaaa ctgtggtgat tccatgtctc gggtccattt 480caaatctcaa
cgtgtcactt tgtgcaagat acccagaaaa gagatttgtt cctgatggta
540acagaatttc ctgggacagc aagaagggct ttactattcc cagctacatg
atcagctatg 600ctggcatggt cttctgtgaa gcaaaaatta atgatgaaag
ttaccagtct attatgtaca 660tagttgtcgt tgtagggtat aggatttatg
atgtggttct gagtccgtct catggaattg 720aactatctgt tggagaaaag
cttgtcttaa attgtacagc aagaactgaa ctaaatgtgg 780ggattgactt
caactgggaa tacccttctt cgaagcatca gcataagaaa cttgtaaacc
840gagacctaaa aacccagtct gggagtgaga tgaagaaatt tttgagcacc
ttaactatag 900atggtgtaac ccggagtgac caaggattgt acacctgtgc
agcatccagt gggctgatga 960ccaagaagaa cagcacattt gtcagggtcc
atgaaaaacc ttttgttgct tttggaagtg 1020gcatggaatc tctggtggaa
gccacggtgg gggagcgtgt cagaatccct gcgaagtacc 1080ttggttaccc
acccccagaa ataaaatggt ataaaaatgg aatacccctt gagtccaatc
1140acacaattaa agcggggcat gtactgacga ttatggaagt gagtgaaaga
gacacaggaa 1200attacactgt catccttacc aatcccattt caaaggagaa
gcagagccat gtggtctctc 1260tggttgtgta tgtcccaccc cagattggtg
agaaatctct aatctctcct gtggattcct 1320accagtacgg caccactcaa
acgctgacat gtacggtcta tgccattcct cccccgcatc 1380acatccactg
gtattggcag ttggaggaag agtgcgccaa cgagcccagc caagctgtct
1440cagtgacaaa cccataccct tgtgaagaat ggagaagtgt ggaggacttc
cagggaggaa 1500ataaaattga agttaataaa aatcaatttg ctctaattga
aggaaaaaac aaaactgtaa 1560gtacccttgt tatccaagcg gcaaatgtgt
cagctttgta caaatgtgaa gcggtcaaca 1620aagtcgggag aggagagagg
gtgatctcct tccacgtgac caggggtcct gaaattactt 1680tgcaacctga
catgcagccc actgagcagg agagcgtgtc tttgtggtgc actgcagaca
1740gatctacgtt tgagaacctc acatggtaca agcttggccc acagcctctg
ccaatccatg 1800tgggagagtt gcccacacct gtttgcaaga acttggatac
tctttggaaa ttgaatgcca 1860ccatgttctc taatagcaca aatgacattt
tgatcatgga gcttaagaat gcatccttgc 1920aggaccaagg agactatgtc
tgccttgctc aagacaggaa gaccaagaaa agacattgcg 1980tggtcaggca
gctcacagtc ctagagcgtg tggcacccac gatcacagga aacctggaga
2040atcagacgac aagtattggg gaaagcatcg aagtctcatg cacggcatct
gggaatcccc 2100ctccacagat catgtggttt aaagataatg agacccttgt
agaagactca ggcattgtat 2160tgaaggatgg gaaccggaac ctcactatcc
gcagagtgag gaaggaggac gaaggcctct 2220acacctgcca ggcatgcagt
gttcttggct gtgcaaaagt ggaggcattt ttcataatag 2280aaggtgccca
ggaaaagacg aacttggaag ggtgcgtatc cggtgacacc attgtaatga
2340ctagtggcgg tccgcgcact gtggctgaac tggagggcaa accgttcacc
gcactgattc 2400gcggctctgg ctacccatgc ccctcaggtt tcttccgcac
ctgtgaacgt gacgtatatg 2460atctgcgtac acgtgagggt cattgcttac
gtttgaccca tgatcaccgt gttctggtga 2520tggatggtgg cctggaatgg
cgtgccgcgg gtgaactgga acgcggcgac cgcctggtga 2580tggatgatgc
agctggcgag tttccggcac tggcaacctt ccgtggcctg cgtggcgctg
2640gccgccagga tgtttatgac gctactgttt acggtgctag cgcattcact
gctaatggct 2700tcattgtaca cgcatgtggc gagcagcccg ggaccggtct
gaactcaggc ctcacgacaa 2760atcctggtgt atccgcttgg caggtcaaca
cagcttatac tgcgggacaa ttggtcacat 2820ataacggcaa gacgtataaa
tgtttgcagc cccacacctc cttggcagga tgggaaccat 2880ccaacgttcc
tgccttgtgg cagcttcaat gaagatctgt taac 2924242957DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 24gttaacgaat tcccaccatg cagcggggcg ccgcgctgtg
cctgcgactg tggctctgcc 60tgggactcct ggacggcctg gtgagtggct actccatgac
ccccccgacc ttgaacatca 120cggaggagtc acacgtcatc gacaccggtg
acagcctgtc catctcctgc aggggacagc 180accccctcga gtgggcttgg
ccaggagctc aggaggcgcc agccaccgga gacaaggaca 240gcgaggacac
gggggtggtg cgagactgcg agggcacaga cgccaggccc tactgcaagg
300tgttgctgct gcacgaggta catgccaacg acacaggcag ctacgtctgc
tactacaagt 360acatcaaggc acgcatcgag ggcaccacgg ccgccagctc
ctacgtgttc gtgagagact 420ttgagcagcc attcatcaac aagcctgaca
cgctcttggt caacaggaag gacgccatgt 480gggtgccctg tctggtgtcc
atccccggcc tcaatgtcac gctgcgctcg caaagctcgg 540tgctgtggcc
agacgggcag gaggtggtgt gggatgaccg gcggggcatg ctcgtgtcca
600cgccactgct gcacgatgcc ctgtacctgc agtgcgagac cacctgggga
gaccaggact 660tcctttccaa ccccttcctg gtgcacatca caggcaacga
gctctatgac atccagctgt 720tgcccaggaa gtcgctggag ctgctggtag
gggagaagct ggtcctgaac tgcaccgtgt 780gggctgagtt taactcaggt
gtcacctttg actgggacta cccagggaag caggcagagc 840ggggtaagtg
ggtgcccgag cgacgctccc agcagaccca cacagaactc tccagcatcc
900tgaccatcca caacgtcagc cagcacgacc tgggctcgta tgtgtgcaag
gccaacaacg 960gcatccagcg atttcgggag agcaccgagg tcattgtgca
tgaaaatccc ttcatcagcg 1020tcgagtggct caaaggaccc atcctggagg
ccacggcagg agacgagctg gtgaagctgc 1080ccgtgaagct ggcagcgtac
cccccgcccg agttccagtg gtacaaggat ggaaaggcac 1140tgtccgggcg
ccacagtcca catgccctgg tgctcaagga ggtgacagag gccagcacag
1200gcacctacac cctcgccctg tggaactccg ctgctggcct gaggcgcaac
atcagcctgg 1260agctggtggt gaatgtgccc ccccagatac atgagaagga
ggcctcctcc cccagcatct 1320actcgcgtca cagccgccag gccctcacct
gcacggccta cggggtgccc ctgcctctca 1380gcatccagtg gcactggcgg
ccctggacac cctgcaagat gtttgcccag cgtagtctcc 1440ggcggcggca
gcagcaagac ctcatgccac agtgccgtga ctggagggcg gtgaccacgc
1500aggatgccgt gaaccccatc gagagcctgg acacctggac cgagtttgtg
gagggaaaga 1560ataagactgt gagcaagctg gtgatccaga atgccaacgt
gtctgccatg tacaagtgtg 1620tggtctccaa caaggtgggc caggatgagc
ggctcatcta cttctatgtg accaccatcc 1680ccgacggctt caccatcgaa
tccaagccat ccgaggagct actagagggc cagccggtgc 1740tcctgagctg
ccaagccgac agctacaagt acgagcatct gcgctggtac cgcctcaacc
1800tgtccacgct gcacgatgcg cacgggaacc cgcttctgct cgactgcaag
aacgtgcatc 1860tgttcgccac ccctctggcc gccagcctgg aggaggtggc
acctggggcg cgccacgcca 1920cgctcagcct gagtatcccc cgcgtcgcgc
ccgagcacga gggccactat gtgtgcgaag 1980tgcaagaccg gcgcagccat
gacaagcact gccacaagaa gtacctgtcg gtgcaggccc 2040tggaagcccc
tcggctcacg cagaacttga ccgacctcct ggtgaacgtg agcgactcgc
2100tggagatgca gtgcttggtg gccggagcgc acgcgcccag catcgtgtgg
tacaaagacg 2160agaggctgct ggaggaaaag tctggagtcg acttggcgga
ctccaaccag aagctgagca 2220tccagcgcgt gcgcgaggag gatgcgggac
gctatctgtg cagcgtgtgc aacgccaagg 2280gctgcgtcaa ctcctccgcc
agcgtggccg tggaaggctc cgaggataag ggcagcatgg 2340aggggtgcgt
atccggtgac accattgtaa tgactagtgg cggtccgcgc actgtggctg
2400aactggaggg caaaccgttc accgcactga ttcgcggctc tggctaccca
tgcccctcag 2460gtttcttccg cacctgtgaa cgtgacgtat atgatctgcg
tacacgtgag ggtcattgct 2520tacgtttgac ccatgatcac cgtgttctgg
tgatggatgg tggcctggaa tggcgtgccg 2580cgggtgaact ggaacgcggc
gaccgcctgg tgatggatga tgcagctggc gagtttccgg 2640cactggcaac
cttccgtggc ctgcgtggcg ctggccgcca ggatgtttat gacgctactg
2700tttacggtgc tagcgcattc actgctaatg gcttcattgt acacgcatgt
ggcgagcagc 2760ccgggaccgg tctgaactca ggcctcacga caaatcctgg
tgtatccgct tggcaggtca 2820acacagctta tactgcggga caattggtca
catataacgg caagacgtat aaatgtttgc 2880agccccacac ctccttggca
ggatgggaac catccaacgt tcctgccttg tggcagcttc 2940aatgaagatc tgttaac
2957252567DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 25gttaacgaat tcccaccatg cgaccctccg
ggacggccgg ggcagcgctc ctggcgctgc 60tggctgcgct ctgcccggcg agtcgggctc
tggaggaaaa gaaagtttgc caaggcacga 120gtaacaagct cacgcagttg
ggcacttttg aagatcattt tctcagcctc cagaggatgt 180tcaataactg
tgaggtggtc cttgggaatt tggaaattac ctatgtgcag aggaattatg
240atctttcctt cttaaagacc atccaggagg tggctggtta tgtcctcatt
gccctcaaca 300cagtggagcg aattcctttg gaaaacctgc agatcatcag
aggaaatatg tactacgaaa 360attcctatgc cttagcagtc ttatctaact
atgatgcaaa taaaaccgga ctgaaggagc 420tgcccatgag aaatttacag
gaaatcctgc atggcgccgt gcggttcagc aacaaccctg 480ccctgtgcaa
cgtggagagc atccagtggc gggacatagt cagcagtgac tttctcagca
540acatgtcgat ggacttccag aaccacctgg gcagctgcca aaagtgtgat
ccaagctgtc 600ccaatgggag ctgctggggt gcaggagagg agaactgcca
gaaactgacc aaaatcatct 660gtgcccagca gtgctccggg cgctgccgtg
gcaagtcccc cagtgactgc tgccacaacc 720agtgtgctgc aggctgcaca
ggcccccggg agagcgactg cctggtctgc cgcaaattcc 780gagacgaagc
cacgtgcaag gacacctgcc ccccactcat gctctacaac cccaccacgt
840accagatgga tgtgaacccc gagggcaaat acagctttgg tgccacctgc
gtgaagaagt 900gtccccgtaa ttatgtggtg acagatcacg gctcgtgcgt
ccgagcctgt ggggccgaca 960gctatgagat ggaggaagac ggcgtccgca
agtgtaagaa gtgcgaaggg ccttgccgca 1020aagtgtgtaa cggaataggt
attggtgaat ttaaagactc actctccata aatgctacga 1080atattaaaca
cttcaaaaac tgcacctcca tcagtggcga tctccacatc ctgccggtgg
1140catttagggg tgactccttc acacatactc ctcctctgga tccacaggaa
ctggatattc 1200tgaaaaccgt aaaggaaatc acagggtttt tgctgattca
ggcttggcct gaaaacagga 1260cggacctcca tgcctttgag aacctagaaa
tcatacgcgg caggaccaag caacatggtc 1320agttttctct tgcagtcgtc
agcctgaaca taacatcctt gggattacgc tccctcaagg 1380agataagtga
tggagatgtg ataatttcag gaaacaaaaa tttgtgctat gcaaatacaa
1440taaactggaa aaaactgttt gggacctccg gtcagaaaac caaaattata
agcaacagag 1500gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc
cttgtgctcc cccgagggct 1560gctggggccc ggagcccagg gactgcgtct
cttgccggaa tgtcagccga ggcagggaat 1620gcgtggacaa gtgcaagctt
ctggagggtg agccaaggga gtttgtggag aactctgagt 1680gcatacagtg
ccacccagag tgcctgcctc aggccatgaa catcacctgc acaggacggg
1740gaccagacaa ctgtatccag tgtgcccact acattgacgg cccccactgc
gtcaagacct 1800gcccggcagg agtcatggga gaaaacaaca ccctggtctg
gaagtacgca gacgccggcc 1860atgtgtgcca cctgtgccat ccaaactgca
cctacggatg cactgggcca ggtcttgaag 1920gctgtccaac gaatgggcct
aagatcccgt ccgggtgcgt atccggtgac accattgtaa 1980tgactagtgg
cggtccgcgc actgtggctg aactggaggg caaaccgttc accgcactga
2040ttcgcggctc tggctaccca tgcccctcag gtttcttccg cacctgtgaa
cgtgacgtat 2100atgatctgcg tacacgtgag ggtcattgct tacgtttgac
ccatgatcac cgtgttctgg 2160tgatggatgg tggcctggaa tggcgtgccg
cgggtgaact ggaacgcggc gaccgcctgg 2220tgatggatga tgcagctggc
gagtttccgg cactggcaac cttccgtggc ctgcgtggcg 2280ctggccgcca
ggatgtttat gacgctactg tttacggtgc tagcgcattc actgctaatg
2340gcttcattgt acacgcatgt ggcgagcagc ccgggaccgg tctgaactca
ggcctcacga 2400caaatcctgg tgtatccgct tggcaggtca acacagctta
tactgcggga caattggtca 2460catataacgg caagacgtat aaatgtttgc
agccccacac ctccttggca ggatgggaac 2520catccaacgt tcctgccttg
tggcagcttc aatgaagatc tgttaac 2567262588DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 26gttaacgaat tcccaccatg gagctggcgg ccttgtgccg
ctgggggctc ctcctcgccc 60tcttgccccc cggagccgcg agcacccaag tgtgcaccgg
cacagacatg aagctgcggc 120tccctgccag tcccgagacc cacctggaca
tgctccgcca cctctaccag ggctgccagg 180tggtgcaggg aaacctggaa
ctcacctacc tgcccaccaa tgccagcctg tccttcctgc 240aggatatcca
ggaggtgcag ggctacgtgc tcatcgctca caaccaagtg aggcaggtcc
300cactgcagag gctgcggatt gtgcgaggca cccagctctt tgaggacaac
tatgccctgg 360ccgtgctaga caatggagac ccgctgaaca ataccacccc
tgtcacaggg gcctccccag 420gaggcctgcg ggagctgcag cttcgaagcc
tcacagagat cttgaaagga ggggtcttga 480tccagcggaa cccccagctc
tgctaccagg acacgatttt gtggaaggac atcttccaca 540agaacaacca
gctggctctc acactgatag acaccaaccg ctctcgggcc tgccacccct
600gttctccgat gtgtaagggc tcccgctgct ggggagagag ttctgaggat
tgtcagagcc 660tgacgcgcac tgtctgtgcc ggtggctgtg cccgctgcaa
ggggccactg cccactgact 720gctgccatga gcagtgtgct gccggctgca
cgggccccaa gcactctgac tgcctggcct 780gcctccactt caaccacagt
ggcatctgtg agctgcactg cccagccctg gtcacctaca 840acacagacac
gtttgagtcc atgcccaatc ccgagggccg gtatacattc ggcgccagct
900gtgtgactgc ctgtccctac aactaccttt ctacggacgt gggatcctgc
accctcgtct 960gccccctgca caaccaagag gtgacagcag aggatggaac
acagcggtgt gagaagtgca 1020gcaagccctg tgcccgagtg tgctatggtc
tgggcatgga gcacttgcga gaggtgaggg 1080cagttaccag tgccaatatc
caggagtttg ctggctgcaa gaagatcttt gggagcctgg 1140catttctgcc
ggagagcttt gatggggacc cagcctccaa cactgccccg ctccagccag
1200agcagctcca agtgtttgag actctggaag agatcacagg ttacctatac
atctcagcat 1260ggccggacag cctgcctgac ctcagcgtct tccagaacct
gcaagtaatc cggggacgaa 1320ttctgcacaa tggcgcctac tcgctgaccc
tgcaagggct gggcatcagc tggctggggc 1380tgcgctcact gagggaactg
ggcagtggac tggccctcat ccaccataac acccacctct 1440gcttcgtgca
cacggtgccc tgggaccagc tctttcggaa cccgcaccaa gctctgctcc
1500acactgccaa ccggccagag gacgagtgtg tgggcgaggg cctggcctgc
caccagctgt 1560gcgcccgagg gcactgctgg ggtccagggc ccacccagtg
tgtcaactgc agccagttcc 1620ttcggggcca ggagtgcgtg gaggaatgcc
gagtactgca ggggctcccc agggagtatg 1680tgaatgccag gcactgtttg
ccgtgccacc ctgagtgtca gccccagaat ggctcagtga 1740cctgttttgg
accggaggct gaccagtgtg tggcctgtgc ccactataag gaccctccct
1800tctgcgtggc ccgctgcccc agcggtgtga aacctgacct ctcctacatg
cccatctgga 1860agtttccaga tgaggagggc gcatgccagc cttgccccat
caactgcacc cactcctgtg 1920tggacctgga tgacaagggc tgccccgccg
agcagagagc cagccctctg acggggtgcg 1980tatccggtga caccattgta
atgactagtg gcggtccgcg cactgtggct gaactggagg 2040gcaaaccgtt
caccgcactg attcgcggct ctggctaccc atgcccctca ggtttcttcc
2100gcacctgtga acgtgacgta tatgatctgc gtacacgtga gggtcattgc
ttacgtttga 2160cccatgatca ccgtgttctg gtgatggatg gtggcctgga
atggcgtgcc gcgggtgaac 2220tggaacgcgg cgaccgcctg gtgatggatg
atgcagctgg cgagtttccg gcactggcaa 2280ccttccgtgg cctgcgtggc
gctggccgcc aggatgttta tgacgctact gtttacggtg 2340ctagcgcatt
cactgctaat ggcttcattg tacacgcatg tggcgagcag cccgggaccg
2400gtctgaactc aggcctcacg acaaatcctg gtgtatccgc ttggcaggtc
aacacagctt 2460atactgcggg acaattggtc acatataacg gcaagacgta
taaatgtttg cagccccaca 2520cctccttggc aggatgggaa ccatccaacg
ttcctgcctt gtggcagctt caatgaagat 2580ctgttaac
2588272561DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 27gttaacgaat tcccaccatg agggcgaacg
acgctctgca ggtgctgggc ttgcttttca 60gcctggcccg gggctccgag gtgggcaact
ctcaggcagt gtgtcctggg actctgaatg 120gcctgagtgt gaccggcgat
gctgagaacc aataccagac actgtacaag ctctacgaga 180ggtgtgaggt
ggtgatgggg aaccttgaga ttgtgctcac gggacacaat gccgacctct
240ccttcctgca gtggattcga gaagtgacag gctatgtcct cgtggccatg
aatgaattct 300ctactctacc attgcccaac ctccgcgtgg tgcgagggac
ccaggtctac gatgggaagt 360ttgccatctt cgtcatgttg aactataaca
ccaactccag ccacgctctg cgccagctcc 420gcttgactca gctcaccgag
attctgtcag ggggtgttta tattgagaag aacgataagc 480tttgtcacat
ggacacaatt gactggaggg acatcgtgag ggaccgagat gctgagatag
540tggtgaagga caatggcaga agctgtcccc cctgtcatga ggtttgcaag
gggcgatgct 600ggggtcctgg atcagaagac tgccagacat tgaccaagac
catctgtgct
cctcagtgta 660atggtcactg ctttgggccc aaccccaacc agtgctgcca
tgatgagtgt gccgggggct 720gctcaggccc tcaggacaca gactgctttg
cctgccggca cttcaatgac agtggagcct 780gtgtacctcg ctgtccacag
cctcttgtct acaacaagct aactttccag ctggaaccca 840atccccacac
caagtatcag tatggaggag tttgtgtagc cagctgtccc cataactttg
900tggtggatca aacatcctgt gtcagggcct gtcctcctga caagatggaa
gtagataaaa 960atgggctcaa gatgtgtgag ccttgtgggg gactatgtcc
caaagcctgt gagggaacag 1020gctctgggag ccgcttccag actgtggact
cgagcaacat tgatggattt gtgaactgca 1080ccaagatcct gggcaacctg
gactttctga tcaccggcct caatggagac ccctggcaca 1140agatccctgc
cctggaccca gagaagctca atgtcttccg gacagtacgg gagatcacag
1200gttacctgaa catccagtcc tggccgcccc acatgcacaa cttcagtgtt
ttttccaatt 1260tgacaaccat tggaggcaga agcctctaca accggggctt
ctcattgttg atcatgaaga 1320acttgaatgt cacatctctg ggcttccgat
ccctgaagga aattagtgct gggcgtatct 1380atataagtgc caataggcag
ctctgctacc accactcttt gaactggacc aaggtgcttc 1440gggggcctac
ggaagagcga ctagacatca agcataatcg gccgcgcaga gactgcgtgg
1500cagagggcaa agtgtgtgac ccactgtgct cctctggggg atgctggggc
ccaggccctg 1560gtcagtgctt gtcctgtcga aattatagcc gaggaggtgt
ctgtgtgacc cactgcaact 1620ttctgaatgg ggagcctcga gaatttgccc
atgaggccga atgcttctcc tgccacccgg 1680aatgccaacc catggagggc
actgccacat gcaatggctc gggctctgat acttgtgctc 1740aatgtgccca
ttttcgagat gggccccact gtgtgagcag ctgcccccat ggagtcctag
1800gtgccaaggg cccaatctac aagtacccag atgttcagaa tgaatgtcgg
ccctgccatg 1860agaactgcac ccaggggtgt aaaggaccag agcttcaaga
ctgtttagga caaacactgg 1920tgctgatcgg caaaacccat ctgacagggt
gcgtatccgg tgacaccatt gtaatgacta 1980gtggcggtcc gcgcactgtg
gctgaactgg agggcaaacc gttcaccgca ctgattcgcg 2040gctctggcta
cccatgcccc tcaggtttct tccgcacctg tgaacgtgac gtatatgatc
2100tgcgtacacg tgagggtcat tgcttacgtt tgacccatga tcaccgtgtt
ctggtgatgg 2160atggtggcct ggaatggcgt gccgcgggtg aactggaacg
cggcgaccgc ctggtgatgg 2220atgatgcagc tggcgagttt ccggcactgg
caaccttccg tggcctgcgt ggcgctggcc 2280gccaggatgt ttatgacgct
actgtttacg gtgctagcgc attcactgct aatggcttca 2340ttgtacacgc
atgtggcgag cagcccggga ccggtctgaa ctcaggcctc acgacaaatc
2400ctggtgtatc cgcttggcag gtcaacacag cttatactgc gggacaattg
gtcacatata 2460acggcaagac gtataaatgt ttgcagcccc acacctcctt
ggcaggatgg gaaccatcca 2520acgttcctgc cttgtggcag cttcaatgaa
gatctgttaa c 2561282585DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 28gttaacgaat tcccaccatg
aagccggcga caggactttg ggtctgggtg agccttctcg 60tggcggcggg gaccgtccag
cccagcgatt ctcagtcagt gtgtgcagga acggagaata 120aactgagctc
tctctctgac ctggaacagc agtaccgagc cttgcgcaag tactatgaaa
180actgtgaggt tgtcatgggc aacctggaga taaccagcat tgagcacaac
cgggacctct 240ccttcctgcg gtctgttcga gaagtcacag gctacgtgtt
agtggctctt aatcagtttc 300gttacctgcc tctggagaat ttacgcatta
ttcgtgggac aaaactttat gaggatcgat 360atgccttggc aatattttta
aactacagaa aagatggaaa ctttggactt caagaacttg 420gattaaagaa
cttgacagaa atcctaaatg gtggagtcta tgtagaccag aacaaattcc
480tttgttatgc agacaccatt cattggcaag atattgttcg gaacccatgg
ccttccaact 540tgactcttgt gtcaacaaat ggtagttcag gatgtggacg
ttgccataag tcctgtactg 600gccgttgctg gggacccaca gaaaatcatt
gccagacttt gacaaggacg gtgtgtgcag 660aacaatgtga cggcagatgc
tacggacctt acgtcagtga ctgctgccat cgagaatgtg 720ctggaggctg
ctcaggacct aaggacacag actgctttgc ctgcatgaat ttcaatgaca
780gtggagcatg tgttactcag tgtccccaaa cctttgtcta caatccaacc
acctttcaac 840tggagcacaa tttcaatgca aagtacacat atggagcatt
ctgtgtcaag aaatgtccac 900ataactttgt ggtagattcc agttcttgtg
tgcgtgcctg ccctagttcc aagatggaag 960tagaagaaaa tgggattaaa
atgtgtaaac cttgcactga catttgccca aaagcttgtg 1020atggcattgg
cacaggatca ttgatgtcag ctcagactgt ggattccagt aacattgaca
1080aattcataaa ctgtaccaag atcaatggga atttgatctt tctagtcact
ggtattcatg 1140gggaccctta caatgcaatt gaagccatag acccagagaa
actgaacgtc tttcggacag 1200tcagagagat aacaggtttc ctgaacatac
agtcatggcc accaaacatg actgacttca 1260gtgttttttc taacctggtg
accattggtg gaagagtact ctatagtggc ctgtccttgc 1320ttatcctcaa
gcaacagggc atcacctctc tacagttcca gtccctgaag gaaatcagcg
1380caggaaacat ctatattact gacaacagca acctgtgtta ttatcatacc
attaactgga 1440caacactctt cagcacaatc aaccagagaa tagtaatccg
ggacaacaga aaagctgaaa 1500attgtactgc tgaaggaatg gtgtgcaacc
atctgtgttc cagtgatggc tgttggggac 1560ctgggccaga ccaatgtctg
tcgtgtcgcc gcttcagtag aggaaggatc tgcatagagt 1620cttgtaacct
ctatgatggt gaatttcggg agtttgagaa tggctccatc tgtgtggagt
1680gtgaccccca gtgtgagaag atggaagatg gcctcctcac atgccatgga
ccgggtcctg 1740acaactgtac aaagtgctct cattttaaag atggcccaaa
ctgtgtggaa aaatgtccag 1800atggcttaca gggggcaaac agtttcattt
tcaagtatgc tgatccagat cgggagtgcc 1860acccatgcca tccaaactgc
acccaagggt gtaacggtcc cactagtcat gactgcattt 1920actacccatg
gacgggccat tccactttac cacaacatgc tagaactccc gggtgcgtat
1980ccggtgacac cattgtaatg actagtggcg gtccgcgcac tgtggctgaa
ctggagggca 2040aaccgttcac cgcactgatt cgcggctctg gctacccatg
cccctcaggt ttcttccgca 2100cctgtgaacg tgacgtatat gatctgcgta
cacgtgaggg tcattgctta cgtttgaccc 2160atgatcaccg tgttctggtg
atggatggtg gcctggaatg gcgtgccgcg ggtgaactgg 2220aacgcggcga
ccgcctggtg atggatgatg cagctggcga gtttccggca ctggcaacct
2280tccgtggcct gcgtggcgct ggccgccagg atgtttatga cgctactgtt
tacggtgcta 2340gcgcattcac tgctaatggc ttcattgtac acgcatgtgg
cgagcagccc gggaccggtc 2400tgaactcagg cctcacgaca aatcctggtg
tatccgcttg gcaggtcaac acagcttata 2460ctgcgggaca attggtcaca
tataacggca agacgtataa atgtttgcag ccccacacct 2520ccttggcagg
atgggaacca tccaacgttc ctgccttgtg gcagcttcaa tgaagatctg 2580ttaac
258529785DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 29aagcttgaat tcccaccatg ctgctgctgg
cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgtg
acaaaactca cacatgccca ccgtgcccag 120cacctgaact cctggggggg
ccctcagtct tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc
ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
240ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc 300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc 360aggactggct gaatggcaag gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc
aaagccaaag ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc
ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
540gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
gagaacaact 600acaagaccac gcctcccgtg ctggactccg acggctcctt
cttcctctac agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg
cagaagagcc tctccctgtc tccgggtaaa tgactcgagc 780ggccg
78530785DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 30aagcttgaat tcccaccatg gccttgacct
ttgctttact ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgtg
acaaaactca cacatgccca ccgtgcccag 120cacctgaact cctggggggg
ccctcagtct tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc
ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
240ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc 300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc 360aggactggct gaatggcaag gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc
aaagccaaag ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc
ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
540gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
gagaacaact 600acaagaccac gcctcccgtg ctggactccg acggctcctt
cttcctctac agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg
cagaagagcc tctccctgtc tccgggtaaa tgactcgagc 780ggccg
78531767DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 31aagcttgaat tcccaccatg ctggctgcca
cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg tgacaaaact cacacatgcc
caccgtgccc agcacctgaa ctcctggggg 120ggccctcagt cttcctcttc
cccccaaaac ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac
atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc aagttcaact
240ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
gagcagtaca 300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag
ccctcccagc ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc
cgagaaccac aggtgtacac cctgccccca tcccgggatg 480agctgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca
540tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
acgcctcccg 600tgctggactc cgacggctcc ttcttcctct acagcaagct
caccgtggac aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac aaccactaca 720cgcagaagag cctctccctg
tctccgggta aatgactcga gcggccg 76732251PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 32Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu
Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Asp Lys Thr
His Thr Cys Pro Pro20 25 30Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr50 55 60Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg85 90 95Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val100 105 110Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser115 120 125Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys130 135
140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp145 150 155 160Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe195 200 205Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly210 215 220Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys245 25033251PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 33Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu
Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys Asp Lys Thr
His Thr Cys Pro Pro20 25 30Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr50 55 60Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg85 90 95Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val100 105 110Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser115 120 125Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys130 135
140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp145 150 155 160Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe195 200 205Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly210 215 220Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys245 25034245PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 34Met Leu Ala Ala Thr Val Leu Thr Leu Ala Leu Leu
Gly Asn Ala His1 5 10 15Ala Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu20 25 30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val50 55 60Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser85 90 95Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu100 105 110Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala115 120 125Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro130 135
140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu195 200 205Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser210 215 220Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu
Ser Pro Gly Lys24535228PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 35Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75
80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85
90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200
205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu210 215 220Ser Pro Gly Lys22536228PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 36Xaa Asp Lys Thr His Thr Xaa Pro Pro Xaa Pro Ala
Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135
140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Gly
Lys22537222PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 37Xaa Pro Pro Xaa Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val35 40 45Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr50 55
60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val65
70 75 80Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys85 90 95Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser100 105 110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro115 120 125Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His195 200
205Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys210 215
22038219PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 38Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn35 40 45Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105 110Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp115 120
125Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe130 135 140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly180 185 190Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys210 21539224PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 39Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp35 40 45Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Tyr Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly100 105 110Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu115 120 125Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr130 135
140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn180 185 190Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr195 200 205Gln Lys Ser Leu Ser Leu
Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys210 215
22040235PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 40Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp35 40 45Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly100 105 110Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu115 120
125Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn180 185 190Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr195 200 205Gln Lys Ser Leu
Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Xaa210 215 220Ala Glu
Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230 23541234PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 41Xaa Asp Lys Thr His Thr Xaa Pro Pro Xaa Pro Ala
Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135
140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Glu Leu
Gln Leu Glu Glu Ser Cys225 23042228PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 42Xaa Pro Pro Xaa
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val35 40 45Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr50 55
60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val65
70 75 80Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys85 90 95Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser100 105 110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro115 120 125Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His195 200
205Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln
Leu210 215 220Glu Glu Ser Cys22543225PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 43Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn35 40 45Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105 110Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp115 120 125Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe130 135
140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly180 185 190Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln Lys Ser Leu Ser
Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser210 215
220Cys22544245PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 44Xaa Asp Lys Thr His Thr Xaa Pro
Pro Xaa Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215
220Ser Pro Glu Leu Gln Leu Glu Glu Ser Xaa Ala Glu Ala Gln Asp
Gly225 230 235 240Glu Leu Asp Gly Xaa24545239PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 45Xaa Pro Pro Xaa Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val35 40 45Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr50 55 60Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys85 90 95Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser100 105 110Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro115 120 125Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val130 135
140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp165 170 175Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp180 185 190Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His195 200 205Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu210 215 220Glu Glu Ser Xaa
Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230
23546236PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 46Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn35 40 45Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105 110Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp115 120
125Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe130 135 140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly180 185 190Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln Lys Ser
Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser210 215 220Xaa Ala
Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230
23547776DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 47aagcttgaat tcccaccatg ctgctgctgg
cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgtt
gtgtcgagtg cccaccgtgc ccagcaccac 120ctgtggcagg accgtcagtc
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct 180cccggacccc
tgaggtcacg tgcgtggtgg tggacgtgag ccacgaagac cccgaggtcc
240agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag
ccacgggagg 300agcagttcaa cagcacgttc cgtgtggtca gcgtcctcac
cgttgtgcac caggactggc 360tgaacggcaa ggagtacaag tgcaaggtct
ccaacaaagg cctcccagcc cccatcgaga 420aaaccatctc caaaaccaaa
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat 480cccgggagga
gatgaccaag aaccaggtca gcctgacctg cctggtcaaa ggcttctacc
540ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac
tacaagacca 600cacctcccat gctggactcc gacggctcct tcttcctcta
cagcaagctc accgtggaca 660agagcaggtg gcagcagggg aacgtcttct
catgctccgt gatgcatgag gctctgcaca 720accactacac gcagaagagc
ctctccctgt ctccgggtaa atgactcgag cggccg 77648776DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 48aagcttgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgtt gtgtcgagtg
cccaccgtgc ccagcaccac 120ctgtggcagg accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct 180cccggacccc tgaggtcacg
tgcgtggtgg tggacgtgag ccacgaagac cccgaggtcc 240agttcaactg
gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccacgggagg
300agcagttcaa cagcacgttc cgtgtggtca gcgtcctcac cgttgtgcac
caggactggc 360tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg
cctcccagcc cccatcgaga 420aaaccatctc caaaaccaaa gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat 480cccgggagga gatgaccaag
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc 540ccagcgacat
cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca
600cacctcccat gctggactcc gacggctcct tcttcctcta cagcaagctc
accgtggaca 660agagcaggtg gcagcagggg aacgtcttct
catgctccgt gatgcatgag gctctgcaca 720accactacac gcagaagagc
ctctccctgt ctccgggtaa atgactcgag cggccg 77649758DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 49aagcttgaat tcccaccatg ctggctgcca cagtcctgac
cctggccctg ctgggcaatg 60cccatgcctg ttgtgtcgag tgcccaccgt gcccagcacc
acctgtggca ggaccgtcag 120tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc cctgaggtca 180cgtgcgtggt ggtggacgtg
agccacgaag accccgaggt ccagttcaac tggtacgtgg 240acggcgtgga
ggtgcataat gccaagacaa agccacggga ggagcagttc aacagcacgt
300tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc
aaggagtaca 360agtgcaaggt ctccaacaaa ggcctcccag cccccatcga
gaaaaccatc tccaaaacca 420aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggag gagatgacca 480agaaccaggt cagcctgacc
tgcctggtca aaggcttcta ccccagcgac atcgccgtgg 540agtgggagag
caatgggcag ccggagaaca actacaagac cacacctccc atgctggact
600ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
tggcagcagg 660ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac acgcagaaga 720gcctctccct gtctccgggt aaatgactcg agcggccg
75850248PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 50Met Leu Leu Leu Ala Arg Cys Leu Leu Leu
Val Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Cys
Val Glu Cys Pro Pro Cys Pro20 25 30Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro35 40 45Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val50 55 60Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val65 70 75 80Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln85 90 95Phe Asn Ser
Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln100 105 110Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly115 120
125Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro130 135 140Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr145 150 155 160Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser165 170 175Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr180 185 190Lys Thr Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr195 200 205Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe210 215 220Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys225 230 235
240Ser Leu Ser Leu Ser Pro Gly Lys24551248PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 51Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu
Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys Cys Val Glu
Cys Pro Pro Cys Pro20 25 30Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro35 40 45Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val50 55 60Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val65 70 75 80Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln85 90 95Phe Asn Ser Thr Phe
Arg Val Val Ser Val Leu Thr Val Val His Gln100 105 110Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly115 120 125Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro130 135
140Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr145 150 155 160Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser165 170 175Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr180 185 190Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr195 200 205Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe210 215 220Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys225 230 235 240Ser
Leu Ser Leu Ser Pro Gly Lys24552242PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 52Met Leu Ala Ala
Thr Val Leu Thr Leu Ala Leu Leu Gly Asn Ala His1 5 10 15Ala Cys Cys
Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly20 25 30Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile35 40 45Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu50 55
60Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His65
70 75 80Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg85 90 95Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys100 105 110Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile Glu115 120 125Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr130 135 140Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu145 150 155 160Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp165 170 175Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met180 185 190Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp195 200
205Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His210 215 220Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro225 230 235 240Gly Lys53225PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 53Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
Val Ala Gly Pro1 5 10 15Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser20 25 30Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp35 40 45Pro Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn50 55 60Ala Lys Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Phe Arg Val65 70 75 80Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn Gly Lys Glu85 90 95Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys100 105 110Thr Ile Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr115 120 125Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr130 135
140Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu145 150 155 160Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu165 170 175Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys180 185 190Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu195 200 205Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly210 215
220Lys22554225PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 54Xaa Xaa Val Glu Xaa Pro Pro Xaa
Pro Ala Pro Pro Val Ala Gly Pro1 5 10 15Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser20 25 30Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp35 40 45Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn50 55 60Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val65 70 75 80Val Ser
Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu85 90 95Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys100 105
110Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr115 120 125Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr130 135 140Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu145 150 155 160Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Met Leu165 170 175Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys180 185 190Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu195 200 205Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly210 215
220Lys22555224PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 55Xaa Val Glu Xaa Pro Pro Xaa Pro
Ala Pro Pro Val Ala Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg20 25 30Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro35 40 45Glu Val Gln Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala50 55 60Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val65 70 75 80Ser Val
Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr85 90 95Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr100 105
110Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu115 120 125Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys130 135 140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp165 170 175Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser180 185 190Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala195 200 205Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys210 215
22056221PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 56Xaa Pro Pro Xaa Pro Ala Pro Pro Val Ala
Gly Pro Ser Val Phe Leu1 5 10 15Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu20 25 30Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln35 40 45Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys50 55 60Pro Arg Glu Glu Gln Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu65 70 75 80Thr Val Val His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys85 90 95Val Ser Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys100 105 110Thr
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser115 120
125Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys130 135 140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln145 150 155 160Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu Asp Ser Asp Gly165 170 175Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln180 185 190Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn195 200 205His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys210 215 22057218PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 57Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Asn Trp35 40 45Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val65 70 75 80His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Gly Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly100 105 110Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu115 120 125Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr130 135
140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn180 185 190Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr195 200 205Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys210 21558223PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 58Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val20 25 30Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr35 40 45Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu50 55
60Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His65
70 75 80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys85 90 95Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln100 105 110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Glu Glu Met115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro130 135 140Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu165 170 175Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val180 185 190Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln195 200
205Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys210
215 22059234PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 59Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val20 25 30Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr35 40 45Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu50 55 60Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val His65 70 75 80Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys85 90 95Gly Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln100 105
110Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
Met115 120 125Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro130 135 140Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn145 150 155 160Tyr Lys Thr Thr Pro Pro Met Leu
Asp Ser Asp Gly Ser Phe Phe Leu165 170 175Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val180 185 190Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln195 200 205Lys Ser
Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Xaa Ala210 215
220Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 23060231PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 60Xaa Xaa Val Glu Xaa Pro Pro Xaa Pro Ala Pro Pro
Val Ala Gly Pro1 5 10 15Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser20 25 30Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp35 40
45Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn50
55 60Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val65 70 75 80Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu85 90 95Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
Pro Ile Glu Lys100 105 110Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr115 120 125Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr130 135 140Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu145 150 155 160Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu165 170 175Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys180 185
190Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu195 200 205Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Glu210 215 220Leu Gln Leu Glu Glu Ser Cys225
23061230PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 61Xaa Val Glu Xaa Pro Pro Xaa Pro Ala Pro
Pro Val Ala Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg20 25 30Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro35 40 45Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala50 55 60Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val65 70 75 80Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr85 90 95Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr100 105 110Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu115 120
125Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys130 135 140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Met Leu Asp165 170 175Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser180 185 190Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala195 200 205Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu210 215 220Gln Leu
Glu Glu Ser Cys225 23062227PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 62Xaa Pro Pro Xaa
Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu1 5 10 15Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu20 25 30Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln35 40 45Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys50 55
60Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu65
70 75 80Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys85 90 95Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys100 105 110Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser115 120 125Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys130 135 140Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln145 150 155 160Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly165 170 175Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln180 185 190Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn195 200
205His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu
Glu210 215 220Glu Ser Cys22563224PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 63Cys Pro Ala Pro
Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys20 25 30Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp35 40 45Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu50 55
60Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val65
70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn85 90 95Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr130 135 140Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe165 170 175Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn180 185 190Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr195 200
205Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser
Cys210 215 22064242PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 64Xaa Xaa Val Glu Xaa Pro
Pro Xaa Pro Ala Pro Pro Val Ala Gly Pro1 5 10 15Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser20 25 30Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp35 40 45Pro Glu Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn50 55 60Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val65 70 75
80Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu85
90 95Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu
Lys100 105 110Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr115 120 125Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr130 135 140Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu145 150 155 160Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu165 170 175Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys180 185 190Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu195 200
205Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Glu210 215 220Leu Gln Leu Glu Glu Ser Xaa Ala Glu Ala Gln Asp Gly
Glu Leu Asp225 230 235 240Gly Xaa65241PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 65Xaa Val Glu Xaa Pro Pro Xaa Pro Ala Pro Pro Val
Ala Gly Pro Ser1 5 10 15Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg20 25 30Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro35 40 45Glu Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala50 55 60Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val65 70 75 80Ser Val Leu Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr85 90 95Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr100 105 110Ile Ser Lys
Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu115 120 125Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys130 135
140Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser145 150 155 160Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp165 170 175Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser180 185 190Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala195 200 205Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu210 215 220Gln Leu Glu Glu
Ser Xaa Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly225 230 235
240Xaa66238PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 66Xaa Pro Pro Xaa Pro Ala Pro Pro Val
Ala Gly Pro Ser Val Phe Leu1 5 10 15Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu20 25 30Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Gln35 40 45Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys50 55 60Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu65 70 75 80Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys85 90 95Val Ser
Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys100 105
110Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser115 120 125Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys130 135 140Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln145 150 155 160Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly165 170 175Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln180 185 190Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn195 200 205His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu210 215
220Glu Ser Xaa Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230
23567235PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 67Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp35 40 45Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Val65 70 75 80His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Gly Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly100 105 110Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu115 120
125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn180 185 190Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr195 200 205Gln Lys Ser Leu
Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Xaa210 215 220Ala Glu
Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230 23568902DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 68aagcttgaat tcccaccatg ctgctgctgg cgagatgtct
gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgcc cacggtgccc
agagcccaaa tcttgtgaca 120cacctccccc gtgcccacgg tgcccagagc
ccaaatcttg tgacacacct cccccatgcc 180cacggtgccc agagcccaaa
tcttgtgaca cacctccccc gtgcccaagg tgcccagcac 240ctgaactcct
gggaggaccg tcagtcttcc tcttcccccc aaaacccaag gataccctta
300tgatttcccg gacccctgag gtcacgtgcg tggtggtgga cgtgagccac
gaagaccccg 360aggtccagtt caagtggtac gtggacggcg tggaggtgca
taatgccaag acaaagccgc 420gggaggagca gtacaacagc acgttccgtg
tggtcagcgt cctcaccgtc ctgcaccagg 480actggctgaa cggcaaggag
tacaagtgca aggtctccaa caaagccctc ccagccccca 540tcgagaaaac
catctccaaa accaaaggac agccccgaga accacaggtg tacaccctgc
600ccccatcccg ggaggagatg accaagaacc aggtcagcct gacctgcctg
gtcaaaggct 660tctaccccag cgacatcgcc gtggagtggg agagcagcgg
gcagccggag aacaactaca 720acaccacgcc tcccatgctg gactccgacg
gctccttctt cctctacagc aagctcaccg 780tggacaagag caggtggcag
caggggaaca tcttctcatg ctccgtgatg catgaggctc 840tgcacaaccg
cttcacgcag aagagcctct ccctgtctcc gggtaaatga ctcgagcggc 900cg
90269902DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 69aagcttgaat tcccaccatg gccttgacct
ttgctttact ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc
cacggtgccc agagcccaaa tcttgtgaca 120cacctccccc gtgcccacgg
tgcccagagc ccaaatcttg tgacacacct cccccatgcc 180cacggtgccc
agagcccaaa tcttgtgaca cacctccccc gtgcccaagg tgcccagcac
240ctgaactcct gggaggaccg tcagtcttcc tcttcccccc aaaacccaag
gataccctta 300tgatttcccg gacccctgag gtcacgtgcg tggtggtgga
cgtgagccac gaagaccccg 360aggtccagtt caagtggtac gtggacggcg
tggaggtgca taatgccaag acaaagccgc 420gggaggagca gtacaacagc
acgttccgtg tggtcagcgt cctcaccgtc ctgcaccagg 480actggctgaa
cggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca
540tcgagaaaac catctccaaa accaaaggac agccccgaga accacaggtg
tacaccctgc 600ccccatcccg ggaggagatg accaagaacc aggtcagcct
gacctgcctg gtcaaaggct 660tctaccccag cgacatcgcc gtggagtggg
agagcagcgg gcagccggag aacaactaca 720acaccacgcc tcccatgctg
gactccgacg gctccttctt cctctacagc aagctcaccg 780tggacaagag
caggtggcag caggggaaca tcttctcatg ctccgtgatg catgaggctc
840tgcacaaccg cttcacgcag aagagcctct ccctgtctcc gggtaaatga
ctcgagcggc 900cg 90270884DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 70aagcttgaat tcccaccatg
ctggctgcca cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg cccacggtgc
ccagagccca aatcttgtga cacacctccc ccgtgcccac 120ggtgcccaga
gcccaaatct tgtgacacac ctcccccatg cccacggtgc ccagagccca
180aatcttgtga cacacctccc ccgtgcccaa ggtgcccagc acctgaactc
ctgggaggac 240cgtcagtctt cctcttcccc ccaaaaccca aggataccct
tatgatttcc cggacccctg 300aggtcacgtg cgtggtggtg gacgtgagcc
acgaagaccc cgaggtccag ttcaagtggt 360acgtggacgg cgtggaggtg
cataatgcca agacaaagcc gcgggaggag cagtacaaca 420gcacgttccg
tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aacggcaagg
480agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa
accatctcca 540aaaccaaagg acagccccga gaaccacagg tgtacaccct
gcccccatcc cgggaggaga 600tgaccaagaa ccaggtcagc ctgacctgcc
tggtcaaagg cttctacccc agcgacatcg 660ccgtggagtg ggagagcagc
gggcagccgg agaacaacta caacaccacg cctcccatgc 720tggactccga
cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc
780agcaggggaa catcttctca tgctccgtga tgcatgaggc tctgcacaac
cgcttcacgc 840agaagagcct ctccctgtct ccgggtaaat gactcgagcg gccg
88471290PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 71Met Leu Leu Leu Ala Arg Cys Leu Leu Leu
Val Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Pro
Arg Cys Pro Glu Pro Lys Ser20 25 30Cys Asp Thr Pro Pro Pro Cys Pro
Arg Cys Pro Glu Pro Lys Ser Cys35 40 45Asp Thr Pro Pro Pro Cys Pro
Arg Cys Pro Glu Pro Lys Ser Cys Asp50 55 60Thr Pro Pro Pro Cys Pro
Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly65 70 75 80Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile85 90 95Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu100 105 110Asp
Pro Glu Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His115 120
125Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
Arg130 135
140Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys145 150 155 160Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu165 170 175Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr180 185 190Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu195 200 205Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp210 215 220Glu Ser Ser Gly
Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met225 230 235 240Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp245 250
255Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met
His260 265 270Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser
Leu Ser Pro275 280 285Gly Lys29072290PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 72Met Ala Leu Thr Phe Ala Leu Leu Val Ala Leu Leu
Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys Pro Arg Cys
Pro Glu Pro Lys Ser20 25 30Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro Glu Pro Lys Ser Cys35 40 45Asp Thr Pro Pro Pro Cys Pro Arg Cys
Pro Glu Pro Lys Ser Cys Asp50 55 60Thr Pro Pro Pro Cys Pro Arg Cys
Pro Ala Pro Glu Leu Leu Gly Gly65 70 75 80Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile85 90 95Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu100 105 110Asp Pro Glu
Val Gln Phe Lys Trp Tyr Val Asp Gly Val Glu Val His115 120 125Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg130 135
140Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys145 150 155 160Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu165 170 175Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr180 185 190Thr Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu195 200 205Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp210 215 220Glu Ser Ser Gly
Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met225 230 235 240Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp245 250
255Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met
His260 265 270Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser
Leu Ser Pro275 280 285Gly Lys29073284PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 73Met Leu Ala Ala Thr Val Leu Thr Leu Ala Leu Leu
Gly Asn Ala His1 5 10 15Ala Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro20 25 30Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys35 40 45Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro50 55 60Arg Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe65 70 75 80Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val85 90 95Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe100 105 110Lys Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro115 120 125Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr130 135
140Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val145 150 155 160Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Thr165 170 175Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg180 185 190Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly195 200 205Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Ser Gly Gln Pro210 215 220Glu Asn Asn Tyr
Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser225 230 235 240Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln245 250
255Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
Arg260 265 270Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys275
28074267PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 74Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys1 5 10 15Pro Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro20 25 30Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg35 40 45Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro50 55 60Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr65 70 75 80Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys85 90 95Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg100 105 110Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val115 120
125Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser130 135 140Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys145 150 155 160Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu165 170 175Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe180 185 190Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu195 200 205Asn Asn Tyr Asn
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe210 215 220Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly225 230 235
240Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Phe245 250 255Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys260
26575267PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 75Xaa Pro Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa1 5 10 15Pro Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa Pro20 25 30Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa Pro Arg35 40 45Xaa Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro50 55 60Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr65 70 75 80Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys85 90 95Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg100 105 110Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val115 120
125Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser130 135 140Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys145 150 155 160Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu165 170 175Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe180 185 190Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu195 200 205Asn Asn Tyr Asn
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe210 215 220Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly225 230 235
240Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Phe245 250 255Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys260
26576264PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 76Xaa Pro Glu Pro Lys Ser Xaa Asp Thr Pro
Pro Pro Xaa Pro Arg Xaa1 5 10 15Pro Glu Pro Lys Ser Xaa Asp Thr Pro
Pro Pro Xaa Pro Arg Xaa Pro20 25 30Glu Pro Lys Ser Xaa Asp Thr Pro
Pro Pro Xaa Pro Arg Xaa Pro Ala35 40 45Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro50 55 60Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val65 70 75 80Val Asp Val Ser
His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val85 90 95Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln100 105 110Phe
Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln115 120
125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala130 135 140Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys
Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr165 170 175Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser180 185 190Asp Ile Ala Val Glu Trp
Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr195 200 205Asn Thr Thr Pro
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr210 215 220Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe225 230 235
240Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln
Lys245 250 255Ser Leu Ser Leu Ser Pro Gly Lys26077224PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 77Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Gln Phe Lys Trp35 40 45Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly100 105 110Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu115 120 125Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr130 135
140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu
Asn145 150 155 160Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn180 185 190Ile Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn Arg Phe Thr195 200 205Gln Lys Ser Leu Ser Leu
Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys210 215
22078235PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 78Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Gln Phe Lys Trp35 40 45Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly100 105 110Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu115 120
125Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr130 135 140Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln
Pro Glu Asn145 150 155 160Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp
Ser Asp Gly Ser Phe Phe165 170 175Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn180 185 190Ile Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn Arg Phe Thr195 200 205Gln Lys Ser Leu
Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Xaa210 215 220Ala Glu
Ala Gln Gly Asp Glu Leu Asp Gly Xaa225 230 23579273PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 79Xaa Pro Arg Xaa Pro Glu Pro Lys Ser Xaa Asp Thr
Pro Pro Pro Xaa1 5 10 15Pro Arg Xaa Pro Glu Pro Lys Ser Xaa Asp Thr
Pro Pro Pro Xaa Pro20 25 30Arg Xaa Pro Glu Pro Lys Ser Xaa Asp Thr
Pro Pro Pro Xaa Pro Arg35 40 45Xaa Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro50 55 60Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr65 70 75 80Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Lys85 90 95Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg100 105 110Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val115 120 125Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser130 135
140Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys145 150 155 160Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu165 170 175Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe180 185 190Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Ser Gly Gln Pro Glu195 200 205Asn Asn Tyr Asn Thr Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe210 215 220Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly225 230 235 240Asn
Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe245 250
255Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu
Ser260 265 270Cys80270PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 80Xaa Pro Glu Pro Lys Ser
Xaa Asp Thr Pro Pro Pro Xaa Pro Arg Xaa1 5 10 15Pro Glu Pro Lys Ser
Xaa Asp Thr Pro Pro Pro Xaa Pro Arg Xaa Pro20 25 30Glu Pro Lys Ser
Xaa Asp Thr Pro Pro Pro Xaa Pro Arg Xaa Pro Ala35 40 45Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro50 55 60Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65 70 75
80Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val85
90 95Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln100 105 110Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val
Leu His Gln115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala130 135 140Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr165 170 175Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser180 185 190Asp Ile
Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr195 200
205Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr210 215 220Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Ile Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu His
Asn Arg Phe Thr Gln Lys245 250 255Ser Leu Ser Leu Ser Pro Glu Leu
Gln Leu Glu Glu Ser Cys260 265
27081284PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 81Xaa Pro Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa1 5 10 15Pro Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa Pro20 25 30Arg Xaa Pro Glu Pro Lys Ser Xaa
Asp Thr Pro Pro Pro Xaa Pro Arg35 40 45Xaa Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro50 55 60Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr65 70 75 80Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys85 90 95Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg100 105 110Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val115 120
125Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser130 135 140Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys145 150 155 160Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu165 170 175Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe180 185 190Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Ser Gly Gln Pro Glu195 200 205Asn Asn Tyr Asn
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe210 215 220Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly225 230 235
240Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Phe245 250 255Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu
Glu Glu Ser260 265 270Xaa Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly
Xaa275 28082281PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 82Xaa Pro Glu Pro Lys Ser Xaa Asp
Thr Pro Pro Pro Xaa Pro Arg Xaa1 5 10 15Pro Glu Pro Lys Ser Xaa Asp
Thr Pro Pro Pro Xaa Pro Arg Xaa Pro20 25 30Glu Pro Lys Ser Xaa Asp
Thr Pro Pro Pro Xaa Pro Arg Xaa Pro Ala35 40 45Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro50 55 60Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val65 70 75 80Val Asp
Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val85 90 95Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln100 105
110Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His
Gln115 120 125Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala130 135 140Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Thr Lys Gly Gln Pro145 150 155 160Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr165 170 175Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser180 185 190Asp Ile Ala Val
Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr195 200 205Asn Thr
Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr210 215
220Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile
Phe225 230 235 240Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg
Phe Thr Gln Lys245 250 255Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu
Glu Glu Ser Xaa Ala Glu260 265 270Ala Gln Asp Gly Glu Leu Asp Gly
Xaa275 28083767DNAArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 83aagcttgaat tcccaccatg ctgctgctgg
cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgcc
catcatgccc agcacctgag ttcctggggg 120gaccatcagt cttcctgttc
cccccaaaac ccaaggacac tctcatgatc tcccggaccc 180ctgaggtcac
gtgcgtggtg gtggacgtga gccaggaaga ccccgaggtc cagttcaact
240ggtacgtgga tggcgtggag gtgcataatg ccaagacaaa gccgcgggag
gagcagttca 300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg ctgaacggca 360aggagtacaa gtgcaaggtc tccaacaaag
gcctcccgtc ctccatcgag aaaaccatct 420ccaaagccaa agggcagccc
cgagagccac aggtgtacac cctgccccca tcccaggagg 480agatgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctac cccagcgaca
540tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
acgcctcccg 600tgctggactc cgacggctcc ttcttcctct acagcaggct
aaccgtggac aagagcaggt 660ggcaggaggg gaatgtcttc tcatgctccg
tgatgcatga ggctctgcac aaccactaca 720cacagaagag cctctccctg
tctctgggta aatgactcga gcggccg 76784767DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 84aagcttgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc catcatgccc
agcacctgag ttcctggggg 120gaccatcagt cttcctgttc cccccaaaac
ccaaggacac tctcatgatc tcccggaccc 180ctgaggtcac gtgcgtggtg
gtggacgtga gccaggaaga ccccgaggtc cagttcaact 240ggtacgtgga
tggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagttca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaacggca 360aggagtacaa gtgcaaggtc tccaacaaag gcctcccgtc
ctccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagagccac
aggtgtacac cctgccccca tcccaggagg 480agatgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctac cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaggct aaccgtggac
aagagcaggt 660ggcaggaggg gaatgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cacagaagag cctctccctg tctctgggta
aatgactcga gcggccg 76785749DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 85aagcttgaat
tcccaccatg ctggctgcca cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg
cccatcatgc ccagcacctg agttcctggg gggaccatca gtcttcctgt
120tccccccaaa acccaaggac actctcatga tctcccggac ccctgaggtc
acgtgcgtgg 180tggtggacgt gagccaggaa gaccccgagg tccagttcaa
ctggtacgtg gatggcgtgg 240aggtgcataa tgccaagaca aagccgcggg
aggagcagtt caacagcacg taccgtgtgg 300tcagcgtcct caccgtcctg
caccaggact ggctgaacgg caaggagtac aagtgcaagg 360tctccaacaa
aggcctcccg tcctccatcg agaaaaccat ctccaaagcc aaagggcagc
420cccgagagcc acaggtgtac accctgcccc catcccagga ggagatgacc
aagaaccagg 480tcagcctgac ctgcctggtc aaaggcttct accccagcga
catcgccgtg gagtgggaga 540gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac tccgacggct 600ccttcttcct ctacagcagg
ctaaccgtgg acaagagcag gtggcaggag gggaatgtct 660tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacacagaag agcctctccc
720tgtctctggg taaatgactc gagcggccg 74986245PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 86Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu
Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Pro Ser Cys
Pro Ala Pro Glu Phe20 25 30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val50 55 60Ser Gln Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser85 90 95Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu100 105 110Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser115 120 125Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro130 135
140Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Arg Leu195 200 205Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser210 215 220Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu
Ser Leu Gly Lys24587245PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 87Met Ala Leu Thr Phe Ala
Leu Leu Val Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser
Val Gly Cys Pro Ser Cys Pro Ala Pro Glu Phe20 25 30Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val50 55 60Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val65 70 75
80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser85
90 95Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser115 120 125Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala165 170 175Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu195 200
205Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser210 215 220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser225 230 235 240Leu Ser Leu Gly Lys24588239PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 88Met Leu Ala Ala Thr Val Leu Thr Leu Ala Leu Leu
Gly Asn Ala His1 5 10 15Ala Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val20 25 30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr35 40 45Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu50 55 60Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys65 70 75 80Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser85 90 95Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys100 105 110Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile115 120 125Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro130 135
140Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu145 150 155 160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn165 170 175Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser180 185 190Asp Gly Ser Phe Phe Leu Tyr Ser
Arg Leu Thr Val Asp Lys Ser Arg195 200 205Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu210 215 220His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys225 230
23589222PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 89Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val35 40 45Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr50 55 60Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys85 90 95Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser100 105 110Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro115 120
125Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His195 200 205Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys210 215
22090222PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 90Xaa Pro Ser Xaa Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val35 40 45Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr50 55 60Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys85 90 95Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser100 105 110Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro115 120
125Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His195 200 205Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys210 215
22091219PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 91Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro Glu Val Gln Phe Asn35 40 45Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105 110Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu115 120
125Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe130 135 140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly180 185 190Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys210 21592224PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 92Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro1 5 10 15Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys20 25 30Val Val Val Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp35 40 45Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu50 55 60Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu65 70 75 80His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn85 90 95Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Gln Glu Glu115 120 125Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr130 135 140Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe165 170
175Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
Asn180 185 190Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr195 200 205Gln Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln
Leu Glu Glu Ser Cys210 215 22093235PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 93Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro1 5 10 15Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys20 25 30Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp35 40 45Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu50 55
60Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu65
70 75 80His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn85 90 95Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly100 105 110Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu115 120 125Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr130 135 140Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn145 150 155 160Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe165 170 175Leu Tyr Ser
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn180 185 190Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr195 200
205Gln Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln Leu Glu Glu Ser
Xaa210 215 220Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230
23594228PRTArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 94Xaa Pro Ser Xaa Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val35 40 45Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr50 55 60Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys85 90 95Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser100 105 110Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro115 120
125Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His195 200 205Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln Leu210 215 220Glu Glu
Ser Cys22595225PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 95Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn35 40 45Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105
110Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu115 120 125Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe130 135 140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly180 185 190Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln
Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln Leu Glu Glu Ser210 215
220Cys22596239PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 96Xaa Pro Ser Xaa Pro Ala Pro Glu
Phe Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu Val35 40 45Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr50 55 60Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys85 90 95Lys
Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser100 105
110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro115 120 125Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His195 200 205Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln Leu210 215
220Glu Glu Ser Xaa Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225
230 23597236PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 97Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn35 40 45Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys
Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105
110Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu115 120 125Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe130 135 140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly180 185 190Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln
Lys Ser Leu Ser Leu Ser Leu Glu Leu Gln Leu Glu Glu Ser210 215
220Xaa Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Xaa225 230
23598767DNAArtificial Sequenceartificial construct relating to Homo
Sapiens immunoglobulin 98aagcttgaat tcccaccatg ctgctgctgg
cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt atgctcggga ctggcgtgcc
caccgtgccc agcacctgaa ctcctggggg 120ggccctcagt cttcctcttc
cccccaaaac ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac
atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc aagttcaact
240ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
gagcagtaca 300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag
ccctcccagc ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc
cgagaaccac aggtgtacac cctgccccca tcccgggatg 480agctgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca
540tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
acgcctcccg 600tgctggactc cgacggctcc ttcttcctct acagcaagct
caccgtggac aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac aaccactaca 720cgcagaagag cctctccctg
tctccgggta aatgactcga gcggccg 76799767DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 99aagcttgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc caccgtgccc
agcacctgaa ctcctggggg 120ggccctcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc tcccggaccc 180ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc aagttcaact 240ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag gagcagtaca
300acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
ctgaatggca 360aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc
ccccatcgag aaaaccatct 420ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca tcccgggatg 480agctgaccaa gaaccaggtc
agcctgacct gcctggtcaa aggcttctat cccagcgaca 540tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg
600tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
aagagcaggt 660ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac aaccactaca 720cgcagaagag cctctccctg tctccgggta
aatgactcga gcggccg 767100749DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 100aagcttgaat
tcccaccatg ctggctgcca cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg
cccaccgtgc ccagcacctg aactcctggg ggggccctca gtcttcctct
120tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc
acatgcgtgg 180tggtggacgt gagccacgaa gaccctgagg tcaagttcaa
ctggtacgtg gacggcgtgg 240aggtgcataa tgccaagaca aagccgcggg
aggagcagta caacagcacg taccgtgtgg 300tcagcgtcct caccgtcctg
caccaggact ggctgaatgg caaggagtac aagtgcaagg 360tctccaacaa
agccctccca gcccccatcg agaaaaccat ctccaaagcc aaagggcagc
420cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc
aagaaccagg 480tcagcctgac ctgcctggtc aaaggcttct atcccagcga
catcgccgtg gagtgggaga 540gcaatgggca gccggagaac aactacaaga
ccacgcctcc cgtgctggac tccgacggct 600ccttcttcct ctacagcaag
ctcaccgtgg acaagagcag gtggcagcag gggaacgtct 660tctcatgctc
cgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc
720tgtctccggg taaatgactc gagcggccg 749101245PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 101Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu
Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Pro Pro Cys
Pro Ala Pro Glu Leu20 25 30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val50 55 60Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser85 90 95Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu100 105 110Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala115 120 125Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro130 135
140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu195 200 205Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser210 215 220Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu
Ser Pro Gly Lys245102245PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 102Met Ala Leu Thr Phe Ala
Leu Leu Val Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser
Val Gly Cys Pro Pro Cys Pro Ala Pro Glu Leu20 25 30Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val50 55 60Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val65 70 75
80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser85
90 95Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala165 170 175Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu195 200
205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser210 215 220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser225 230 235 240Leu Ser Pro Gly Lys245103239PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 103Met Leu Ala Ala Thr Val Leu Thr Leu Ala Leu Leu
Gly Asn Ala His1 5 10 15Ala Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val20 25 30Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr35 40 45Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu50 55 60Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys65 70 75 80Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser85 90 95Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys100 105 110Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile115 120 125Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro130 135
140Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu145 150 155 160Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn165 170 175Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser180 185 190Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg195 200 205Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu210 215 220His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys225 230
235104222PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 104Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val35 40 45Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr50 55
60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val65
70 75 80Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys85 90 95Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser100 105 110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro115 120 125Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp180 185 190Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His195 200
205Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys210 215
220105228PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 105Xaa Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215
220Ser Pro Gly Lys225106758DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 106aagcttgaat
tcccaccatg ctgctgctgg cgagatgtct gctgctagtc ctcgtctcct 60cgctgctggt
atgctcggga ctggcgtgcc cagcacctga actcctgggg gggccctcag
120tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
cctgaggtca 180catgcgtggt ggtggacgtg agccacgaag accctgaggt
caagttcaac tggtacgtgg 240acggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagtac aacagcacgt 300accgtgtggt cagcgtcctc
accgtcctgc accaggactg gctgaatggc aaggagtaca 360agtgcaaggt
ctccaacaaa gccctcccag cccccatcga gaaaaccatc tccaaagcca
420aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat
gagctgacca 480agaaccaggt cagcctgacc tgcctggtca aaggcttcta
tcccagcgac atcgccgtgg 540agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc gtgctggact 600ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga caagagcagg tggcagcagg 660ggaacgtctt
ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga
720gcctctccct gtctccgggt aaatgactcg agcggccg 758107758DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 107aagcttgaat tcccaccatg gccttgacct ttgctttact
ggtggccctc ctggtgctca 60gctgcaagtc aagctgctct gtgggctgcc cagcacctga
actcctgggg gggccctcag 120tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc cctgaggtca 180catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac tggtacgtgg 240acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac aacagcacgt
300accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
aaggagtaca 360agtgcaaggt ctccaacaaa gccctcccag cccccatcga
gaaaaccatc tccaaagcca 420aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atcccgggat gagctgacca 480agaaccaggt cagcctgacc
tgcctggtca aaggcttcta tcccagcgac atcgccgtgg 540agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc gtgctggact
600ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
tggcagcagg 660ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac acgcagaaga 720gcctctccct gtctccgggt aaatgactcg agcggccg
758108740DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 108aagcttgaat tcccaccatg ctggctgcca
cagtcctgac cctggccctg ctgggcaatg 60cccatgcctg cccagcacct gaactcctgg
gggggccctc agtcttcctc ttccccccaa 120aacccaagga caccctcatg
atctcccgga cccctgaggt cacatgcgtg gtggtggacg 180tgagccacga
agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata
240atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg
gtcagcgtcc 300tcaccgtcct gcaccaggac tggctgaatg gcaaggagta
caagtgcaag gtctccaaca 360aagccctccc agcccccatc gagaaaacca
tctccaaagc caaagggcag ccccgagaac 420cacaggtgta caccctgccc
ccatcccggg atgagctgac caagaaccag gtcagcctga 480cctgcctggt
caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc
540agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc
tccttcttcc 600tctacagcaa gctcaccgtg gacaagagca ggtggcagca
ggggaacgtc ttctcatgct 660ccgtgatgca tgaggctctg cacaaccact
acacgcagaa gagcctctcc ctgtctccgg 720gtaaatgact cgagcggccg
740109242PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 109Met Leu Leu Leu Ala Arg Cys Leu Leu
Leu Val Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys
Pro Ala Pro Glu Leu Leu Gly Gly20 25 30Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile35 40 45Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu50 55 60Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His65 70 75 80Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg85 90 95Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys100 105
110Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu115 120 125Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr130 135 140Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu145 150 155 160Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp165 170 175Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val180 185 190Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp195 200 205Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His210 215
220Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro225 230 235 240Gly Lys110242PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 110Met Ala Leu
Thr Phe Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser
Ser Cys Ser Val Gly Cys Pro Ala Pro Glu Leu Leu Gly Gly20 25 30Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile35 40
45Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu50
55 60Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His65 70 75 80Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg85 90 95Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys100 105 110Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu115 120 125Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr130 135 140Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu145 150 155 160Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp165 170 175Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val180 185
190Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp195 200 205Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His210 215 220Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro225 230 235 240Gly Lys111236PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 111Met Leu Ala Ala Thr Val Leu Thr Leu Ala Leu Leu
Gly Asn Ala His1 5 10 15Ala Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe20 25 30Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val35 40 45Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe50 55 60Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro65 70 75 80Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr85 90 95Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val100 105 110Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala115 120 125Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg130 135
140Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly145 150 155 160Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro165 170 175Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser180 185 190Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln195 200 205Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His210 215 220Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys225 230 235112219PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 112Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro1 5 10 15Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr20 25 30Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn35 40 45Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg50 55 60Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val65 70 75 80Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser85 90 95Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys100 105 110Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp115 120 125Glu
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe130 135
140Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu145 150 155 160Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe165 170 175Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly180 185 190Asn Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr195 200 205Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly Lys210 215113222PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 113Xaa Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val35 40
45Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr50
55 60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val65 70 75 80Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys85 90 95Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser100 105 110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro115 120 125Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp180 185
190Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His195 200 205Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys210 215 2201141427DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 114aagcttgaat tcccaccatg
agctttccat gtaaatttgt agccagcttc cttctgattt 60tcaatgtttc ttccaaaggt
gcagtctcca aaactcacac atgcccaccg tgcccagcac 120ctgaactcct
gggggggccc tcagtcttcc tcttcccccc aaaacccaag gacaccctca
180tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac
gaagaccctg 240aggtcaagtt caactggtac gtggacggcg tggaggtgca
taatgccaag acaaagccgc 300gggaggagca gtacaacagc acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg 360actggctgaa tggcaaggag
tacaagtgca aggtctccaa caaagccctc ccagccccca 420tcgagaaaac
catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc
480ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg
gtcaaaggct 540tctatcccag cgacatcgcc gtggagtggg agagcaatgg
gcagccggag aacaactaca 600agaccacgcc tcccgtgctg gactccgacg
gctccttctt cctctacagc aagctcaccg 660tggacaagag caggtggcag
caggggaacg tcttctcatg ctccgtgatg catgaggctc 720tgcacaacca
ctacacgcag aagagcctct ccctgtctcc ggagctgcaa ctggaggaga
780gctgtgcgga ggcgcaggac ggggagctgg acgggtgcgt atccggtgac
accattgtaa 840tgactagtgg cggtccgcgc actgtggctg aactggaggg
caaaccgttc accgcactga 900ttcgcggctc tggctaccca tgcccctcag
gtttcttccg cacctgtgaa cgtgacgtat 960atgatctgcg tacacgtgag
ggtcattgct tacgtttgac ccatgatcac cgtgttctgg 1020tgatggatgg
tggcctggaa tggcgtgccg cgggtgaact ggaacgcggc gaccgcctgg
1080tgatggatga tgcagctggc gagtttccgg cactggcaac cttccgtggc
ctgcgtggcg 1140ctggccgcca ggatgtttat gacgctactg tttacggtgc
tagcgcattc actgctaatg 1200gcttcattgt acacgcatgt ggcgagcagc
ccgggaccgg tctgaactca ggcctcacga 1260caaatcctgg tgtatccgct
tggcaggtca acacagctta tactgcggga caattggtca 1320catataacgg
caagacgtat aaatgtttgc agccccacac ctccttggca ggatgggaac
1380catccaacgt tcctgccttg tggcagcttc aatgactcga gcggccg
14271151430DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 115aagcttgaat tcccaccatg aaccggggag
tcccttttag gcacttgctt ctggtgctgc 60aactggcgct cctcccagca gccactcagg
gaaaaactca cacatgccca ccgtgcccag 120cacctgaact cctggggggg
ccctcagtct tcctcttccc cccaaaaccc aaggacaccc 180tcatgatctc
ccggacccct gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
240ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc 300cgcgggagga gcagtacaac agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc 360aggactggct gaatggcaag gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc 420ccatcgagaa aaccatctcc
aaagccaaag ggcagccccg agaaccacag gtgtacaccc 480tgcccccatc
ccgggatgag ctgaccaaga accaggtcag cctgacctgc ctggtcaaag
540gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg
gagaacaact 600acaagaccac gcctcccgtg ctggactccg acggctcctt
cttcctctac agcaagctca 660ccgtggacaa gagcaggtgg cagcagggga
acgtcttctc atgctccgtg atgcatgagg 720ctctgcacaa ccactacacg
cagaagagcc tctccctgtc tccggagctg caactggagg 780agagctgtgc
ggaggcgcag gacggggagc tggacgggtg cgtatccggt gacaccattg
840taatgactag tggcggtccg cgcactgtgg ctgaactgga gggcaaaccg
ttcaccgcac 900tgattcgcgg ctctggctac ccatgcccct caggtttctt
ccgcacctgt gaacgtgacg 960tatatgatct gcgtacacgt gagggtcatt
gcttacgttt gacccatgat caccgtgttc 1020tggtgatgga tggtggcctg
gaatggcgtg ccgcgggtga actggaacgc ggcgaccgcc 1080tggtgatgga
tgatgcagct ggcgagtttc cggcactggc aaccttccgt ggcctgcgtg
1140gcgctggccg ccaggatgtt tatgacgcta ctgtttacgg tgctagcgca
ttcactgcta 1200atggcttcat tgtacacgca tgtggcgagc agcccgggac
cggtctgaac tcaggcctca 1260cgacaaatcc tggtgtatcc gcttggcagg
tcaacacagc ttatactgcg ggacaattgg 1320tcacatataa cggcaagacg
tataaatgtt tgcagcccca cacctccttg gcaggatggg 1380aaccatccaa
cgttcctgcc ttgtggcagc ttcaatgact cgagcggccg 1430116465PRTArtificial
Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 116Met Ser Phe Pro Cys Lys Phe Val
Ala Ser Phe Leu Leu Ile Phe Asn1 5 10 15Val Ser Ser Lys Gly Ala Val
Ser Lys Thr His Thr Cys Pro Pro Cys20 25 30Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro35 40 45Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys50 55 60Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp65 70 75 80Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu85 90 95Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu100 105
110His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn115 120 125Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly130 135 140Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu145 150 155 160Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr165 170 175Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn180 185 190Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe195 200 205Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn210 215
220Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr225 230 235 240Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu
Glu Glu Ser Cys245 250 255Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly
Cys Val Ser Gly Asp Thr260 265 270Ile Val Met Thr Ser Gly Gly Pro
Arg Thr Val Ala Glu Leu Glu Gly275 280 285Lys Pro Phe Thr Ala Leu
Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser290 295 300Gly Phe Phe Arg
Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg305 310 315 320Glu
Gly His Cys Leu Arg Leu Thr His Asp His Arg Val Leu Val Met325 330
335Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly
Asp340 345 350Arg Leu Val Met Asp Asp Ala Ala Gly Glu Phe Pro Ala
Leu Ala Thr355 360 365Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp
Val Tyr Asp Ala Thr370 375 380Val Tyr Gly Ala Ser Ala Phe Thr Ala
Asn Gly Phe Ile Val His Ala385 390 395 400Cys Gly Glu Gln Pro Gly
Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn405 410 415Pro Gly Val Ser
Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln420 425 430Leu Val
Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro His Thr435 440
445Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp Gln
Leu450 455 460Gln465117466PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 117Met Asn Arg
Gly Val Pro Phe Arg His Leu Leu Leu Val Leu Gln Leu1 5 10 15Ala Leu
Leu Pro Ala Ala Thr Gln Gly Lys Thr His Thr Cys Pro Pro20 25 30Cys
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro35 40
45Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr50
55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn65 70 75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg85 90 95Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val100 105 110Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser115 120 125Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys130 135 140Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp145 150 155 160Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe165 170 175Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu180 185
190Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe195 200 205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly210 215 220Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr225 230 235 240Thr Gln Lys Ser Leu Ser Leu Ser
Pro Glu Leu Gln Leu Glu Glu Ser245 250 255Cys Ala Glu Ala Gln Asp
Gly Glu Leu Asp Gly Cys Val Ser Gly Asp260 265 270Thr Ile Val Met
Thr Ser Gly Gly Pro Arg Thr Val Ala Glu Leu Glu275 280 285Gly Lys
Pro Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys Pro290 295
300Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg
Thr305 310 315 320Arg Glu Gly His Cys Leu Arg Leu Thr His Asp His
Arg Val Leu Val325 330 335Met Asp Gly Gly Leu Glu Trp Arg Ala Ala
Gly Glu Leu Glu Arg Gly340 345 350Asp Arg Leu Val Met Asp Asp Ala
Ala Gly Glu Phe Pro Ala Leu Ala355 360 365Thr Phe Arg Gly Leu Arg
Gly Ala Gly Arg Gln Asp Val Tyr Asp Ala370 375 380Thr Val Tyr Gly
Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile Val His385 390 395 400Ala
Cys Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu Thr Thr405 410
415Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala
Gly420 425 430Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu
Gln Pro His435 440 445Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val
Pro Ala Leu Trp Gln450 455 460Leu Gln465118441PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 118Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile20 25 30Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu35 40 45Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His50 55 60Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75 80Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys85 90 95Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu100 105 110Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr115 120 125Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu130 135
140Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp145 150 155 160Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val165 170 175Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp180 185 190Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His195 200 205Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro210 215 220Glu Leu Gln Leu
Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu225 230 235 240Asp
Gly Cys Val Ser Gly Asp Thr Ile Val Met Thr Ser Gly Gly Pro245 250
255Arg Thr Val Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile
Arg260 265 270Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr
Cys Glu Arg275 280 285Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His
Cys Leu Arg Leu Thr290 295 300His Asp His Arg Val Leu Val Met Asp
Gly Gly Leu Glu Trp Arg Ala305 310 315 320Ala Gly Glu Leu Glu Arg
Gly Asp Arg Leu Val Met Asp Asp Ala Ala325 330 335Gly Glu Phe Pro
Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly340 345 350Arg Gln
Asp Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala Phe Thr355 360
365Ala Asn Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro Gly Thr
Gly370 375 380Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val Ser Ala
Trp Gln Val385 390 395 400Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val
Thr Tyr Asn Gly Lys Thr405 410 415Tyr Lys Cys Leu Gln Pro His Thr
Ser Leu Ala Gly Trp Glu Pro Ser420 425 430Asn Val Pro Ala Leu Trp
Gln Leu Gln435 440119242PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 119Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile20 25 30Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu35 40 45Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His50 55 60Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75
80Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys85
90 95Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu100 105 110Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr115 120 125Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu130 135 140Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp145 150 155 160Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val165 170 175Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp180 185 190Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His195 200
205Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro210 215 220Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp
Gly Glu Leu225 230 235 240Asp Xaa120243PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 120Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile20 25 30Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu35 40 45Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His50 55 60Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75 80Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys85 90 95Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu100 105 110Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr115 120 125Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu130 135
140Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp145 150 155 160Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val165 170 175Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp180 185 190Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His195 200 205Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro210 215 220Glu Leu Gln Leu
Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu Leu225 230 235 240Asp
Gly Xaa121800DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 121aagcttgaat tcccaccatg agctttccat
gtaaatttgt agccagcttc cttctgattt 60tcaatgtttc ttccaaaggt gcagtctcca
aaactcacac atgcccaccg tgcccagcac 120ctgaactcct gggggggccc
tcagtcttcc tcttcccccc aaaacccaag gacaccctca 180tgatctcccg
gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
240aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag
acaaagccgc 300gggaggagca gtacaacagc acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg 360actggctgaa tggcaaggag tacaagtgca
aggtctccaa caaagccctc ccagccccca 420tcgagaaaac catctccaaa
gccaaagggc agccccgaga accacaggtg tacaccctgc 480ccccatcccg
ggatgagctg accaagaacc aggtcagcct gacctgcctg gtcaaaggct
540tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag
aacaactaca 600agaccacgcc tcccgtgctg gactccgacg gctccttctt
cctctacagc aagctcaccg 660tggacaagag caggtggcag caggggaacg
tcttctcatg ctccgtgatg catgaggctc 720tgcacaacca ctacacgcag
aagagcctct ccctgtctcc ggagctgcaa ctggaggaga 780gctgttgact
cgagcggccg 800122803DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 122aagcttgaat tcccaccatg
aaccggggag tcccttttag gcacttgctt ctggtgctgc 60aactggcgct cctcccagca
gccactcagg gaaaaactca cacatgccca ccgtgcccag 120cacctgaact
cctggggggg ccctcagtct tcctcttccc cccaaaaccc aaggacaccc
180tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc 240ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc 300cgcgggagga gcagtacaac agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc 360aggactggct gaatggcaag
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc 420ccatcgagaa
aaccatctcc aaagccaaag ggcagccccg agaaccacag gtgtacaccc
480tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc
ctggtcaaag 540gcttctatcc cagcgacatc gccgtggagt gggagagcaa
tgggcagccg gagaacaact 600acaagaccac gcctcccgtg ctggactccg
acggctcctt cttcctctac agcaagctca 660ccgtggacaa gagcaggtgg
cagcagggga acgtcttctc atgctccgtg atgcatgagg 720ctctgcacaa
ccactacacg cagaagagcc tctccctgtc tccggagctg caactggagg
780agagctgttg actcgagcgg ccg 803123256PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 123Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu
Leu Ile Phe Asn1 5 10 15Val Ser Ser Lys Gly Ala Val Ser Lys Thr His
Thr Cys Pro Pro Cys20 25 30Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro35 40 45Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys50 55 60Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp65 70 75 80Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu85 90 95Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu100 105 110His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn115 120 125Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly130 135
140Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu145 150 155 160Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr165 170 175Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn180 185 190Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe195 200 205Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn210 215 220Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr225 230 235 240Gln
Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys245 250
255124257PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 124Met Asn Arg Gly Val Pro Phe Arg His
Leu Leu Leu Val Leu Gln Leu1 5 10 15Ala Leu Leu Pro Ala Ala Thr Gln
Gly Lys Thr His Thr Cys Pro Pro20 25 30Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro35 40 45Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr50 55 60Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg85 90 95Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val100 105
110Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser115 120 125Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys130 135
140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp145 150 155 160Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe195 200 205Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly210 215 220Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser245 250
255Cys125232PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 125Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile20 25 30Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu35 40 45Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His50 55 60Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75 80Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys85 90 95Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu100 105
110Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr115 120 125Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu130 135 140Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp145 150 155 160Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val165 170 175Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp180 185 190Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His195 200 205Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro210 215
220Glu Leu Gln Leu Glu Glu Ser Cys225 230126466PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 126Met Leu Leu Leu Ala Arg Cys Leu Leu Leu Val Leu
Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys Asp Lys Thr
His Thr Cys Pro Pro20 25 30Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr50 55 60Cys Val Val Val Asp Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg85 90 95Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val100 105 110Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser115 120 125Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys130 135
140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp145 150 155 160Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe195 200 205Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly210 215 220Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr
Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser245 250
255Cys Ala Glu Ala Gln Asp Gly Glu Leu Asp Gly Cys Val Ser Gly
Asp260 265 270Thr Ile Val Met Thr Ser Gly Gly Pro Arg Thr Val Ala
Glu Leu Glu275 280 285Gly Lys Pro Phe Thr Ala Leu Ile Arg Gly Ser
Gly Tyr Pro Cys Pro290 295 300Ser Gly Phe Phe Arg Thr Cys Glu Arg
Asp Val Tyr Asp Leu Arg Thr305 310 315 320Arg Glu Gly His Cys Leu
Arg Leu Thr His Asp His Arg Val Leu Val325 330 335Met Asp Gly Gly
Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly340 345 350Asp Arg
Leu Val Met Asp Asp Ala Ala Gly Glu Phe Pro Ala Leu Ala355 360
365Thr Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp Val Tyr Asp
Ala370 375 380Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn Gly Phe
Ile Val His385 390 395 400Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu
Asn Ser Gly Leu Thr Thr405 410 415Asn Pro Gly Val Ser Ala Trp Gln
Val Asn Thr Ala Tyr Thr Ala Gly420 425 430Gln Leu Val Thr Tyr Asn
Gly Lys Thr Tyr Lys Cys Leu Gln Pro His435 440 445Thr Ser Leu Ala
Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp Gln450 455 460Leu
Gln465127466PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 127Met Ala Leu Thr Phe Ala Leu Leu Val
Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys
Asp Lys Thr His Thr Cys Pro Pro20 25 30Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro35 40 45Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr50 55 60Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg85 90 95Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val100 105
110Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys130 135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp145 150 155 160Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe165 170 175Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu180 185 190Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe195 200 205Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly210 215
220Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr225 230 235 240Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu Gln
Leu Glu Glu Ser245 250 255Cys Ala Glu Ala Gln Asp Gly Glu Leu Asp
Gly Cys Val Ser Gly Asp260 265 270Thr Ile Val Met Thr Ser Gly Gly
Pro Arg Thr Val Ala Glu Leu Glu275 280 285Gly Lys Pro Phe Thr Ala
Leu Ile Arg Gly Ser Gly Tyr Pro Cys Pro290 295 300Ser Gly Phe Phe
Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg Thr305 310 315 320Arg
Glu Gly His Cys Leu Arg Leu Thr His Asp His Arg Val Leu Val325 330
335Met Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu Arg
Gly340 345 350Asp Arg Leu Val Met Asp Asp Ala Ala Gly Glu Phe Pro
Ala Leu Ala355 360 365Thr Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln
Asp Val Tyr Asp Ala370 375 380Thr Val Tyr Gly Ala Ser Ala Phe Thr
Ala Asn Gly Phe Ile Val His385 390 395 400Ala Cys Gly Glu Gln Pro
Gly Thr Gly Leu Asn Ser Gly Leu Thr Thr405 410 415Asn Pro Gly Val
Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly420 425 430Gln Leu
Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro His435 440
445Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp
Gln450 455 460Leu Gln465128460PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 128Met Leu Ala
Ala Thr Val Leu Thr Leu Ala Leu Leu Gly Asn Ala His1 5 10 15Ala Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu20 25 30Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr35 40
45Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val50
55 60Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val65 70 75 80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser85 90 95Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala115 120 125Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro130 135 140Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala165 170 175Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr180 185
190Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu195 200 205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser210 215 220Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser225 230 235 240Leu Ser Pro Glu Leu Gln Leu Glu
Glu Ser Cys Ala Glu Ala Gln Asp245 250 255Gly Glu Leu Asp Gly Cys
Val Ser Gly Asp Thr Ile Val Met Thr Ser260 265 270Gly Gly Pro Arg
Thr Val Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala275 280 285Leu Ile
Arg Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr290 295
300Cys Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys
Leu305 310 315 320Arg Leu Thr His Asp His Arg Val Leu Val Met Asp
Gly Gly Leu Glu325 330 335Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly
Asp Arg Leu Val Met Asp340 345 350Asp Ala Ala Gly Glu Phe Pro Ala
Leu Ala Thr Phe Arg Gly Leu Arg355 360 365Gly Ala Gly Arg Gln Asp
Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser370 375 380Ala Phe Thr Ala
Asn Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro385 390 395 400Gly
Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val Ser Ala405 410
415Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr
Asn420 425 430Gly Lys Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu
Ala Gly Trp435 440 445Glu Pro Ser Asn Val Pro Ala Leu Trp Gln Leu
Gln450 455 460129443PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 129Cys Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75
80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85
90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200
205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu210 215 220Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala
Gln Asp Gly225 230 235 240Glu Leu Asp Gly Cys Val Ser Gly Asp Thr
Ile Val Met Thr Ser Gly245 250 255Gly Pro Arg Thr Val Ala Glu Leu
Glu Gly Lys Pro Phe Thr Ala Leu260 265 270Ile Arg Gly Ser Gly Tyr
Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys275 280 285Glu Arg Asp Val
Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg290 295 300Leu Thr
His Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu Trp305 310 315
320Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met Asp
Asp325 330 335Ala Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly
Leu Arg Gly340 345 350Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr Val
Tyr Gly Ala Ser Ala355 360 365Phe Thr Ala Asn Gly Phe Ile Val His
Ala Cys Gly Glu Gln Pro Gly370 375 380Thr Gly Leu Asn Ser Gly Leu
Thr Thr Asn Pro Gly Val Ser Ala Trp385 390 395 400Gln Val Asn Thr
Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly405 410 415Lys Thr
Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp Glu420 425
430Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln435
440130244PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 130Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215
220Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp
Gly225 230 235 240Glu Leu Asp Xaa131245PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 131Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys
Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155
160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Glu Leu Gln Leu Glu
Glu Ser Cys Ala Glu Ala Gln Asp Gly225 230 235 240Glu Leu Asp Gly
Xaa245132460PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 132Met Leu Leu Leu Ala Arg Cys Leu Leu
Leu Val Leu Val Ser Ser Leu1 5 10 15Leu Val Cys Ser Gly Leu Ala Cys
Pro Pro Cys Pro Ala Pro Glu Leu20 25 30Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val50 55 60Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser85 90 95Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu100 105
110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala165 170 175Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu195 200 205Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser210 215
220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser225 230 235 240Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala
Glu Ala Gln Asp245 250 255Gly Glu Leu Asp Gly Cys Val Ser Gly Asp
Thr Ile Val Met Thr Ser260 265 270Gly Gly Pro Arg Thr Val Ala Glu
Leu Glu Gly Lys Pro Phe Thr Ala275 280 285Leu Ile Arg Gly Ser Gly
Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr290 295 300Cys Glu Arg Asp
Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu305 310 315 320Arg
Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu325 330
335Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met
Asp340 345 350Asp Ala Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg
Gly Leu Arg355 360 365Gly Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr
Val Tyr Gly Ala Ser370 375 380Ala Phe Thr Ala Asn Gly Phe Ile Val
His Ala Cys Gly Glu Gln Pro385 390 395 400Gly Thr Gly Leu Asn Ser
Gly Leu Thr Thr Asn Pro Gly Val Ser Ala405 410 415Trp Gln Val Asn
Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn420 425 430Gly Lys
Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp435 440
445Glu Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln450 455
460133460PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 133Met Ala Leu Thr Phe Ala Leu Leu Val
Ala Leu Leu Val Leu Ser Cys1 5 10 15Lys Ser Ser Cys Ser Val Gly Cys
Pro Pro Cys Pro Ala Pro Glu Leu20 25 30Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr35 40 45Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val50 55 60Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser85 90 95Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu100 105
110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala165 170 175Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr180 185 190Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu195 200 205Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser210 215
220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser225 230 235 240Leu Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala
Glu Ala Gln Asp245 250 255Gly Glu Leu Asp Gly Cys Val Ser Gly Asp
Thr Ile Val Met Thr Ser260 265 270Gly Gly Pro Arg Thr Val Ala Glu
Leu Glu Gly Lys Pro Phe Thr Ala275 280 285Leu Ile Arg Gly Ser Gly
Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr290 295 300Cys Glu Arg Asp
Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu305 310 315 320Arg
Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu325 330
335Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met
Asp340 345 350Asp Ala Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg
Gly Leu Arg355 360 365Gly Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr
Val Tyr Gly Ala Ser370 375 380Ala Phe Thr Ala Asn Gly Phe Ile Val
His Ala Cys Gly Glu Gln Pro385 390 395 400Gly Thr Gly Leu Asn Ser
Gly Leu Thr Thr Asn Pro Gly Val Ser Ala405 410 415Trp Gln Val Asn
Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn420 425 430Gly Lys
Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp435 440
445Glu Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln450 455
460134454PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 134Met Leu Ala Ala Thr Val Leu Thr Leu
Ala Leu Leu Gly Asn Ala His1 5 10 15Ala Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val20 25 30Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr35 40 45Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu50 55 60Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys65 70 75 80Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser85 90 95Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys100 105
110Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile115 120 125Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro130 135 140Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu145 150 155 160Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn165 170 175Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser180 185 190Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg195 200 205Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu210 215
220His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu Leu
Gln225 230 235 240Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu
Leu Asp Gly Cys245 250 255Val Ser Gly Asp Thr Ile Val Met Thr Ser
Gly Gly Pro Arg Thr Val260 265 270Ala Glu Leu Glu Gly Lys Pro Phe
Thr Ala Leu Ile Arg Gly Ser Gly275 280 285Tyr Pro Cys Pro Ser Gly
Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr290 295 300Asp Leu Arg Thr
Arg Glu Gly His Cys Leu Arg Leu Thr His Asp His305 310 315 320Arg
Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu325 330
335Leu Glu Arg Gly Asp Arg Leu Val Met Asp Asp Ala Ala Gly Glu
Phe340 345 350Pro Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly
Arg Gln Asp355 360 365Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala
Phe Thr Ala Asn Gly370 375 380Phe Ile Val His Ala Cys Gly Glu Gln
Pro Gly Thr Gly Leu Asn Ser385 390 395 400Gly Leu Thr Thr Asn Pro
Gly Val Ser Ala Trp Gln Val Asn Thr Ala405 410 415Tyr Thr Ala Gly
Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys420 425 430Leu Gln
Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro435 440
445Ala Leu Trp Gln Leu Gln450135437PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 135Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe1 5 10 15Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro20 25 30Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val35 40
45Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr50
55 60Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val65 70 75 80Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys85 90 95Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser100 105 110Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro115 120 125Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val130 135 140Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly145 150 155 160Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp165 170 175Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp180 185
190Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His195 200 205Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Glu
Leu Gln Leu210 215 220Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly Glu
Leu Asp Gly Cys Val225 230 235 240Ser Gly Asp Thr Ile Val Met Thr
Ser Gly Gly Pro Arg Thr Val Ala245 250 255Glu Leu Glu Gly Lys Pro
Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr260 265 270Pro Cys Pro Ser
Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp275 280 285Leu Arg
Thr Arg Glu Gly His Cys Leu Arg Leu Thr His Asp His Arg290 295
300Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu
Leu305 310 315 320Glu Arg Gly Asp Arg Leu Val Met Asp Asp Ala Ala
Gly Glu Phe Pro325 330 335Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly
Ala Gly Arg Gln Asp Val340 345 350Tyr Asp Ala Thr Val Tyr Gly Ala
Ser Ala Phe Thr Ala Asn Gly Phe355 360 365Ile Val His Ala Cys Gly
Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly370 375 380Leu Thr Thr Asn
Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr385 390 395 400Thr
Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu405 410
415Gln Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro
Ala420 425 430Leu Trp Gln Leu Gln435136443PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 136Xaa Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105 110Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln115 120 125Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val130 135
140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215 220Ser Pro Glu Leu
Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp Gly225 230 235 240Glu
Leu Asp Gly Cys Val Ser Gly Asp Thr Ile Val Met Thr Ser Gly245 250
255Gly Pro Arg Thr Val Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala
Leu260 265 270Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe
Arg Thr Cys275 280 285Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu
Gly His Cys Leu Arg290 295 300Leu Thr His Asp His Arg Val Leu Val
Met Asp Gly Gly Leu Glu Trp305 310 315 320Arg Ala Ala Gly Glu Leu
Glu Arg Gly Asp Arg Leu Val Met Asp Asp325 330 335Ala Ala Gly Glu
Phe Pro Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly340 345 350Ala Gly
Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala355 360
365Phe Thr Ala Asn Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro
Gly370 375 380Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val
Ser Ala Trp385 390 395 400Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln
Leu Val Thr Tyr Asn Gly405 410 415Lys Thr Tyr Lys Cys Leu Gln Pro
His Thr Ser Leu Ala Gly Trp Glu420 425 430Pro Ser Asn Val Pro Ala
Leu Trp Gln Leu Gln435 440137244PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 137Xaa Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser35 40
45His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50
55 60Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr65 70 75 80Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn85 90 95Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro100 105 110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro165 170
175Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185
190Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val195 200 205Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu210 215 220Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala
Glu Ala Gln Asp Gly225 230 235 240Glu Leu Asp
Xaa138245PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 138Xaa Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu1 5 10 15Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu20 25 30Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser35 40 45His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu50 55 60Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr65 70 75 80Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn85 90 95Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro100 105
110Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln115 120 125Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro165 170 175Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr180 185 190Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val195 200 205Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu210 215
220Ser Pro Glu Leu Gln Leu Glu Glu Ser Cys Ala Glu Ala Gln Asp
Gly225 230 235 240Glu Leu Asp Gly Xaa2451391820DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 139aagcttgaat tcccaccatg aaccggggag tcccttttag
gcacttgctt ctggtgctgc 60aactggcgct cctcccagca gccactcagg gaaagaaagt
ggtgctgggc aaaaaagggg 120atacagtgga actgacctgt acagcttccc
agaagaagag catacaattc cactggaaaa 180actccaacca gataaagatt
ctgggaaatc agggctcctt cttaactaaa ggtccatcca 240agctgaatga
tcgcgctgac tcaagaagaa gcctttggga ccaaggaaac ttccccctga
300tcatcaagaa tcttaagata gaagactcag atacttacat ctgtgaagtg
gaggaccaga 360aggaggaggt gcaattgcta gtgttcggat tgactgccaa
ctctgacacc cacctgcttc 420aggggcagag cctgaccctg accttggaga
gcccccctgg tagtagcccc tcagtgcaat 480gtaggagtcc aaggggtaaa
aacatacagg gggggaagac cctctccgtg tctcagctgg 540agctccagga
tagtggcacc tggacatgca ctgtcttgca gaaccagaag aaggtggagt
600tcaaaataga catcgtggtg ctagctttcc agaaggcctc cagcatagtc
tataagaaag 660agggggaaca ggtggagttc tccttcccac tcgcctttac
agttgaaaag ctgacgggca 720gtggcgagct gtggtggcag gcggagaggg
cttcctcctc caagtcttgg atcacctttg 780acctgaagaa caaggaagtg
tctgtaaaac gggttaccca ggaccctaag ctccagatgg 840gcaagaagct
cccgctccac ctcaccctgc cccaggcctt gcctcagtat gctggctctg
900gaaacctcac cctggccctt gaagcgaaaa caggaaagtt gcatcaggaa
gtgaacctgg 960tggtgatgag agccactcag ctccagaaaa atttgacctg
tgaggtgtgg ggacccacct 1020cccctaagct gatgctgagc ttgaaactgg
agaacaagga ggcaaaggtc tcgaagcggg 1080agaaggcggt gtgggtgctg
aaccctgagg cggggatgtg gcagtgtctg ctgagtgact 1140cgggacaggt
cctgctggaa tccaacatca aggttctgcc cacatggtcc accccggtgc
1200agccagggtg cgtatccggt gacaccattg taatgactag tggcgggccc
cgcactgtgg 1260ctgaactgga gggcaaaccg ttcaccgcac tgattcgcgg
ctctggctac ccatgcccct 1320caggtttctt ccgcacctgt gaacgtgacg
tatatgatct gcgtacacgt gagggtcatt 1380gcttacgttt gacccatgat
caccgtgttc tggtgatgga tggtggcctg gaatggcgtg 1440ccgcgggtga
actggaacgc ggcgaccgcc tggtgatgga tgatgcagct ggcgagtttc
1500cggcactggc aaccttccgt ggcctgcgtg gcgctggccg ccaggatgtt
tatgacgcta 1560ctgtttacgg tgctagcgca ttcactgcta atggcttcat
tgtacacgca tgtggcgagc 1620agcccgggac cggtctgaac tcaggcctca
cgacaaatcc tggtgtatcc gcttggcagg 1680tcaacacagc ttatactgcg
ggacaattgg tcacatataa cggcaagacg tataaatgtt 1740tgcagcccca
cacctccttg gcaggatggg aaccatccaa cgttcctgcc ttgtggcagc
1800ttcaatgact cgagcggccg 1820140596PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 140Met Asn Arg Gly Val Pro Phe Arg His Leu Leu Leu
Val Leu Gln Leu1 5 10 15Ala Leu Leu Pro Ala Ala Thr Gln Gly Lys Lys
Val Val Leu Gly Lys20 25 30Lys Gly Asp Thr Val Glu Leu Thr Cys Thr
Ala Ser Gln Lys Lys Ser35 40 45Ile Gln Phe His Trp Lys Asn Ser Asn
Gln Ile Lys Ile Leu Gly Asn50 55 60Gln Gly Ser Phe Leu Thr Lys Gly
Pro Ser Lys Leu Asn Asp Arg Ala65 70 75 80Asp Ser Arg Arg Ser Leu
Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile85 90 95Lys Asn Leu Lys Ile
Glu Asp Ser Asp Thr Tyr Ile Cys Glu Val Glu100 105 110Asp Gln Lys
Glu Glu Val Gln Leu Leu Val Phe Gly Leu Thr Ala Asn115 120 125Ser
Asp Thr His Leu Leu Gln Gly Gln Ser Leu Thr Leu Thr Leu Glu130 135
140Ser Pro Pro Gly Ser Ser Pro Ser Val Gln Cys Arg Ser Pro Arg
Gly145 150 155 160Lys Asn Ile Gln Gly Gly Lys Thr Leu Ser Val Ser
Gln Leu Glu Leu165 170 175Gln Asp Ser Gly Thr Trp Thr Cys Thr Val
Leu Gln Asn Gln Lys Lys180 185 190Val Glu Phe Lys Ile Asp Ile Val
Val Leu Ala Phe Gln Lys Ala Ser195 200 205Ser Ile Val Tyr Lys Lys
Glu Gly Glu Gln Val Glu Phe Ser Phe Pro210 215 220Leu Ala Phe Thr
Val Glu Lys Leu Thr Gly Ser Gly Glu Leu Trp Trp225 230 235 240Gln
Ala Glu Arg Ala Ser Ser Ser Lys Ser Trp Ile Thr Phe Asp Leu245 250
255Lys Asn Lys Glu Val Ser Val Lys Arg Val Thr Gln Asp Pro Lys
Leu260 265 270Gln Met Gly Lys Lys Leu Pro Leu His Leu Thr Leu Pro
Gln Ala Leu275 280 285Pro Gln Tyr Ala Gly Ser Gly Asn Leu Thr Leu
Ala Leu Glu Ala Lys290 295 300Thr Gly Lys Leu His Gln Glu Val Asn
Leu Val Val Met Arg Ala Thr305 310 315 320Gln Leu Gln Lys Asn Leu
Thr Cys Glu Val Trp Gly Pro Thr Ser Pro325 330 335Lys Leu Met Leu
Ser Leu Lys Leu Glu Asn Lys Glu Ala Lys Val Ser340 345 350Lys Arg
Glu Lys Ala Val Trp Val Leu Asn Pro Glu Ala Gly Met Trp355 360
365Gln Cys Leu Leu Ser Asp Ser Gly Gln Val Leu Leu Glu Ser Asn
Ile370 375 380Lys Val Leu Pro Thr Trp Ser Thr Pro Val Gln Pro Gly
Cys Val Ser385 390 395 400Gly Asp Thr Ile Val Met Thr Ser Gly Gly
Pro Arg Thr Val Ala Glu405 410 415Leu Glu Gly Lys Pro Phe Thr Ala
Leu Ile Arg Gly Ser Gly Tyr Pro420 425 430Cys Pro Ser Gly Phe Phe
Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu435 440 445Arg Thr Arg Glu
Gly His Cys Leu Arg Leu Thr His Asp His Arg Val450 455 460Leu Val
Met Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu465 470 475
480Arg Gly Asp Arg Leu Val Met Asp Asp Ala Ala Gly Glu Phe Pro
Ala485 490 495Leu Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln
Asp Val Tyr500 505 510Asp Ala Thr Val Tyr Gly Ala Ser Ala Phe Thr
Ala Asn Gly Phe Ile515 520 525Val His Ala Cys Gly Glu Gln Pro Gly
Thr Gly Leu Asn Ser Gly Leu530 535 540Thr Thr Asn Pro Gly Val Ser
Ala Trp Gln Val Asn Thr Ala Tyr Thr545 550 555 560Ala Gly Gln Leu
Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln565 570 575Pro His
Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu580 585
590Trp Gln Leu Gln595141571PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 141Lys Lys Val
Val Leu Gly Lys Lys Gly Asp Thr Val Glu Leu Thr Cys1 5 10 15Thr Ala
Ser Gln Lys Lys Ser Ile Gln Phe His Trp Lys Asn Ser Asn20 25 30Gln
Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro35 40
45Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln50
55 60Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser
Asp65 70 75 80Thr Tyr Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val
Gln Leu Leu85 90 95Val Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu
Leu Gln Gly Gln100 105 110Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro
Gly Ser Ser Pro Ser Val115 120 125Gln Cys Arg Ser Pro Arg Gly Lys
Asn Ile Gln Gly Gly Lys Thr Leu130 135 140Ser Val Ser Gln Leu Glu
Leu Gln Asp Ser Gly Thr Trp Thr Cys Thr145 150 155 160Val Leu Gln
Asn Gln Lys Lys Val Glu Phe Lys Ile Asp Ile Val Val165 170 175Leu
Ala Phe Gln Lys Ala Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu180 185
190Gln Val Glu Phe Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu
Thr195 200 205Gly Ser Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser
Ser Ser Lys210 215 220Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu
Val Ser Val Lys Arg225 230 235 240Val Thr Gln Asp Pro Lys Leu Gln
Met Gly Lys Lys Leu Pro Leu His245 250 255Leu Thr Leu Pro Gln Ala
Leu Pro Gln Tyr Ala Gly Ser Gly Asn Leu260 265 270Thr Leu Ala Leu
Glu Ala Lys Thr Gly Lys Leu His Gln Glu Val Asn275 280 285Leu Val
Val Met Arg Ala Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu290 295
300Val Trp Gly Pro Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu
Glu305 310 315 320Asn Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala
Val Trp Val Leu325 330 335Asn Pro Glu Ala Gly Met Trp Gln Cys Leu
Leu Ser Asp Ser Gly Gln340 345 350Val Leu Leu Glu Ser Asn Ile Lys
Val Leu Pro Thr Trp Ser Thr Pro355 360 365Val Gln Pro Gly Cys Val
Ser Gly Asp Thr Ile Val Met Thr Ser Gly370 375 380Gly Pro Arg Thr
Val Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala Leu385 390 395 400Ile
Arg Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys405 410
415Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu
Arg420 425 430Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly
Leu Glu Trp435 440 445Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg
Leu Val Met Asp Asp450 455 460Ala Ala Gly Glu Phe Pro Ala Leu Ala
Thr Phe Arg Gly Leu Arg Gly465 470 475 480Ala Gly Arg Gln Asp Val
Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala485 490 495Phe Thr Ala Asn
Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro Gly500 505 510Thr Gly
Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val Ser Ala Trp515 520
525Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn
Gly530 535 540Lys Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala
Gly Trp Glu545 550 555 560Pro Ser Asn Val Pro Ala Leu Trp Gln Leu
Gln565 570142372PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 142Lys Lys Val Val Leu Gly Lys Lys
Gly Asp Thr Val Glu Leu Thr Cys1 5 10 15Thr Ala Ser Gln Lys Lys Ser
Ile Gln Phe His Trp Lys Asn Ser Asn20 25 30Gln Ile Lys Ile Leu Gly
Asn Gln Gly Ser Phe Leu Thr Lys Gly Pro35 40 45Ser Lys Leu Asn Asp
Arg Ala Asp Ser Arg Arg Ser Leu Trp Asp Gln50 55 60Gly Asn Phe Pro
Leu Ile Ile Lys Asn Leu Lys Ile Glu Asp Ser Asp65 70 75 80Thr Tyr
Ile Cys Glu Val Glu Asp Gln Lys Glu Glu Val Gln Leu Leu85 90 95Val
Phe Gly Leu Thr Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln100 105
110Ser Leu Thr Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser
Val115 120 125Gln Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly
Lys Thr Leu130 135 140Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly
Thr Trp Thr Cys Thr145 150 155 160Val Leu Gln Asn Gln Lys Lys Val
Glu Phe Lys Ile Asp Ile Val Val165 170 175Leu Ala Phe Gln Lys Ala
Ser Ser Ile Val Tyr Lys Lys Glu Gly Glu180 185 190Gln Val Glu Phe
Ser Phe Pro Leu Ala Phe Thr Val Glu Lys Leu Thr195 200 205Gly Ser
Gly Glu Leu Trp Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys210 215
220Ser Trp Ile Thr Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys
Arg225 230 235 240Val Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys
Leu Pro Leu His245 250 255Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr
Ala Gly Ser Gly Asn Leu260 265 270Thr Leu Ala Leu Glu Ala Lys Thr
Gly Lys Leu His Gln Glu Val Asn275 280 285Leu Val Val Met Arg Ala
Thr Gln Leu Gln Lys Asn Leu Thr Cys Glu290 295 300Val Trp Gly Pro
Thr Ser Pro Lys Leu Met Leu Ser Leu Lys Leu Glu305 310 315 320Asn
Lys Glu Ala Lys Val Ser Lys Arg Glu Lys Ala Val Trp Val Leu325 330
335Asn Pro Glu Ala Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly
Gln340 345 350Val Leu Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp
Ser Thr Pro355 360 365Val Gln Pro Xaa370143373PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 143Lys Lys Val Val Leu Gly Lys Lys Gly Asp Thr Val
Glu Leu Thr Cys1 5 10 15Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His
Trp Lys Asn Ser Asn20 25 30Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser
Phe Leu Thr Lys Gly Pro35 40 45Ser Lys Leu Asn Asp Arg Ala Asp Ser
Arg Arg Ser Leu Trp Asp Gln50 55 60Gly Asn Phe Pro Leu Ile Ile Lys
Asn Leu Lys Ile Glu Asp Ser Asp65 70 75 80Thr Tyr Ile Cys Glu Val
Glu Asp Gln Lys Glu Glu Val Gln Leu Leu85 90 95Val Phe Gly Leu Thr
Ala Asn Ser Asp Thr His Leu Leu Gln Gly Gln100 105 110Ser Leu Thr
Leu Thr Leu Glu Ser Pro Pro Gly Ser Ser Pro Ser Val115 120 125Gln
Cys Arg Ser Pro Arg Gly Lys Asn Ile Gln Gly Gly Lys Thr Leu130 135
140Ser Val Ser Gln Leu Glu Leu Gln Asp Ser Gly Thr Trp Thr Cys
Thr145 150 155 160Val Leu Gln Asn Gln Lys Lys Val Glu Phe Lys Ile
Asp Ile Val Val165 170 175Leu Ala Phe Gln Lys Ala Ser Ser Ile Val
Tyr Lys Lys Glu Gly Glu180 185 190Gln Val Glu Phe Ser Phe Pro Leu
Ala Phe Thr Val Glu Lys Leu Thr195 200 205Gly Ser Gly Glu Leu Trp
Trp Gln Ala Glu Arg Ala Ser Ser Ser Lys210 215 220Ser Trp Ile Thr
Phe Asp Leu Lys Asn Lys Glu Val Ser Val Lys Arg225 230 235 240Val
Thr Gln Asp Pro Lys Leu Gln Met Gly Lys Lys Leu Pro Leu His245 250
255Leu Thr Leu Pro Gln Ala Leu Pro Gln Tyr Ala Gly Ser Gly Asn
Leu260 265 270Thr Leu Ala Leu Glu Ala Lys Thr Gly Lys Leu His Gln
Glu Val Asn275 280 285Leu Val Val Met Arg Ala Thr Gln Leu Gln Lys
Asn Leu Thr Cys Glu290 295 300Val Trp Gly Pro Thr Ser Pro Lys Leu
Met Leu Ser Leu Lys Leu Glu305 310 315 320Asn Lys Glu Ala Lys Val
Ser Lys Arg Glu Lys Ala Val Trp Val Leu325 330 335Asn Pro Glu Ala
Gly Met Trp Gln Cys Leu Leu Ser Asp Ser Gly Gln340 345 350Val Leu
Leu Glu Ser Asn Ile Lys Val Leu Pro Thr Trp Ser Thr Pro355 360
365Val Gln Pro Gly Xaa3701441364DNAArtificial Sequenceartificial
construct relating to Homo Sapiens
immunoglobulin 144aagcttgaat tcccaccatg gcttgggtgt ggaccttgct
tttcctgatg gcagctgccc 60aaagtatcca agcacagatc cagttggtcc agtctggacc
tgagctgaag aagcctggag 120agacagtcaa gatctcctgc aaggcttcag
gatatacctt cacacactat ggaatgaact 180gggtgaagca ggctccagga
aagggtttaa agtggatggg ctggataaac acctacactg 240gagagccaac
atatgctgat gacttcaagg aacactttgc cttctctttg gaaacctctg
300ccagcactgt ctttttgcag atcaacaacc tcaaaaatga ggacacggcc
acatatttct 360gtgcaagaga acggggggat gctatggact actggggtca
gggaacctcc gtcaccgtct 420cctcagcctc caccaagggg ccatcggtct
tccccctggc accctcctcc aagagcacct 480ctgggggcac agcggccctg
ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg 540tgtcgtggaa
ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt
600cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc
ttgggcaccc 660agacctacat ctgcaacgtg aatcacaagc ccagcaacac
caaggtggac aagaaagttg 720agcccaaatc ttgtgacaaa actcacacag
ggtgcgtatc cggtgacacc attgtaatga 780ctagtggcgg gccccgcact
gtggctgaac tggagggcaa accgttcacc gcactgattc 840gcggctctgg
ctacccatgc ccctcaggtt tcttccgcac ctgtgaacgt gacgtatatg
900atctgcgtac acgtgagggt cattgcttac gtttgaccca tgatcaccgt
gttctggtga 960tggatggtgg cctggaatgg cgtgccgcgg gtgaactgga
acgcggcgac cgcctggtga 1020tggatgatgc agctggcgag tttccggcac
tggcaacctt ccgtggcctg cgtggcgctg 1080gccgccagga tgtttatgac
gctactgttt acggtgctag cgcattcact gctaatggct 1140tcattgtaca
cgcatgtggc gagcagcccg ggaccggtct gaactcaggc ctcacgacaa
1200atcctggtgt atccgcttgg caggtcaaca cagcttatac tgcgggacaa
ttggtcacat 1260ataacggcaa gacgtataaa tgtttgcagc cccacacctc
cttggcagga tgggaaccat 1320ccaacgttcc tgccttgtgg cagcttcaat
gactcgagcg gccg 1364145444PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 145Met Ala Trp
Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser1 5 10 15Ile Gln
Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys20 25 30Pro
Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe35 40
45Thr His Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu50
55 60Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr
Ala65 70 75 80Asp Asp Phe Lys Glu His Phe Ala Phe Ser Leu Glu Thr
Ser Ala Ser85 90 95Thr Val Phe Leu Gln Ile Asn Asn Leu Lys Asn Glu
Asp Thr Ala Thr100 105 110Tyr Phe Cys Ala Arg Glu Arg Gly Asp Ala
Met Asp Tyr Trp Gly Gln115 120 125Gly Thr Ser Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val130 135 140Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala145 150 155 160Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser165 170 175Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val180 185
190Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro195 200 205Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys210 215 220Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp225 230 235 240Lys Thr His Thr Gly Cys Val Ser
Gly Asp Thr Ile Val Met Thr Ser245 250 255Gly Gly Pro Arg Thr Val
Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala260 265 270Leu Ile Arg Gly
Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr275 280 285Cys Glu
Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu290 295
300Arg Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly Leu
Glu305 310 315 320Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg
Leu Val Met Asp325 330 335Asp Ala Ala Gly Glu Phe Pro Ala Leu Ala
Thr Phe Arg Gly Leu Arg340 345 350Gly Ala Gly Arg Gln Asp Val Tyr
Asp Ala Thr Val Tyr Gly Ala Ser355 360 365Ala Phe Thr Ala Asn Gly
Phe Ile Val His Ala Cys Gly Glu Gln Pro370 375 380Gly Thr Gly Leu
Asn Ser Gly Leu Thr Thr Asn Pro Gly Val Ser Ala385 390 395 400Trp
Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn405 410
415Gly Lys Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly
Trp420 425 430Glu Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln435
440146425PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 146Gln Ile Gln Leu Val Gln Ser Gly Pro
Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr His Tyr20 25 30Gly Met Asn Trp Val Lys Gln
Ala Pro Gly Lys Gly Leu Lys Trp Met35 40 45Gly Trp Ile Asn Thr Tyr
Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe50 55 60Lys Glu His Phe Ala
Phe Ser Leu Glu Thr Ser Ala Ser Thr Val Phe65 70 75 80Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys85 90 95Ala Arg
Glu Arg Gly Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser100 105
110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu115 120 125Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys130 135 140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser165 170 175Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His210 215
220Thr Gly Cys Val Ser Gly Asp Thr Ile Val Met Thr Ser Gly Gly
Pro225 230 235 240Arg Thr Val Ala Glu Leu Glu Gly Lys Pro Phe Thr
Ala Leu Ile Arg245 250 255Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe
Phe Arg Thr Cys Glu Arg260 265 270Asp Val Tyr Asp Leu Arg Thr Arg
Glu Gly His Cys Leu Arg Leu Thr275 280 285His Asp His Arg Val Leu
Val Met Asp Gly Gly Leu Glu Trp Arg Ala290 295 300Ala Gly Glu Leu
Glu Arg Gly Asp Arg Leu Val Met Asp Asp Ala Ala305 310 315 320Gly
Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly325 330
335Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala Phe
Thr340 345 350Ala Asn Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro
Gly Thr Gly355 360 365Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val
Ser Ala Trp Gln Val370 375 380Asn Thr Ala Tyr Thr Ala Gly Gln Leu
Val Thr Tyr Asn Gly Lys Thr385 390 395 400Tyr Lys Cys Leu Gln Pro
His Thr Ser Leu Ala Gly Trp Glu Pro Ser405 410 415Asn Val Pro Ala
Leu Trp Gln Leu Gln420 425147226PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 147Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr His Tyr20 25 30Gly
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met35 40
45Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe50
55 60Lys Glu His Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Val
Phe65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
Tyr Phe Cys85 90 95Ala Arg Glu Arg Gly Asp Ala Met Asp Tyr Trp Gly
Gln Gly Thr Ser100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu115 120 125Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys130 135 140Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185
190Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn195 200 205Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His210 215 220Thr Xaa225148227PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 148Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys
Lys Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr His Tyr20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly
Lys Gly Leu Lys Trp Met35 40 45Gly Trp Ile Asn Thr Tyr Thr Gly Glu
Pro Thr Tyr Ala Asp Asp Phe50 55 60Lys Glu His Phe Ala Phe Ser Leu
Glu Thr Ser Ala Ser Thr Val Phe65 70 75 80Leu Gln Ile Asn Asn Leu
Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys85 90 95Ala Arg Glu Arg Gly
Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser100 105 110Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135
140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser180 185 190Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn195 200 205Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His210 215 220Thr Gly
Xaa225149731DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 149aagcttgaat tcccaccatg aagtcacaga
cccaggtctt cgtatttcta ctgctctgtg 60tgtctggtgc tcatgggagt attgtgatga
cccagactcc caaattcctg cttgtatcag 120caggagacag ggttaccata
acctgcacgg ccagtcagag tgtgagtaat gatgtagttt 180ggtaccaaca
gaagccaggg cagtctccta aaatgctgat gtattctgca ttcaatcgct
240acactggagt ccctgatcgt ttcactggca gaggatacgg gacggatttc
actttcacca 300tcagctctgt gcaggctgaa gacctggcag tttatttctg
tcagcaggat tataactctc 360ctcggacgtt cggtggaggc accaagctgg
agatcaaacg aactgtggct gcaccatctg 420tcttcatctt cccgccatct
gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc 480tgctgaataa
cttctatccc agagaggcca aagtacagtg gaaggtggat aacgccctcc
540aatcgggtaa ctcccaggag agtgtcacag agcaggacag caaggacagc
acctacagcc 600tcagcagcac cctgacgctg agcaaagcag actacgagaa
acacaaagtc tacgcctgcg 660aagtcaccca tcagggcctg agctcgcccg
tcacaaagag cttcaacagg ggagagtgtt 720agtgactcga g
731150234PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 150Met Lys Ser Gln Thr Gln Val Phe Val
Phe Leu Leu Leu Cys Val Ser1 5 10 15Gly Ala His Gly Ser Ile Val Met
Thr Gln Thr Pro Lys Phe Leu Leu20 25 30Val Ser Ala Gly Asp Arg Val
Thr Ile Thr Cys Thr Ala Ser Gln Ser35 40 45Val Ser Asn Asp Val Val
Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro50 55 60Lys Met Leu Met Tyr
Ser Ala Phe Asn Arg Tyr Thr Gly Val Pro Asp65 70 75 80Arg Phe Thr
Gly Arg Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile Ser85 90 95Ser Val
Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr100 105
110Asn Ser Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg115 120 125Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser165 170 175Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr180 185 190Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys195 200 205His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro210 215
220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225
230151214PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 151Ser Ile Val Met Thr Gln Thr Pro Lys
Phe Leu Leu Val Ser Ala Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Thr
Ala Ser Gln Ser Val Ser Asn Asp20 25 30Val Val Trp Tyr Gln Gln Lys
Pro Gly Gln Ser Pro Lys Met Leu Met35 40 45Tyr Ser Ala Phe Asn Arg
Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly50 55 60Arg Gly Tyr Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Ala65 70 75 80Glu Asp Leu
Ala Val Tyr Phe Cys Gln Gln Asp Tyr Asn Ser Pro Arg85 90 95Thr Phe
Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205Phe Asn
Arg Gly Glu Cys2101521268DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 152aagcttgaat tcccaccatg
ggcctctcca ccgtgcctga cctgctgctg ccgctggtgc 60tcctggagct gttggtggga
atatacccct caggggttat tggactggtc cctcacctag 120gggacaggga
gaagagagat agtgtgtgtc cccaaggaaa atatatccac cctcaaaata
180attcgatttg ctgtaccaag tgccacaaag gaacctactt gtacaatgac
tgtccaggcc 240cggggcagga tacggactgc agggagtgtg agagcggctc
cttcaccgct tcagaaaacc 300acctcagaca ctgcctcagc tgctccaaat
gccgaaagga aatgggtcag gtggagatct 360cttcttgcac agtggaccgg
gacaccgtgt gtggctgcag gaagaaccag taccggcatt 420attggagtga
aaaccttttc cagtgcttca attgcagcct ctgcctcaat gggaccgtgc
480acctctcctg ccaggagaaa cagaacaccg tgtgcacctg ccatgcaggt
ttctttctaa 540gagaaaacga gtgtgtctcc tgtagtaact gtaagaaaag
cctggagtgc acgaagttgt 600gcctacccca gattgagaat gttaagggca
ctgaggactc aggcaccaca gtggggtgcg 660tatccggtga caccattgta
atgactagtg gcgggccccg cactgtggct gaactggagg 720gcaaaccgtt
caccgcactg attcgcggct ctggctaccc atgcccctca ggtttcttcc
780gcacctgtga acgtgacgta tatgatctgc gtacacgtga gggtcattgc
ttacgtttga 840cccatgatca ccgtgttctg gtgatggatg gtggcctgga
atggcgtgcc gcgggtgaac 900tggaacgcgg cgaccgcctg gtgatggatg
atgcagctgg cgagtttccg gcactggcaa 960ccttccgtgg cctgcgtggc
gctggccgcc aggatgttta tgacgctact gtttacggtg 1020ctagcgcatt
cactgctaat ggcttcattg tacacgcatg tggcgagcag cccgggaccg
1080gtctgaactc aggcctcacg acaaatcctg gtgtatccgc ttggcaggtc
aacacagctt 1140atactgcggg acaattggtc acatataacg gcaagacgta
taaatgtttg cagccccaca 1200cctccttggc aggatgggaa ccatccaacg
ttcctgcctt gtggcagctt caatgactcg 1260agcggccg
1268153411PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 153Met Gly Leu Ser Thr Val Pro Asp Leu
Leu Leu Pro Leu Val Leu Leu1 5 10 15Glu Leu Leu Val Gly Ile Tyr Pro
Ser Gly Val Ile Gly Leu Val Pro20 25 30His Leu Gly Asp Arg Glu Lys
Arg Asp Ser Val Cys Pro Gln Gly Lys35 40 45Tyr Ile His Pro Gln Asn
Asn Ser Ile Cys Cys Thr Lys Cys His Lys50 55 60Gly Thr Tyr Leu Tyr
Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp65 70 75 80Cys Arg Glu
Cys Glu Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu85 90 95Arg His
Cys Leu Ser Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val100 105
110Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr Val Cys Gly Cys
Arg115 120 125Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu Asn Leu
Phe Gln Cys Phe130 135 140Asn Cys Ser Leu Cys Leu Asn Gly Thr Val
His Leu Ser Cys Gln Glu145 150 155 160Lys Gln Asn Thr Val Cys Thr
Cys His Ala Gly Phe Phe Leu Arg Glu165 170 175Asn Glu Cys Val Ser
Cys Ser Asn Cys Lys Lys Ser Leu Glu Cys Thr180 185 190Lys Leu Cys
Leu Pro Gln Ile Glu Asn Val Lys Gly Thr Glu Asp Ser195 200 205Gly
Thr Thr Gly Cys Val Ser Gly Asp Thr Ile Val Met Thr Ser Gly210 215
220Gly Pro Arg Thr Val Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala
Leu225 230 235 240Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe
Phe Arg Thr Cys245 250 255Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg
Glu Gly His Cys Leu Arg260 265 270Leu Thr His Asp His Arg Val Leu
Val Met Asp Gly Gly Leu Glu Trp275 280 285Arg Ala Ala Gly Glu Leu
Glu Arg Gly Asp Arg Leu Val Met Asp Asp290 295 300Ala Ala Gly Glu
Phe Pro Ala Leu Ala Thr Phe Arg Gly Leu Arg Gly305 310 315 320Ala
Gly Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala325 330
335Phe Thr Ala Asn Gly Phe Ile Val His Ala Cys Gly Glu Gln Pro
Gly340 345 350Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val
Ser Ala Trp355 360 365Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu
Val Thr Tyr Asn Gly370 375 380Lys Thr Tyr Lys Cys Leu Gln Pro His
Thr Ser Leu Ala Gly Trp Glu385 390 395 400Pro Ser Asn Val Pro Ala
Leu Trp Gln Leu Gln405 410154390PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 154Ile Tyr Pro
Ser Gly Val Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys
Arg Asp Ser Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn
Asn Ser Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40
45Asn Asp Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50
55 60Ser Gly Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu
Ser65 70 75 80Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile
Ser Ser Cys85 90 95Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys
Asn Gln Tyr Arg100 105 110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys
Phe Asn Cys Ser Leu Cys115 120 125Leu Asn Gly Thr Val His Leu Ser
Cys Gln Glu Lys Gln Asn Thr Val130 135 140Cys Thr Cys His Ala Gly
Phe Phe Leu Arg Glu Asn Glu Cys Val Ser145 150 155 160Cys Ser Asn
Cys Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro165 170 175Gln
Ile Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Thr Thr Gly Cys180 185
190Val Ser Gly Asp Thr Ile Val Met Thr Ser Gly Gly Pro Arg Thr
Val195 200 205Ala Glu Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg
Gly Ser Gly210 215 220Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys
Glu Arg Asp Val Tyr225 230 235 240Asp Leu Arg Thr Arg Glu Gly His
Cys Leu Arg Leu Thr His Asp His245 250 255Arg Val Leu Val Met Asp
Gly Gly Leu Glu Trp Arg Ala Ala Gly Glu260 265 270Leu Glu Arg Gly
Asp Arg Leu Val Met Asp Asp Ala Ala Gly Glu Phe275 280 285Pro Ala
Leu Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp290 295
300Val Tyr Asp Ala Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn
Gly305 310 315 320Phe Ile Val His Ala Cys Gly Glu Gln Pro Gly Thr
Gly Leu Asn Ser325 330 335Gly Leu Thr Thr Asn Pro Gly Val Ser Ala
Trp Gln Val Asn Thr Ala340 345 350Tyr Thr Ala Gly Gln Leu Val Thr
Tyr Asn Gly Lys Thr Tyr Lys Cys355 360 365Leu Gln Pro His Thr Ser
Leu Ala Gly Trp Glu Pro Ser Asn Val Pro370 375 380Ala Leu Trp Gln
Leu Gln385 390155191PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 155Ile Tyr Pro Ser Gly Val
Ile Gly Leu Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys Arg Asp Ser
Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile
Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys
Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50 55 60Ser Gly
Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser65 70 75
80Cys Ser Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys85
90 95Thr Val Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr
Arg100 105 110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys
Ser Leu Cys115 120 125Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu
Lys Gln Asn Thr Val130 135 140Cys Thr Cys His Ala Gly Phe Phe Leu
Arg Glu Asn Glu Cys Val Ser145 150 155 160Cys Ser Asn Cys Lys Lys
Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro165 170 175Gln Ile Glu Asn
Val Lys Gly Thr Glu Asp Ser Gly Thr Thr Xaa180 185
190156192PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 156Ile Tyr Pro Ser Gly Val Ile Gly Leu
Val Pro His Leu Gly Asp Arg1 5 10 15Glu Lys Arg Asp Ser Val Cys Pro
Gln Gly Lys Tyr Ile His Pro Gln20 25 30Asn Asn Ser Ile Cys Cys Thr
Lys Cys His Lys Gly Thr Tyr Leu Tyr35 40 45Asn Asp Cys Pro Gly Pro
Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu50 55 60Ser Gly Ser Phe Thr
Ala Ser Glu Asn His Leu Arg His Cys Leu Ser65 70 75 80Cys Ser Lys
Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys85 90 95Thr Val
Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg100 105
110His Tyr Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser Leu
Cys115 120 125Leu Asn Gly Thr Val His Leu Ser Cys Gln Glu Lys Gln
Asn Thr Val130 135 140Cys Thr Cys His Ala Gly Phe Phe Leu Arg Glu
Asn Glu Cys Val Ser145 150 155 160Cys Ser Asn Cys Lys Lys Ser Leu
Glu Cys Thr Lys Leu Cys Leu Pro165 170 175Gln Ile Glu Asn Val Lys
Gly Thr Glu Asp Ser Gly Thr Thr Gly Xaa180 185
1901571403DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 157aagcttgaat tcccaccatg gcgcccgtcg
ccgtctgggc cgcgctggcc gtcggactgg 60agctctgggc tgcggcgcac gccttgcccg
cccaggtggc atttacaccc tacgccccgg 120agcccgggag cacatgccgg
ctcagagaat actatgacca gacagctcag atgtgctgca 180gcaaatgctc
gccgggccaa catgcaaaag tcttctgtac caagacctcg gacaccgtgt
240gtgactcctg tgaggacagc acatacaccc agctctggaa ctgggttccc
gagtgcttga 300gctgtggctc ccgctgtagc tctgaccagg tggaaactca
agcctgcact cgggaacaga 360accgcatctg cacctgcagg cccggctggt
actgcgcgct gagcaagcag gaggggtgcc 420ggctgtgcgc gccgctgcgc
aagtgccgcc cgggcttcgg cgtggccaga ccaggaactg 480aaacatcaga
cgtggtgtgc aagccctgtg ccccggggac gttctccaac acgacttcat
540ccacggatat ttgcaggccc caccagatct gtaacgtggt ggccatccct
gggaatgcaa 600gcatggatgc agtctgcacg tccacgtccc ccacccggag
tatggcccca ggggcagtac 660acttacccca gccagtgtcc acacgatccc
aacacacgca gccaactcca gaacccagca 720ctgctccaag cacctccttc
ctgctcccaa tgggccccag ccccccagct gaagggagca 780ctggcgacgg
gtgcgtatcc ggtgacacca ttgtaatgac tagtggcggg ccccgcactg
840tggctgaact ggagggcaaa ccgttcaccg cactgattcg cggctctggc
tacccatgcc 900cctcaggttt cttccgcacc tgtgaacgtg acgtatatga
tctgcgtaca cgtgagggtc 960attgcttacg tttgacccat gatcaccgtg
ttctggtgat ggatggtggc ctggaatggc 1020gtgccgcggg tgaactggaa
cgcggcgacc gcctggtgat ggatgatgca gctggcgagt 1080ttccggcact
ggcaaccttc cgtggcctgc gtggcgctgg ccgccaggat gtttatgacg
1140ctactgttta cggtgctagc gcattcactg ctaatggctt cattgtacac
gcatgtggcg 1200agcagcccgg gaccggtctg aactcaggcc tcacgacaaa
tcctggtgta tccgcttggc 1260aggtcaacac agcttatact gcgggacaat
tggtcacata taacggcaag acgtataaat 1320gtttgcagcc ccacacctcc
ttggcaggat gggaaccatc caacgttcct gccttgtggc 1380agcttcaatg
actcgagcgg ccg 1403158457PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 158Met Ala Pro Val Ala Val
Trp Ala Ala Leu Ala Val Gly Leu Glu Leu1 5 10 15Trp Ala Ala Ala His
Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr20 25 30Ala Pro Glu Pro
Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln35 40 45Thr Ala Gln
Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys50 55 60Val Phe
Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp65 70 75
80Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys85
90 95Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr
Arg100 105 110Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr
Cys Ala Leu115 120 125Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro
Leu Arg Lys Cys Arg130 135 140Pro Gly Phe Gly Val Ala Arg Pro Gly
Thr Glu Thr Ser Asp Val Val145 150 155 160Cys Lys Pro Cys Ala Pro
Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr165 170 175Asp Ile Cys Arg
Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly180 185 190Asn Ala
Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser195 200
205Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg
Ser210 215 220Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr Ser225 230 235 240Phe Leu Leu Pro Met Gly Pro Ser Pro Pro
Ala Glu Gly Ser Thr Gly245 250 255Asp Gly Cys Val Ser Gly Asp Thr
Ile Val Met Thr Ser Gly Gly Pro260 265 270Arg Thr Val Ala Glu Leu
Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg275 280 285Gly Ser Gly Tyr
Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg290 295 300Asp Val
Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr305 310 315
320His Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg
Ala325 330 335Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met Asp
Asp Ala Ala340 345 350Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly
Leu Arg Gly Ala Gly355 360 365Arg Gln Asp Val Tyr Asp Ala Thr Val
Tyr Gly Ala Ser Ala Phe Thr370 375 380Ala Asn Gly Phe Ile Val His
Ala Cys Gly Glu Gln Pro Gly Thr Gly385 390 395 400Leu Asn Ser Gly
Leu Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val405 410 415Asn Thr
Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr420 425
430Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp Glu Pro
Ser435 440 445Asn Val Pro Ala Leu Trp Gln Leu Gln450
455159435PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 159Leu Pro Ala Gln Val Ala Phe Thr Pro
Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln
His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp
Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro
Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val
Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys
Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105
110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val
Ala115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro
Cys Ala Pro130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp
Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile
Pro Gly Asn Ala Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser
Pro Thr Arg Ser Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln
Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu
Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215
220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Gly Cys Val Ser
Gly225 230 235 240Asp Thr Ile Val Met Thr Ser Gly Gly Pro Arg Thr
Val Ala Glu Leu245 250 255Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg
Gly Ser Gly Tyr Pro Cys260 265 270Pro Ser Gly Phe Phe Arg Thr Cys
Glu Arg Asp Val Tyr Asp Leu Arg275 280 285Thr Arg Glu Gly His Cys
Leu Arg Leu Thr His Asp His Arg Val Leu290 295 300Val Met Asp Gly
Gly Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu Arg305 310 315 320Gly
Asp Arg Leu Val Met Asp Asp Ala Ala Gly Glu Phe Pro Ala Leu325 330
335Ala Thr Phe Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp Val Tyr
Asp340 345 350Ala Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn Gly
Phe Ile Val355 360 365His Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu
Asn Ser Gly Leu Thr370 375 380Thr Asn Pro Gly Val Ser Ala Trp Gln
Val Asn Thr Ala Tyr Thr Ala385 390 395 400Gly Gln Leu Val Thr Tyr
Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro405 410 415His Thr Ser Leu
Ala Gly Trp Glu Pro Ser Asn Val Pro Ala Leu Trp420 425 430Gln Leu
Gln435160236PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 160Leu Pro Ala Gln Val Ala Phe Thr Pro
Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln
His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp
Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro
Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val
Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys
Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105
110Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val
Ala115 120 125Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro
Cys Ala Pro130 135 140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp
Ile Cys Arg Pro His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile
Pro Gly Asn Ala Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser
Pro Thr Arg Ser Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln
Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu
Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215
220Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Xaa225 230
235161237PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 161Leu Pro Ala Gln Val Ala Phe Thr Pro
Tyr Ala Pro Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr
Asp Gln Thr Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln
His Ala Lys Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp
Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro
Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val
Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys
Arg Pro Gly Trp Tyr Cys Ala Leu Ser
Lys Gln Glu Gly Cys100 105 110Arg Leu Cys Ala Pro Leu Arg Lys Cys
Arg Pro Gly Phe Gly Val Ala115 120 125Arg Pro Gly Thr Glu Thr Ser
Asp Val Val Cys Lys Pro Cys Ala Pro130 135 140Gly Thr Phe Ser Asn
Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His145 150 155 160Gln Ile
Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala165 170
175Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala
Val180 185 190His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr
Gln Pro Thr195 200 205Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe
Leu Leu Pro Met Gly210 215 220Pro Ser Pro Pro Ala Glu Gly Ser Thr
Gly Asp Gly Xaa225 230 2351621499DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 162aagcttgaat
tcccaccatg gcgcccgtcg ccgtctgggc cgcgctggcc gtcggactgg 60agctctgggc
tgcggcgcac gccttgcccg cccaggtggc atttacaccc tacgccccgg
120agcccgggag cacatgccgg ctcagagaat actatgacca gacagctcag
atgtgctgca 180gcaaatgctc gccgggccaa catgcaaaag tcttctgtac
caagacctcg gacaccgtgt 240gtgactcctg tgaggacagc acatacaccc
agctctggaa ctgggttccc gagtgcttga 300gctgtggctc ccgctgtagc
tctgaccagg tggaaactca agcctgcact cgggaacaga 360accgcatctg
cacctgcagg cccggctggt actgcgcgct gagcaagcag gaggggtgcc
420ggctgtgcgc gccgctgcgc aagtgccgcc cgggcttcgg cgtggccaga
ccaggaactg 480aaacatcaga cgtggtgtgc aagccctgtg ccccggggac
gttctccaac acgacttcat 540ccacggatat ttgcaggccc caccagatct
gtaacgtggt ggccatccct gggaatgcaa 600gcatggatgc agtctgcacg
tccacgtccc ccacccggag tatggcccca ggggcagtac 660acttacccca
gccagtgtcc acacgatccc aacacacgca gccaactcca gaacccagca
720ctgctccaag cacctccttc ctgctcccaa tgggccccag ccccccagct
gaagggagca 780ctggcgacga gcccaaatct tgtgacaaaa ctcacacatg
cccaccgtgc ccagcacctg 840aactcctggg ggggccctca gtcttcctct
tccccccaaa acccaaggac accctcatga 900tctcccggac ccctgaggtc
acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 960tcaagttcaa
ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg
1020aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg
caccaggact 1080ggctgaatgg caaggagtac aagtgcaagg tctccaacaa
agccctccca gcccccatcg 1140agaaaaccat ctccaaagcc aaagggcagc
cccgagaacc acaggtgtac accctgcccc 1200catcccggga tgagctgacc
aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 1260atcccagcga
catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga
1320ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag
ctcaccgtgg 1380acaagagcag gtggcagcag gggaacgtct tctcatgctc
cgtgatgcat gaggctctgc 1440acaaccacta cacgcagaag agcctctccc
tgtctccggg taaatgactc gagcggccg 1499163489PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 163Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val
Gly Leu Glu Leu1 5 10 15Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val
Ala Phe Thr Pro Tyr20 25 30Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu
Arg Glu Tyr Tyr Asp Gln35 40 45Thr Ala Gln Met Cys Cys Ser Lys Cys
Ser Pro Gly Gln His Ala Lys50 55 60Val Phe Cys Thr Lys Thr Ser Asp
Thr Val Cys Asp Ser Cys Glu Asp65 70 75 80Ser Thr Tyr Thr Gln Leu
Trp Asn Trp Val Pro Glu Cys Leu Ser Cys85 90 95Gly Ser Arg Cys Ser
Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg100 105 110Glu Gln Asn
Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu115 120 125Ser
Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg130 135
140Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val
Val145 150 155 160Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr
Thr Ser Ser Thr165 170 175Asp Ile Cys Arg Pro His Gln Ile Cys Asn
Val Val Ala Ile Pro Gly180 185 190Asn Ala Ser Met Asp Ala Val Cys
Thr Ser Thr Ser Pro Thr Arg Ser195 200 205Met Ala Pro Gly Ala Val
His Leu Pro Gln Pro Val Ser Thr Arg Ser210 215 220Gln His Thr Gln
Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser225 230 235 240Phe
Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly245 250
255Asp Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro260 265 270Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys275 280 285Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val290 295 300Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr305 310 315 320Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu325 330 335Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His340 345 350Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys355 360
365Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln370 375 380Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu385 390 395 400Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro405 410 415Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn420 425 430Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu435 440 445Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val450 455 460Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln465 470 475
480Lys Ser Leu Ser Leu Ser Pro Gly Lys485164467PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 164Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro
Glu Pro Gly Ser1 5 10 15Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr
Ala Gln Met Cys Cys20 25 30Ser Lys Cys Ser Pro Gly Gln His Ala Lys
Val Phe Cys Thr Lys Thr35 40 45Ser Asp Thr Val Cys Asp Ser Cys Glu
Asp Ser Thr Tyr Thr Gln Leu50 55 60Trp Asn Trp Val Pro Glu Cys Leu
Ser Cys Gly Ser Arg Cys Ser Ser65 70 75 80Asp Gln Val Glu Thr Gln
Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys85 90 95Thr Cys Arg Pro Gly
Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys100 105 110Arg Leu Cys
Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala115 120 125Arg
Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro130 135
140Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro
His145 150 155 160Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala
Ser Met Asp Ala165 170 175Val Cys Thr Ser Thr Ser Pro Thr Arg Ser
Met Ala Pro Gly Ala Val180 185 190His Leu Pro Gln Pro Val Ser Thr
Arg Ser Gln His Thr Gln Pro Thr195 200 205Pro Glu Pro Ser Thr Ala
Pro Ser Thr Ser Phe Leu Leu Pro Met Gly210 215 220Pro Ser Pro Pro
Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys225 230 235 240Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly245 250
255Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met260 265 270Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His275 280 285Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val290 295 300His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr305 310 315 320Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly325 330 335Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile340 345 350Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val355 360
365Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser370 375 380Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu385 390 395 400Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro405 410 415Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val420 425 430Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met435 440 445His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser450 455 460Pro Gly
Lys4651652906DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 165aagcttgaat tcccaccatg gtcagctact
gggacaccgg ggtcctgctg tgcgcgctgc 60tcagctgtct gcttctcaca ggatctagtt
caggttcaaa attaaaagat cctgaactga 120gtttaaaagg cacccagcac
atcatgcaag caggccagac actgcatctc caatgcaggg 180gggaagcagc
ccataaatgg tctttgcctg aaatggtgag taaggaaagc gaaaggctga
240gcataactaa atctgcctgt ggaagaaatg gcaaacaatt ctgcagtact
ttaaccttga 300acacagctca agcaaaccac actggcttct acagctgcaa
atatctagct gtacctactt 360caaagaagaa ggaaacagaa tctgcaatct
atatatttat tagtgataca ggtagacctt 420tcgtagagat gtacagtgaa
atccccgaaa ttatacacat gactgaagga agggagctcg 480tcattccctg
ccgggttacg tcacctaaca tcactgttac tttaaaaaag tttccacttg
540acactttgat ccctgatgga aaacgcataa tctgggacag tagaaagggc
ttcatcatat 600caaatgcaac gtacaaagaa atagggcttc tgacctgtga
agcaacagtc aatgggcatt 660tgtataagac aaactatctc acacatcgac
aaaccaatac aatcatagat gtccaaataa 720gcacaccacg cccagtcaaa
ttacttagag gccatactct tgtcctcaat tgtactgcta 780ccactccctt
gaacacgaga gttcaaatga cctggagtta ccctgatgaa aaaaataaga
840gagcttccgt aaggcgacga attgaccaaa gcaattccca tgccaacata
ttctacagtg 900ttcttactat tgacaaaatg cagaacaaag acaaaggact
ttatacttgt cgtgtaagga 960gtggaccatc attcaaatct gttaacacct
cagtgcatat atatgataaa gcattcatca 1020ctgtgaaaca tcgaaaacag
caggtgcttg aaaccgtagc tggcaagcgg tcttaccggc 1080tctctatgaa
agtgaaggca tttccctcgc cggaagttgt atggttaaaa gatgggttac
1140ctgcgactga gaaatctgct cgctatttga ctcgtggcta ctcgttaatt
atcaaggacg 1200taactgaaga ggatgcaggg aattatacaa tcttgctgag
cataaaacag tcaaatgtgt 1260ttaaaaacct cactgccact ctaattgtca
atgtgaaacc ccagatttac gaaaaggccg 1320tgtcatcgtt tccagacccg
gctctctacc cactgggcag cagacaaatc ctgacttgta 1380ccgcatatgg
tatccctcaa cctacaatca agtggttctg gcacccctgt aaccataatc
1440attccgaagc aaggtgtgac ttttgttcca ataatgaaga gtcctttatc
ctggatgctg 1500acagcaacat gggaaacaga attgagagca tcactcagcg
catggcaata atagaaggaa 1560agaataagat ggctagcacc ttggttgtgg
ctgactctag aatttctgga atctacattt 1620gcatagcttc caataaagtt
gggactgtgg gaagaaacat tagcttttat atcacagatg 1680tgccaaatgg
gtttcatgtt aacttggaaa aaatgccgac ggaaggagag gacctgaaac
1740tgtcttgcac agttaacaag ttcttataca gagacgttac ttggatttta
ctgcggacag 1800ttaataacag aacaatgcac tacagtatta gcaagcaaaa
aatggccatc actaaggagc 1860actccatcac tcttaatctt accatcatga
atgtttccct gcaagattca ggcacctatg 1920cctgcagagc caggaatgta
tacacagggg aagaaatcct ccagaagaaa gaaattacaa 1980tcagagatca
ggaagcacca tacctcctgc gaaacctcag tgatcacaca gtggccatca
2040gcagttccac cactttagac tgtcatgcta atggtgtccc cgagcctcag
atcacttggt 2100ttaaaaacaa ccacaaaata caacaagagc ctggaattat
tttaggacca ggaagcagca 2160cgctgtttat tgaaagagtc acagaagagg
atgaaggtgt ctatcactgc aaagccacca 2220accagaaggg ctctgtggaa
agttcagcat acctcactgt tcaaggaacc tcggacaagt 2280ctaatctgga
ggggtgcgta tccggtgaca ccattgtaat gactagtggc gggccccgca
2340ctgtggctga actggagggc aaaccgttca ccgcactgat tcgcggctct
ggctacccat 2400gcccctcagg tttcttccgc acctgtgaac gtgacgtata
tgatctgcgt acacgtgagg 2460gtcattgctt acgtttgacc catgatcacc
gtgttctggt gatggatggt ggcctggaat 2520ggcgtgccgc gggtgaactg
gaacgcggcg accgcctggt gatggatgat gcagctggcg 2580agtttccggc
actggcaacc ttccgtggcc tgcgtggcgc tggccgccag gatgtttatg
2640acgctactgt ttacggtgct agcgcattca ctgctaatgg cttcattgta
cacgcatgtg 2700gcgagcagcc cgggaccggt ctgaactcag gcctcacgac
aaatcctggt gtatccgctt 2760ggcaggtcaa cacagcttat actgcgggac
aattggtcac atataacggc aagacgtata 2820aatgtttgca gccccacacc
tccttggcag gatgggaacc atccaacgtt cctgccttgt 2880ggcagcttca
atgactcgag cggccg 2906166958PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 166Met Val Ser
Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser1 5 10 15Cys Leu
Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro20 25 30Glu
Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr35 40
45Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro50
55 60Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser
Ala65 70 75 80Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr
Leu Asn Thr85 90 95Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys
Tyr Leu Ala Val100 105 110Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser
Ala Ile Tyr Ile Phe Ile115 120 125Ser Asp Thr Gly Arg Pro Phe Val
Glu Met Tyr Ser Glu Ile Pro Glu130 135 140Ile Ile His Met Thr Glu
Gly Arg Glu Leu Val Ile Pro Cys Arg Val145 150 155 160Thr Ser Pro
Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr165 170 175Leu
Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe180 185
190Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys
Glu195 200 205Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu
Thr His Arg210 215 220Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser
Thr Pro Arg Pro Val225 230 235 240Lys Leu Leu Arg Gly His Thr Leu
Val Leu Asn Cys Thr Ala Thr Thr245 250 255Pro Leu Asn Thr Arg Val
Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys260 265 270Asn Lys Arg Ala
Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His275 280 285Ala Asn
Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys290 295
300Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe
Lys305 310 315 320Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala
Phe Ile Thr Val325 330 335Lys His Arg Lys Gln Gln Val Leu Glu Thr
Val Ala Gly Lys Arg Ser340 345 350Tyr Arg Leu Ser Met Lys Val Lys
Ala Phe Pro Ser Pro Glu Val Val355 360 365Trp Leu Lys Asp Gly Leu
Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu370 375 380Thr Arg Gly Tyr
Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala385 390 395 400Gly
Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys405 410
415Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr
Glu420 425 430Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro
Leu Gly Ser435 440 445Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile
Pro Gln Pro Thr Ile450 455 460Lys Trp Phe Trp His Pro Cys Asn His
Asn His Ser Glu Ala Arg Cys465 470 475 480Asp Phe Cys Ser Asn Asn
Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser485 490 495Asn Met Gly Asn
Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile500 505 510Glu Gly
Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg515 520
525Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr
Val530 535 540Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn
Gly Phe His545 550 555 560Val Asn Leu Glu Lys Met Pro Thr Glu Gly
Glu Asp Leu Lys Leu Ser565 570 575Cys Thr Val Asn Lys Phe Leu Tyr
Arg Asp Val Thr Trp Ile Leu Leu580 585 590Arg Thr Val Asn Asn Arg
Thr Met His Tyr Ser Ile Ser Lys Gln Lys595 600 605Met Ala Ile Thr
Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met610 615 620Asn Val
Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn625 630 635
640Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile
Arg645 650 655Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp
His Thr Val660 665 670Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His
Ala Asn Gly Val Pro675 680 685Glu Pro Gln Ile Thr Trp Phe Lys Asn
Asn His Lys Ile Gln Gln Glu690 695 700Pro Gly Ile Ile Leu Gly
Pro
Gly Ser Ser Thr Leu Phe Ile Glu Arg705 710 715 720Val Thr Glu Glu
Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln725 730 735Lys Gly
Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser740 745
750Asp Lys Ser Asn Leu Glu Gly Cys Val Ser Gly Asp Thr Ile Val
Met755 760 765Thr Ser Gly Gly Pro Arg Thr Val Ala Glu Leu Glu Gly
Lys Pro Phe770 775 780Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys
Pro Ser Gly Phe Phe785 790 795 800Arg Thr Cys Glu Arg Asp Val Tyr
Asp Leu Arg Thr Arg Glu Gly His805 810 815Cys Leu Arg Leu Thr His
Asp His Arg Val Leu Val Met Asp Gly Gly820 825 830Leu Glu Trp Arg
Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val835 840 845Met Asp
Asp Ala Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly850 855
860Leu Arg Gly Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr
Gly865 870 875 880Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile Val His
Ala Cys Gly Glu885 890 895Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu
Thr Thr Asn Pro Gly Val900 905 910Ser Ala Trp Gln Val Asn Thr Ala
Tyr Thr Ala Gly Gln Leu Val Thr915 920 925Tyr Asn Gly Lys Thr Tyr
Lys Cys Leu Gln Pro His Thr Ser Leu Ala930 935 940Gly Trp Glu Pro
Ser Asn Val Pro Ala Leu Trp Gln Leu Gln945 950
955167932PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 167Ser Lys Leu Lys Asp Pro Glu Leu Ser
Leu Lys Gly Thr Gln His Ile1 5 10 15Met Gln Ala Gly Gln Thr Leu His
Leu Gln Cys Arg Gly Glu Ala Ala20 25 30His Lys Trp Ser Leu Pro Glu
Met Val Ser Lys Glu Ser Glu Arg Leu35 40 45Ser Ile Thr Lys Ser Ala
Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser50 55 60Thr Leu Thr Leu Asn
Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser65 70 75 80Cys Lys Tyr
Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser85 90 95Ala Ile
Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met100 105
110Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu
Leu115 120 125Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys130 135 140Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly
Lys Arg Ile Ile Trp145 150 155 160Asp Ser Arg Lys Gly Phe Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile165 170 175Gly Leu Leu Thr Cys Glu
Ala Thr Val Asn Gly His Leu Tyr Lys Thr180 185 190Asn Tyr Leu Thr
His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile195 200 205Ser Thr
Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val Leu210 215
220Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr
Trp225 230 235 240Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val
Arg Arg Arg Ile245 250 255Asp Gln Ser Asn Ser His Ala Asn Ile Phe
Tyr Ser Val Leu Thr Ile260 265 270Asp Lys Met Gln Asn Lys Asp Lys
Gly Leu Tyr Thr Cys Arg Val Arg275 280 285Ser Gly Pro Ser Phe Lys
Ser Val Asn Thr Ser Val His Ile Tyr Asp290 295 300Lys Ala Phe Ile
Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr305 310 315 320Val
Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe325 330
335Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr
Glu340 345 350Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile
Ile Lys Asp355 360 365Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile
Leu Leu Ser Ile Lys370 375 380Gln Ser Asn Val Phe Lys Asn Leu Thr
Ala Thr Leu Ile Val Asn Val385 390 395 400Lys Pro Gln Ile Tyr Glu
Lys Ala Val Ser Ser Phe Pro Asp Pro Ala405 410 415Leu Tyr Pro Leu
Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly420 425 430Ile Pro
Gln Pro Thr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn435 440
445His Ser Glu Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser
Phe450 455 460Ile Leu Asp Ala Asp Ser Asn Met Gly Asn Arg Ile Glu
Ser Ile Thr465 470 475 480Gln Arg Met Ala Ile Ile Glu Gly Lys Asn
Lys Met Ala Ser Thr Leu485 490 495Val Val Ala Asp Ser Arg Ile Ser
Gly Ile Tyr Ile Cys Ile Ala Ser500 505 510Asn Lys Val Gly Thr Val
Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp515 520 525Val Pro Asn Gly
Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly530 535 540Glu Asp
Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp545 550 555
560Val Thr Trp Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His
Tyr565 570 575Ser Ile Ser Lys Gln Lys Met Ala Ile Thr Lys Glu His
Ser Ile Thr580 585 590Leu Asn Leu Thr Ile Met Asn Val Ser Leu Gln
Asp Ser Gly Thr Tyr595 600 605Ala Cys Arg Ala Arg Asn Val Tyr Thr
Gly Glu Glu Ile Leu Gln Lys610 615 620Lys Glu Ile Thr Ile Arg Asp
Gln Glu Ala Pro Tyr Leu Leu Arg Asn625 630 635 640Leu Ser Asp His
Thr Val Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys645 650 655His Ala
Asn Gly Val Pro Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn660 665
670His Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser
Ser675 680 685Thr Leu Phe Ile Glu Arg Val Thr Glu Glu Asp Glu Gly
Val Tyr His690 695 700Cys Lys Ala Thr Asn Gln Lys Gly Ser Val Glu
Ser Ser Ala Tyr Leu705 710 715 720Thr Val Gln Gly Thr Ser Asp Lys
Ser Asn Leu Glu Gly Cys Val Ser725 730 735Gly Asp Thr Ile Val Met
Thr Ser Gly Gly Pro Arg Thr Val Ala Glu740 745 750Leu Glu Gly Lys
Pro Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro755 760 765Cys Pro
Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu770 775
780Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr His Asp His Arg
Val785 790 795 800Leu Val Met Asp Gly Gly Leu Glu Trp Arg Ala Ala
Gly Glu Leu Glu805 810 815Arg Gly Asp Arg Leu Val Met Asp Asp Ala
Ala Gly Glu Phe Pro Ala820 825 830Leu Ala Thr Phe Arg Gly Leu Arg
Gly Ala Gly Arg Gln Asp Val Tyr835 840 845Asp Ala Thr Val Tyr Gly
Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile850 855 860Val His Ala Cys
Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu865 870 875 880Thr
Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr885 890
895Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu
Gln900 905 910Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val
Pro Ala Leu915 920 925Trp Gln Leu Gln930168733PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 168Ser Lys Leu Lys Asp Pro Glu Leu Ser Leu Lys Gly
Thr Gln His Ile1 5 10 15Met Gln Ala Gly Gln Thr Leu His Leu Gln Cys
Arg Gly Glu Ala Ala20 25 30His Lys Trp Ser Leu Pro Glu Met Val Ser
Lys Glu Ser Glu Arg Leu35 40 45Ser Ile Thr Lys Ser Ala Cys Gly Arg
Asn Gly Lys Gln Phe Cys Ser50 55 60Thr Leu Thr Leu Asn Thr Ala Gln
Ala Asn His Thr Gly Phe Tyr Ser65 70 75 80Cys Lys Tyr Leu Ala Val
Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser85 90 95Ala Ile Tyr Ile Phe
Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met100 105 110Tyr Ser Glu
Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu Leu115 120 125Val
Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys130 135
140Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile Ile
Trp145 150 155 160Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr
Tyr Lys Glu Ile165 170 175Gly Leu Leu Thr Cys Glu Ala Thr Val Asn
Gly His Leu Tyr Lys Thr180 185 190Asn Tyr Leu Thr His Arg Gln Thr
Asn Thr Ile Ile Asp Val Gln Ile195 200 205Ser Thr Pro Arg Pro Val
Lys Leu Leu Arg Gly His Thr Leu Val Leu210 215 220Asn Cys Thr Ala
Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr Trp225 230 235 240Ser
Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile245 250
255Asp Gln Ser Asn Ser His Ala Asn Ile Phe Tyr Ser Val Leu Thr
Ile260 265 270Asp Lys Met Gln Asn Lys Asp Lys Gly Leu Tyr Thr Cys
Arg Val Arg275 280 285Ser Gly Pro Ser Phe Lys Ser Val Asn Thr Ser
Val His Ile Tyr Asp290 295 300Lys Ala Phe Ile Thr Val Lys His Arg
Lys Gln Gln Val Leu Glu Thr305 310 315 320Val Ala Gly Lys Arg Ser
Tyr Arg Leu Ser Met Lys Val Lys Ala Phe325 330 335Pro Ser Pro Glu
Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu340 345 350Lys Ser
Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp355 360
365Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile Leu Leu Ser Ile
Lys370 375 380Gln Ser Asn Val Phe Lys Asn Leu Thr Ala Thr Leu Ile
Val Asn Val385 390 395 400Lys Pro Gln Ile Tyr Glu Lys Ala Val Ser
Ser Phe Pro Asp Pro Ala405 410 415Leu Tyr Pro Leu Gly Ser Arg Gln
Ile Leu Thr Cys Thr Ala Tyr Gly420 425 430Ile Pro Gln Pro Thr Ile
Lys Trp Phe Trp His Pro Cys Asn His Asn435 440 445His Ser Glu Ala
Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe450 455 460Ile Leu
Asp Ala Asp Ser Asn Met Gly Asn Arg Ile Glu Ser Ile Thr465 470 475
480Gln Arg Met Ala Ile Ile Glu Gly Lys Asn Lys Met Ala Ser Thr
Leu485 490 495Val Val Ala Asp Ser Arg Ile Ser Gly Ile Tyr Ile Cys
Ile Ala Ser500 505 510Asn Lys Val Gly Thr Val Gly Arg Asn Ile Ser
Phe Tyr Ile Thr Asp515 520 525Val Pro Asn Gly Phe His Val Asn Leu
Glu Lys Met Pro Thr Glu Gly530 535 540Glu Asp Leu Lys Leu Ser Cys
Thr Val Asn Lys Phe Leu Tyr Arg Asp545 550 555 560Val Thr Trp Ile
Leu Leu Arg Thr Val Asn Asn Arg Thr Met His Tyr565 570 575Ser Ile
Ser Lys Gln Lys Met Ala Ile Thr Lys Glu His Ser Ile Thr580 585
590Leu Asn Leu Thr Ile Met Asn Val Ser Leu Gln Asp Ser Gly Thr
Tyr595 600 605Ala Cys Arg Ala Arg Asn Val Tyr Thr Gly Glu Glu Ile
Leu Gln Lys610 615 620Lys Glu Ile Thr Ile Arg Asp Gln Glu Ala Pro
Tyr Leu Leu Arg Asn625 630 635 640Leu Ser Asp His Thr Val Ala Ile
Ser Ser Ser Thr Thr Leu Asp Cys645 650 655His Ala Asn Gly Val Pro
Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn660 665 670His Lys Ile Gln
Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser675 680 685Thr Leu
Phe Ile Glu Arg Val Thr Glu Glu Asp Glu Gly Val Tyr His690 695
700Cys Lys Ala Thr Asn Gln Lys Gly Ser Val Glu Ser Ser Ala Tyr
Leu705 710 715 720Thr Val Gln Gly Thr Ser Asp Lys Ser Asn Leu Glu
Xaa725 730169734PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 169Ser Lys Leu Lys Asp Pro Glu Leu
Ser Leu Lys Gly Thr Gln His Ile1 5 10 15Met Gln Ala Gly Gln Thr Leu
His Leu Gln Cys Arg Gly Glu Ala Ala20 25 30His Lys Trp Ser Leu Pro
Glu Met Val Ser Lys Glu Ser Glu Arg Leu35 40 45Ser Ile Thr Lys Ser
Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser50 55 60Thr Leu Thr Leu
Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr Ser65 70 75 80Cys Lys
Tyr Leu Ala Val Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser85 90 95Ala
Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met100 105
110Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu
Leu115 120 125Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val
Thr Leu Lys130 135 140Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly
Lys Arg Ile Ile Trp145 150 155 160Asp Ser Arg Lys Gly Phe Ile Ile
Ser Asn Ala Thr Tyr Lys Glu Ile165 170 175Gly Leu Leu Thr Cys Glu
Ala Thr Val Asn Gly His Leu Tyr Lys Thr180 185 190Asn Tyr Leu Thr
His Arg Gln Thr Asn Thr Ile Ile Asp Val Gln Ile195 200 205Ser Thr
Pro Arg Pro Val Lys Leu Leu Arg Gly His Thr Leu Val Leu210 215
220Asn Cys Thr Ala Thr Thr Pro Leu Asn Thr Arg Val Gln Met Thr
Trp225 230 235 240Ser Tyr Pro Asp Glu Lys Asn Lys Arg Ala Ser Val
Arg Arg Arg Ile245 250 255Asp Gln Ser Asn Ser His Ala Asn Ile Phe
Tyr Ser Val Leu Thr Ile260 265 270Asp Lys Met Gln Asn Lys Asp Lys
Gly Leu Tyr Thr Cys Arg Val Arg275 280 285Ser Gly Pro Ser Phe Lys
Ser Val Asn Thr Ser Val His Ile Tyr Asp290 295 300Lys Ala Phe Ile
Thr Val Lys His Arg Lys Gln Gln Val Leu Glu Thr305 310 315 320Val
Ala Gly Lys Arg Ser Tyr Arg Leu Ser Met Lys Val Lys Ala Phe325 330
335Pro Ser Pro Glu Val Val Trp Leu Lys Asp Gly Leu Pro Ala Thr
Glu340 345 350Lys Ser Ala Arg Tyr Leu Thr Arg Gly Tyr Ser Leu Ile
Ile Lys Asp355 360 365Val Thr Glu Glu Asp Ala Gly Asn Tyr Thr Ile
Leu Leu Ser Ile Lys370 375 380Gln Ser Asn Val Phe Lys Asn Leu Thr
Ala Thr Leu Ile Val Asn Val385 390 395 400Lys Pro Gln Ile Tyr Glu
Lys Ala Val Ser Ser Phe Pro Asp Pro Ala405 410 415Leu Tyr Pro Leu
Gly Ser Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly420 425 430Ile Pro
Gln Pro Thr Ile Lys Trp Phe Trp His Pro Cys Asn His Asn435 440
445His Ser Glu Ala Arg Cys Asp Phe Cys Ser Asn Asn Glu Glu Ser
Phe450 455 460Ile Leu Asp Ala Asp Ser Asn Met Gly Asn Arg Ile Glu
Ser Ile Thr465 470 475 480Gln Arg Met Ala Ile Ile Glu Gly Lys Asn
Lys Met Ala Ser Thr Leu485 490 495Val Val Ala Asp Ser Arg Ile Ser
Gly Ile Tyr Ile Cys Ile Ala Ser500 505 510Asn Lys Val Gly Thr Val
Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp515 520 525Val Pro Asn Gly
Phe His Val Asn Leu Glu Lys Met Pro Thr Glu Gly530 535 540Glu Asp
Leu Lys Leu Ser Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp545 550 555
560Val Thr Trp Ile Leu Leu Arg Thr Val Asn Asn Arg Thr Met His
Tyr565 570 575Ser Ile Ser Lys Gln Lys Met Ala Ile Thr Lys Glu His
Ser Ile Thr580 585 590Leu Asn Leu Thr Ile Met Asn Val Ser Leu Gln
Asp Ser Gly Thr Tyr595 600 605Ala Cys Arg Ala Arg Asn Val Tyr Thr
Gly Glu Glu Ile Leu Gln Lys610 615 620Lys Glu Ile Thr Ile Arg Asp
Gln Glu Ala Pro Tyr Leu Leu Arg Asn625 630 635 640Leu Ser Asp His
Thr Val Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys645 650 655His Ala
Asn Gly Val Pro Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn660 665
670His Lys Ile Gln Gln Glu Pro Gly Ile Ile Leu Gly Pro Gly Ser
Ser675 680 685Thr Leu Phe Ile Glu Arg Val Thr Glu Glu Asp Glu Gly
Val Tyr His690 695
700Cys Lys Ala Thr Asn Gln Lys Gly Ser Val Glu Ser Ser Ala Tyr
Leu705 710 715 720Thr Val Gln Gly Thr Ser Asp Lys Ser Asn Leu Glu
Gly Xaa725 7301702924DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 170aagcttgaat tcccaccatg
cagagcaagg tgctgctggc cgtcgccctg tggctctgcg 60tggagacccg ggccgcctct
gtgggtttgc ctagtgtttc tcttgatctg cccaggctca 120gcatacaaaa
agacatactt acaattaagg ctaatacaac tcttcaaatt acttgcaggg
180gacagaggga cttggactgg ctttggccca ataatcagag tggcagtgag
caaagggtgg 240aggtgactga gtgcagcgat ggcctcttct gtaagacact
cacaattcca aaagtgatcg 300gaaatgacac tggagcctac aagtgcttct
accgggaaac tgacttggcc tcggtcattt 360atgtctatgt tcaagattac
agatctccat ttattgcttc tgttagtgac caacatggag 420tcgtgtacat
tactgagaac aaaaacaaaa ctgtggtgat tccatgtctc gggtccattt
480caaatctcaa cgtgtcactt tgtgcaagat acccagaaaa gagatttgtt
cctgatggta 540acagaatttc ctgggacagc aagaagggct ttactattcc
cagctacatg atcagctatg 600ctggcatggt cttctgtgaa gcaaaaatta
atgatgaaag ttaccagtct attatgtaca 660tagttgtcgt tgtagggtat
aggatttatg atgtggttct gagtccgtct catggaattg 720aactatctgt
tggagaaaaa cttgtcttaa attgtacagc aagaactgaa ctaaatgtgg
780ggattgactt caactgggaa tacccttctt cgaagcatca gcataagaaa
cttgtaaacc 840gagacctaaa aacccagtct gggagtgaga tgaagaaatt
tttgagcacc ttaactatag 900atggtgtaac ccggagtgac caaggattgt
acacctgtgc agcatccagt gggctgatga 960ccaagaagaa cagcacattt
gtcagggtcc atgaaaaacc ttttgttgct tttggaagtg 1020gcatggaatc
tctggtggaa gccacggtgg gggagcgtgt cagaatccct gcgaagtacc
1080ttggttaccc acccccagaa ataaaatggt ataaaaatgg aatacccctt
gagtccaatc 1140acacaattaa agcggggcat gtactgacga ttatggaagt
gagtgaaaga gacacaggaa 1200attacactgt catccttacc aatcccattt
caaaggagaa gcagagccat gtggtctctc 1260tggttgtgta tgtcccaccc
cagattggtg agaaatctct aatctctcct gtggattcct 1320accagtacgg
caccactcaa acgctgacat gtacggtcta tgccattcct cccccgcatc
1380acatccactg gtattggcag ttggaggaag agtgcgccaa cgagcccagc
caagctgtct 1440cagtgacaaa cccataccct tgtgaagaat ggagaagtgt
ggaggacttc cagggaggaa 1500ataaaattga agttaataaa aatcaatttg
ctctaattga aggaaaaaac aaaactgtaa 1560gtacccttgt tatccaagcg
gcaaatgtgt cagctttgta caaatgtgaa gcggtcaaca 1620aagtcgggag
aggagagagg gtgatctcct tccacgtgac caggggtcct gaaattactt
1680tgcaacctga catgcagccc actgagcagg agagcgtgtc tttgtggtgc
actgcagaca 1740gatctacgtt tgagaacctc acatggtaca aacttggccc
acagcctctg ccaatccatg 1800tgggagagtt gcccacacct gtttgcaaga
acttggatac tctttggaaa ttgaatgcca 1860ccatgttctc taatagcaca
aatgacattt tgatcatgga gcttaagaat gcatccttgc 1920aggaccaagg
agactatgtc tgccttgctc aagacaggaa gaccaagaaa agacattgcg
1980tggtcaggca gctcacagtc ctagagcgtg tggcacccac gatcacagga
aacctggaga 2040atcagacgac aagtattggg gaaagcatcg aagtctcatg
cacggcatct gggaatcccc 2100ctccacagat catgtggttt aaagataatg
agacccttgt agaagactca ggcattgtat 2160tgaaggatgg gaaccggaac
ctcactatcc gcagagtgag gaaggaggac gaaggcctct 2220acacctgcca
ggcatgcagt gttcttggct gtgcaaaagt ggaggcattt ttcataatag
2280aaggtgccca ggaaaagacg aacttggaag ggtgcgtatc cggtgacacc
attgtaatga 2340ctagtggcgg gccccgcact gtggctgaac tggagggcaa
accgttcacc gcactgattc 2400gcggctctgg ctacccatgc ccctcaggtt
tcttccgcac ctgtgaacgt gacgtatatg 2460atctgcgtac acgtgagggt
cattgcttac gtttgaccca tgatcaccgt gttctggtga 2520tggatggtgg
cctggaatgg cgtgccgcgg gtgaactgga acgcggcgac cgcctggtga
2580tggatgatgc agctggcgag tttccggcac tggcaacctt ccgtggcctg
cgtggcgctg 2640gccgccagga tgtttatgac gctactgttt acggtgctag
cgcattcact gctaatggct 2700tcattgtaca cgcatgtggc gagcagcccg
ggaccggtct gaactcaggc ctcacgacaa 2760atcctggtgt atccgcttgg
caggtcaaca cagcttatac tgcgggacaa ttggtcacat 2820ataacggcaa
gacgtataaa tgtttgcagc cccacacctc cttggcagga tgggaaccat
2880ccaacgttcc tgccttgtgg cagcttcaat gactcgagcg gccg
2924171964PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 171Met Gln Ser Lys Val Leu Leu Ala Val
Ala Leu Trp Leu Cys Val Glu1 5 10 15Thr Arg Ala Ala Ser Val Gly Leu
Pro Ser Val Ser Leu Asp Leu Pro20 25 30Arg Leu Ser Ile Gln Lys Asp
Ile Leu Thr Ile Lys Ala Asn Thr Thr35 40 45Leu Gln Ile Thr Cys Arg
Gly Gln Arg Asp Leu Asp Trp Leu Trp Pro50 55 60Asn Asn Gln Ser Gly
Ser Glu Gln Arg Val Glu Val Thr Glu Cys Ser65 70 75 80Asp Gly Leu
Phe Cys Lys Thr Leu Thr Ile Pro Lys Val Ile Gly Asn85 90 95Asp Thr
Gly Ala Tyr Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser100 105
110Val Ile Tyr Val Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala
Ser115 120 125Val Ser Asp Gln His Gly Val Val Tyr Ile Thr Glu Asn
Lys Asn Lys130 135 140Thr Val Val Ile Pro Cys Leu Gly Ser Ile Ser
Asn Leu Asn Val Ser145 150 155 160Leu Cys Ala Arg Tyr Pro Glu Lys
Arg Phe Val Pro Asp Gly Asn Arg165 170 175Ile Ser Trp Asp Ser Lys
Lys Gly Phe Thr Ile Pro Ser Tyr Met Ile180 185 190Ser Tyr Ala Gly
Met Val Phe Cys Glu Ala Lys Ile Asn Asp Glu Ser195 200 205Tyr Gln
Ser Ile Met Tyr Ile Val Val Val Val Gly Tyr Arg Ile Tyr210 215
220Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly
Glu225 230 235 240Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu
Asn Val Gly Ile245 250 255Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys
His Gln His Lys Lys Leu260 265 270Val Asn Arg Asp Leu Lys Thr Gln
Ser Gly Ser Glu Met Lys Lys Phe275 280 285Leu Ser Thr Leu Thr Ile
Asp Gly Val Thr Arg Ser Asp Gln Gly Leu290 295 300Tyr Thr Cys Ala
Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr305 310 315 320Phe
Val Arg Val His Glu Lys Pro Phe Val Ala Phe Gly Ser Gly Met325 330
335Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro
Ala340 345 350Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr
Lys Asn Gly355 360 365Ile Pro Leu Glu Ser Asn His Thr Ile Lys Ala
Gly His Val Leu Thr370 375 380Ile Met Glu Val Ser Glu Arg Asp Thr
Gly Asn Tyr Thr Val Ile Leu385 390 395 400Thr Asn Pro Ile Ser Lys
Glu Lys Gln Ser His Val Val Ser Leu Val405 410 415Val Tyr Val Pro
Pro Gln Ile Gly Glu Lys Ser Leu Ile Ser Pro Val420 425 430Asp Ser
Tyr Gln Tyr Gly Thr Thr Gln Thr Leu Thr Cys Thr Val Tyr435 440
445Ala Ile Pro Pro Pro His His Ile His Trp Tyr Trp Gln Leu Glu
Glu450 455 460Glu Cys Ala Asn Glu Pro Ser Gln Ala Val Ser Val Thr
Asn Pro Tyr465 470 475 480Pro Cys Glu Glu Trp Arg Ser Val Glu Asp
Phe Gln Gly Gly Asn Lys485 490 495Ile Glu Val Asn Lys Asn Gln Phe
Ala Leu Ile Glu Gly Lys Asn Lys500 505 510Thr Val Ser Thr Leu Val
Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr515 520 525Lys Cys Glu Ala
Val Asn Lys Val Gly Arg Gly Glu Arg Val Ile Ser530 535 540Phe His
Val Thr Arg Gly Pro Glu Ile Thr Leu Gln Pro Asp Met Gln545 550 555
560Pro Thr Glu Gln Glu Ser Val Ser Leu Trp Cys Thr Ala Asp Arg
Ser565 570 575Thr Phe Glu Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln
Pro Leu Pro580 585 590Ile His Val Gly Glu Leu Pro Thr Pro Val Cys
Lys Asn Leu Asp Thr595 600 605Leu Trp Lys Leu Asn Ala Thr Met Phe
Ser Asn Ser Thr Asn Asp Ile610 615 620Leu Ile Met Glu Leu Lys Asn
Ala Ser Leu Gln Asp Gln Gly Asp Tyr625 630 635 640Val Cys Leu Ala
Gln Asp Arg Lys Thr Lys Lys Arg His Cys Val Val645 650 655Arg Gln
Leu Thr Val Leu Glu Arg Val Ala Pro Thr Ile Thr Gly Asn660 665
670Leu Glu Asn Gln Thr Thr Ser Ile Gly Glu Ser Ile Glu Val Ser
Cys675 680 685Thr Ala Ser Gly Asn Pro Pro Pro Gln Ile Met Trp Phe
Lys Asp Asn690 695 700Glu Thr Leu Val Glu Asp Ser Gly Ile Val Leu
Lys Asp Gly Asn Arg705 710 715 720Asn Leu Thr Ile Arg Arg Val Arg
Lys Glu Asp Glu Gly Leu Tyr Thr725 730 735Cys Gln Ala Cys Ser Val
Leu Gly Cys Ala Lys Val Glu Ala Phe Phe740 745 750Ile Ile Glu Gly
Ala Gln Glu Lys Thr Asn Leu Glu Gly Cys Val Ser755 760 765Gly Asp
Thr Ile Val Met Thr Ser Gly Gly Pro Arg Thr Val Ala Glu770 775
780Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr
Pro785 790 795 800Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp
Val Tyr Asp Leu805 810 815Arg Thr Arg Glu Gly His Cys Leu Arg Leu
Thr His Asp His Arg Val820 825 830Leu Val Met Asp Gly Gly Leu Glu
Trp Arg Ala Ala Gly Glu Leu Glu835 840 845Arg Gly Asp Arg Leu Val
Met Asp Asp Ala Ala Gly Glu Phe Pro Ala850 855 860Leu Ala Thr Phe
Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp Val Tyr865 870 875 880Asp
Ala Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile885 890
895Val His Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly
Leu900 905 910Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr
Ala Tyr Thr915 920 925Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr
Tyr Lys Cys Leu Gln930 935 940Pro His Thr Ser Leu Ala Gly Trp Glu
Pro Ser Asn Val Pro Ala Leu945 950 955 960Trp Gln Leu
Gln172945PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 172Ala Ser Val Gly Leu Pro Ser Val Ser
Leu Asp Leu Pro Arg Leu Ser1 5 10 15Ile Gln Lys Asp Ile Leu Thr Ile
Lys Ala Asn Thr Thr Leu Gln Ile20 25 30Thr Cys Arg Gly Gln Arg Asp
Leu Asp Trp Leu Trp Pro Asn Asn Gln35 40 45Ser Gly Ser Glu Gln Arg
Val Glu Val Thr Glu Cys Ser Asp Gly Leu50 55 60Phe Cys Lys Thr Leu
Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly65 70 75 80Ala Tyr Lys
Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr85 90 95Val Tyr
Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp100 105
110Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val
Val115 120 125Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser
Leu Cys Ala130 135 140Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly
Asn Arg Ile Ser Trp145 150 155 160Asp Ser Lys Lys Gly Phe Thr Ile
Pro Ser Tyr Met Ile Ser Tyr Ala165 170 175Gly Met Val Phe Cys Glu
Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser180 185 190Ile Met Tyr Ile
Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val195 200 205Leu Ser
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val210 215
220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe
Asn225 230 235 240Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
Leu Val Asn Arg245 250 255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
Lys Lys Phe Leu Ser Thr260 265 270Leu Thr Ile Asp Gly Val Thr Arg
Ser Asp Gln Gly Leu Tyr Thr Cys275 280 285Ala Ala Ser Ser Gly Leu
Met Thr Lys Lys Asn Ser Thr Phe Val Arg290 295 300Val His Glu Lys
Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu305 310 315 320Val
Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu325 330
335Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro
Leu340 345 350Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr
Ile Met Glu355 360 365Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val
Ile Leu Thr Asn Pro370 375 380Ile Ser Lys Glu Lys Gln Ser His Val
Val Ser Leu Val Val Tyr Val385 390 395 400Pro Pro Gln Ile Gly Glu
Lys Ser Leu Ile Ser Pro Val Asp Ser Tyr405 410 415Gln Tyr Gly Thr
Thr Gln Thr Leu Thr Cys Thr Val Tyr Ala Ile Pro420 425 430Pro Pro
His His Ile His Trp Tyr Trp Gln Leu Glu Glu Glu Cys Ala435 440
445Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr Pro Cys
Glu450 455 460Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys
Ile Glu Val465 470 475 480Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly
Lys Asn Lys Thr Val Ser485 490 495Thr Leu Val Ile Gln Ala Ala Asn
Val Ser Ala Leu Tyr Lys Cys Glu500 505 510Ala Val Asn Lys Val Gly
Arg Gly Glu Arg Val Ile Ser Phe His Val515 520 525Thr Arg Gly Pro
Glu Ile Thr Leu Gln Pro Asp Met Gln Pro Thr Glu530 535 540Gln Glu
Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu545 550 555
560Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His
Val565 570 575Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr
Leu Trp Lys580 585 590Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn
Asp Ile Leu Ile Met595 600 605Glu Leu Lys Asn Ala Ser Leu Gln Asp
Gln Gly Asp Tyr Val Cys Leu610 615 620Ala Gln Asp Arg Lys Thr Lys
Lys Arg His Cys Val Val Arg Gln Leu625 630 635 640Thr Val Leu Glu
Arg Val Ala Pro Thr Ile Thr Gly Asn Leu Glu Asn645 650 655Gln Thr
Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys Thr Ala Ser660 665
670Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn Glu Thr
Leu675 680 685Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg
Asn Leu Thr690 695 700Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu
Tyr Thr Cys Gln Ala705 710 715 720Cys Ser Val Leu Gly Cys Ala Lys
Val Glu Ala Phe Phe Ile Ile Glu725 730 735Gly Ala Gln Glu Lys Thr
Asn Leu Glu Gly Cys Val Ser Gly Asp Thr740 745 750Ile Val Met Thr
Ser Gly Gly Pro Arg Thr Val Ala Glu Leu Glu Gly755 760 765Lys Pro
Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser770 775
780Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg Thr
Arg785 790 795 800Glu Gly His Cys Leu Arg Leu Thr His Asp His Arg
Val Leu Val Met805 810 815Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly
Glu Leu Glu Arg Gly Asp820 825 830Arg Leu Val Met Asp Asp Ala Ala
Gly Glu Phe Pro Ala Leu Ala Thr835 840 845Phe Arg Gly Leu Arg Gly
Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr850 855 860Val Tyr Gly Ala
Ser Ala Phe Thr Ala Asn Gly Phe Ile Val His Ala865 870 875 880Cys
Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn885 890
895Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly
Gln900 905 910Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln
Pro His Thr915 920 925Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro
Ala Leu Trp Gln Leu930 935 940Gln945173746PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 173Ala Ser Val Gly Leu Pro Ser Val Ser Leu Asp Leu
Pro Arg Leu Ser1 5 10 15Ile Gln Lys Asp Ile Leu Thr Ile Lys Ala Asn
Thr Thr Leu Gln Ile20 25 30Thr Cys Arg Gly Gln Arg Asp Leu Asp Trp
Leu Trp Pro Asn Asn Gln35 40 45Ser Gly Ser Glu Gln Arg Val Glu Val
Thr Glu Cys Ser Asp Gly Leu50 55 60Phe Cys Lys Thr Leu Thr Ile Pro
Lys Val Ile Gly Asn Asp Thr Gly65 70 75 80Ala Tyr Lys Cys Phe Tyr
Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr85 90 95Val Tyr Val Gln Asp
Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp100 105 110Gln His Gly
Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val Val115 120 125Ile
Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser Leu Cys Ala130
135 140Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly Asn Arg Ile Ser
Trp145 150 155 160Asp Ser Lys Lys Gly Phe Thr Ile Pro Ser Tyr Met
Ile Ser Tyr Ala165 170 175Gly Met Val Phe Cys Glu Ala Lys Ile Asn
Asp Glu Ser Tyr Gln Ser180 185 190Ile Met Tyr Ile Val Val Val Val
Gly Tyr Arg Ile Tyr Asp Val Val195 200 205Leu Ser Pro Ser His Gly
Ile Glu Leu Ser Val Gly Glu Lys Leu Val210 215 220Leu Asn Cys Thr
Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe Asn225 230 235 240Trp
Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn Arg245 250
255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser
Thr260 265 270Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu
Tyr Thr Cys275 280 285Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn
Ser Thr Phe Val Arg290 295 300Val His Glu Lys Pro Phe Val Ala Phe
Gly Ser Gly Met Glu Ser Leu305 310 315 320Val Glu Ala Thr Val Gly
Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu325 330 335Gly Tyr Pro Pro
Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu340 345 350Glu Ser
Asn His Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu355 360
365Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn
Pro370 375 380Ile Ser Lys Glu Lys Gln Ser His Val Val Ser Leu Val
Val Tyr Val385 390 395 400Pro Pro Gln Ile Gly Glu Lys Ser Leu Ile
Ser Pro Val Asp Ser Tyr405 410 415Gln Tyr Gly Thr Thr Gln Thr Leu
Thr Cys Thr Val Tyr Ala Ile Pro420 425 430Pro Pro His His Ile His
Trp Tyr Trp Gln Leu Glu Glu Glu Cys Ala435 440 445Asn Glu Pro Ser
Gln Ala Val Ser Val Thr Asn Pro Tyr Pro Cys Glu450 455 460Glu Trp
Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys Ile Glu Val465 470 475
480Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly Lys Asn Lys Thr Val
Ser485 490 495Thr Leu Val Ile Gln Ala Ala Asn Val Ser Ala Leu Tyr
Lys Cys Glu500 505 510Ala Val Asn Lys Val Gly Arg Gly Glu Arg Val
Ile Ser Phe His Val515 520 525Thr Arg Gly Pro Glu Ile Thr Leu Gln
Pro Asp Met Gln Pro Thr Glu530 535 540Gln Glu Ser Val Ser Leu Trp
Cys Thr Ala Asp Arg Ser Thr Phe Glu545 550 555 560Asn Leu Thr Trp
Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His Val565 570 575Gly Glu
Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr Leu Trp Lys580 585
590Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn Asp Ile Leu Ile
Met595 600 605Glu Leu Lys Asn Ala Ser Leu Gln Asp Gln Gly Asp Tyr
Val Cys Leu610 615 620Ala Gln Asp Arg Lys Thr Lys Lys Arg His Cys
Val Val Arg Gln Leu625 630 635 640Thr Val Leu Glu Arg Val Ala Pro
Thr Ile Thr Gly Asn Leu Glu Asn645 650 655Gln Thr Thr Ser Ile Gly
Glu Ser Ile Glu Val Ser Cys Thr Ala Ser660 665 670Gly Asn Pro Pro
Pro Gln Ile Met Trp Phe Lys Asp Asn Glu Thr Leu675 680 685Val Glu
Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg Asn Leu Thr690 695
700Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu Tyr Thr Cys Gln
Ala705 710 715 720Cys Ser Val Leu Gly Cys Ala Lys Val Glu Ala Phe
Phe Ile Ile Glu725 730 735Gly Ala Gln Glu Lys Thr Asn Leu Glu
Xaa740 745174747PRTArtificial Sequenceartificial construct relating
to Homo Sapiens immunoglobulin 174Ala Ser Val Gly Leu Pro Ser Val
Ser Leu Asp Leu Pro Arg Leu Ser1 5 10 15Ile Gln Lys Asp Ile Leu Thr
Ile Lys Ala Asn Thr Thr Leu Gln Ile20 25 30Thr Cys Arg Gly Gln Arg
Asp Leu Asp Trp Leu Trp Pro Asn Asn Gln35 40 45Ser Gly Ser Glu Gln
Arg Val Glu Val Thr Glu Cys Ser Asp Gly Leu50 55 60Phe Cys Lys Thr
Leu Thr Ile Pro Lys Val Ile Gly Asn Asp Thr Gly65 70 75 80Ala Tyr
Lys Cys Phe Tyr Arg Glu Thr Asp Leu Ala Ser Val Ile Tyr85 90 95Val
Tyr Val Gln Asp Tyr Arg Ser Pro Phe Ile Ala Ser Val Ser Asp100 105
110Gln His Gly Val Val Tyr Ile Thr Glu Asn Lys Asn Lys Thr Val
Val115 120 125Ile Pro Cys Leu Gly Ser Ile Ser Asn Leu Asn Val Ser
Leu Cys Ala130 135 140Arg Tyr Pro Glu Lys Arg Phe Val Pro Asp Gly
Asn Arg Ile Ser Trp145 150 155 160Asp Ser Lys Lys Gly Phe Thr Ile
Pro Ser Tyr Met Ile Ser Tyr Ala165 170 175Gly Met Val Phe Cys Glu
Ala Lys Ile Asn Asp Glu Ser Tyr Gln Ser180 185 190Ile Met Tyr Ile
Val Val Val Val Gly Tyr Arg Ile Tyr Asp Val Val195 200 205Leu Ser
Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu Val210 215
220Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe
Asn225 230 235 240Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys
Leu Val Asn Arg245 250 255Asp Leu Lys Thr Gln Ser Gly Ser Glu Met
Lys Lys Phe Leu Ser Thr260 265 270Leu Thr Ile Asp Gly Val Thr Arg
Ser Asp Gln Gly Leu Tyr Thr Cys275 280 285Ala Ala Ser Ser Gly Leu
Met Thr Lys Lys Asn Ser Thr Phe Val Arg290 295 300Val His Glu Lys
Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu305 310 315 320Val
Glu Ala Thr Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu325 330
335Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro
Leu340 345 350Glu Ser Asn His Thr Ile Lys Ala Gly His Val Leu Thr
Ile Met Glu355 360 365Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr Val
Ile Leu Thr Asn Pro370 375 380Ile Ser Lys Glu Lys Gln Ser His Val
Val Ser Leu Val Val Tyr Val385 390 395 400Pro Pro Gln Ile Gly Glu
Lys Ser Leu Ile Ser Pro Val Asp Ser Tyr405 410 415Gln Tyr Gly Thr
Thr Gln Thr Leu Thr Cys Thr Val Tyr Ala Ile Pro420 425 430Pro Pro
His His Ile His Trp Tyr Trp Gln Leu Glu Glu Glu Cys Ala435 440
445Asn Glu Pro Ser Gln Ala Val Ser Val Thr Asn Pro Tyr Pro Cys
Glu450 455 460Glu Trp Arg Ser Val Glu Asp Phe Gln Gly Gly Asn Lys
Ile Glu Val465 470 475 480Asn Lys Asn Gln Phe Ala Leu Ile Glu Gly
Lys Asn Lys Thr Val Ser485 490 495Thr Leu Val Ile Gln Ala Ala Asn
Val Ser Ala Leu Tyr Lys Cys Glu500 505 510Ala Val Asn Lys Val Gly
Arg Gly Glu Arg Val Ile Ser Phe His Val515 520 525Thr Arg Gly Pro
Glu Ile Thr Leu Gln Pro Asp Met Gln Pro Thr Glu530 535 540Gln Glu
Ser Val Ser Leu Trp Cys Thr Ala Asp Arg Ser Thr Phe Glu545 550 555
560Asn Leu Thr Trp Tyr Lys Leu Gly Pro Gln Pro Leu Pro Ile His
Val565 570 575Gly Glu Leu Pro Thr Pro Val Cys Lys Asn Leu Asp Thr
Leu Trp Lys580 585 590Leu Asn Ala Thr Met Phe Ser Asn Ser Thr Asn
Asp Ile Leu Ile Met595 600 605Glu Leu Lys Asn Ala Ser Leu Gln Asp
Gln Gly Asp Tyr Val Cys Leu610 615 620Ala Gln Asp Arg Lys Thr Lys
Lys Arg His Cys Val Val Arg Gln Leu625 630 635 640Thr Val Leu Glu
Arg Val Ala Pro Thr Ile Thr Gly Asn Leu Glu Asn645 650 655Gln Thr
Thr Ser Ile Gly Glu Ser Ile Glu Val Ser Cys Thr Ala Ser660 665
670Gly Asn Pro Pro Pro Gln Ile Met Trp Phe Lys Asp Asn Glu Thr
Leu675 680 685Val Glu Asp Ser Gly Ile Val Leu Lys Asp Gly Asn Arg
Asn Leu Thr690 695 700Ile Arg Arg Val Arg Lys Glu Asp Glu Gly Leu
Tyr Thr Cys Gln Ala705 710 715 720Cys Ser Val Leu Gly Cys Ala Lys
Val Glu Ala Phe Phe Ile Ile Glu725 730 735Gly Ala Gln Glu Lys Thr
Asn Leu Glu Gly Xaa740 7451752957DNAArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 175aagcttgaat
tcccaccatg cagcggggcg ccgcgctgtg cctgcgactg tggctctgcc 60tgggactcct
ggacggcctg gtgagtggct actccatgac ccccccgacc ttgaacatca
120cggaggagtc acacgtcatc gacaccggtg acagcctgtc catctcctgc
aggggacagc 180accccctgga gtgggcttgg ccaggagctc aggaggcgcc
agccaccgga gacaaggaca 240gcgaggacac gggggtggtg cgagactgcg
agggcacaga cgccaggccc tactgcaagg 300tgttgctgct gcacgaggta
catgccaacg acacaggcag ctacgtctgc tactacaagt 360acatcaaggc
acgcatcgag ggcaccacgg ccgccagctc ctacgtgttc gtgagagact
420ttgagcagcc attcatcaac aagcctgaca cgctcttggt caacaggaag
gacgccatgt 480gggtgccctg tctggtgtcc atccccggcc tcaatgtcac
gctgcgctcg caaagctcgg 540tgctgtggcc agacgggcag gaggtggtgt
gggatgaccg gcggggcatg ctcgtgtcca 600cgccactgct gcacgatgcc
ctgtacctgc agtgcgagac cacctgggga gaccaggact 660tcctttccaa
ccccttcctg gtgcacatca caggcaacga gctctatgac atccagctgt
720tgcccaggaa gtcgctggag ctgctggtag gggagaagct ggtcctgaac
tgcaccgtgt 780gggctgagtt taactcaggt gtcacctttg actgggacta
cccagggaag caggcagagc 840ggggtaagtg ggtgcccgag cgacgctccc
agcagaccca cacagaactc tccagcatcc 900tgaccatcca caacgtcagc
cagcacgacc tgggctcgta tgtgtgcaag gccaacaacg 960gcatccagcg
atttcgggag agcaccgagg tcattgtgca tgaaaatccc ttcatcagcg
1020tcgagtggct caaaggaccc atcctggagg ccacggcagg agacgagctg
gtgaagctgc 1080ccgtgaagct ggcagcgtac cccccgcccg agttccagtg
gtacaaggat ggaaaggcac 1140tgtccgggcg ccacagtcca catgccctgg
tgctcaagga ggtgacagag gccagcacag 1200gcacctacac cctcgccctg
tggaactccg ctgctggcct gaggcgcaac atcagcctgg 1260agctggtggt
gaatgtgccc ccccagatac atgagaagga ggcctcctcc cccagcatct
1320actcgcgtca cagccgccag gccctcacct gcacggccta cggggtgccc
ctgcctctca 1380gcatccagtg gcactggcgg ccctggacac cctgcaagat
gtttgcccag cgtagtctcc 1440ggcggcggca gcagcaagac ctcatgccac
agtgccgtga ctggagggcg gtgaccacgc 1500aggatgccgt gaaccccatc
gagagcctgg acacctggac cgagtttgtg gagggaaaga 1560ataagactgt
gagcaagctg gtgatccaga atgccaacgt gtctgccatg tacaagtgtg
1620tggtctccaa caaggtgggc caggatgagc ggctcatcta cttctatgtg
accaccatcc 1680ccgacggctt caccatcgaa tccaagccat ccgaggagct
actagagggc cagccggtgc 1740tcctgagctg ccaagccgac agctacaagt
acgagcatct gcgctggtac cgcctcaacc 1800tgtccacgct gcacgatgcg
cacgggaacc cgcttctgct cgactgcaag aacgtgcatc 1860tgttcgccac
ccctctggcc gccagcctgg aggaggtggc acctggggcg cgccacgcca
1920cgctcagcct gagtatcccc cgcgtcgcgc ccgagcacga gggccactat
gtgtgcgaag 1980tgcaagaccg gcgcagccat gacaagcact gccacaagaa
gtacctgtcg gtgcaggccc 2040tggaagcccc tcggctcacg cagaacttga
ccgacctcct ggtgaacgtg agcgactcgc 2100tggagatgca gtgcttggtg
gccggagcgc acgcgcccag catcgtgtgg tacaaagacg 2160agaggctgct
ggaggaaaag tctggagtcg acttggcgga ctccaaccag aagctgagca
2220tccagcgcgt gcgcgaggag gatgcgggac gctatctgtg cagcgtgtgc
aacgccaagg 2280gctgcgtcaa ctcctccgcc agcgtggccg tggaaggctc
cgaggataag ggcagcatgg 2340aggggtgcgt atccggtgac accattgtaa
tgactagtgg cgggccccgc actgtggctg 2400aactggaggg caaaccgttc
accgcactga ttcgcggctc tggctaccca tgcccctcag 2460gtttcttccg
cacctgtgaa cgtgacgtat atgatctgcg tacacgtgag ggtcattgct
2520tacgtttgac ccatgatcac cgtgttctgg tgatggatgg tggcctggaa
tggcgtgccg 2580cgggtgaact ggaacgcggc gaccgcctgg tgatggatga
tgcagctggc gagtttccgg 2640cactggcaac cttccgtggc ctgcgtggcg
ctggccgcca ggatgtttat gacgctactg 2700tttacggtgc tagcgcattc
actgctaatg gcttcattgt acacgcatgt ggcgagcagc 2760ccgggaccgg
tctgaactca ggcctcacga caaatcctgg tgtatccgct tggcaggtca
2820acacagctta tactgcggga caattggtca catataacgg caagacgtat
aaatgtttgc 2880agccccacac ctccttggca ggatgggaac catccaacgt
tcctgccttg tggcagcttc 2940aatgactcga gcggccg
2957176975PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 176Met Gln Arg Gly Ala Ala Leu Cys Leu
Arg Leu Trp Leu Cys Leu Gly1 5 10 15Leu Leu Asp Gly Leu Val Ser Gly
Tyr Ser Met Thr Pro Pro Thr Leu20 25 30Asn Ile Thr Glu Glu Ser His
Val Ile Asp Thr Gly Asp Ser Leu Ser35 40 45Ile Ser Cys Arg Gly Gln
His Pro Leu Glu Trp Ala Trp Pro Gly Ala50 55 60Gln Glu Ala Pro Ala
Thr Gly Asp Lys Asp Ser Glu Asp Thr Gly Val65 70 75 80Val Arg Asp
Cys Glu Gly Thr Asp Ala Arg Pro Tyr Cys Lys Val Leu85 90 95Leu Leu
His Glu Val His Ala Asn Asp Thr Gly Ser Tyr Val Cys Tyr100 105
110Tyr Lys Tyr Ile Lys Ala Arg Ile Glu Gly Thr Thr Ala Ala Ser
Ser115 120 125Tyr Val Phe Val Arg Asp Phe Glu Gln Pro Phe Ile Asn
Lys Pro Asp130 135 140Thr Leu Leu Val Asn Arg Lys Asp Ala Met Trp
Val Pro Cys Leu Val145 150 155 160Ser Ile Pro Gly Leu Asn Val Thr
Leu Arg Ser Gln Ser Ser Val Leu165 170 175Trp Pro Asp Gly Gln Glu
Val Val Trp Asp Asp Arg Arg Gly Met Leu180 185 190Val Ser Thr Pro
Leu Leu His Asp Ala Leu Tyr Leu Gln Cys Glu Thr195 200 205Thr Trp
Gly Asp Gln Asp Phe Leu Ser Asn Pro Phe Leu Val His Ile210 215
220Thr Gly Asn Glu Leu Tyr Asp Ile Gln Leu Leu Pro Arg Lys Ser
Leu225 230 235 240Glu Leu Leu Val Gly Glu Lys Leu Val Leu Asn Cys
Thr Val Trp Ala245 250 255Glu Phe Asn Ser Gly Val Thr Phe Asp Trp
Asp Tyr Pro Gly Lys Gln260 265 270Ala Glu Arg Gly Lys Trp Val Pro
Glu Arg Arg Ser Gln Gln Thr His275 280 285Thr Glu Leu Ser Ser Ile
Leu Thr Ile His Asn Val Ser Gln His Asp290 295 300Leu Gly Ser Tyr
Val Cys Lys Ala Asn Asn Gly Ile Gln Arg Phe Arg305 310 315 320Glu
Ser Thr Glu Val Ile Val His Glu Asn Pro Phe Ile Ser Val Glu325 330
335Trp Leu Lys Gly Pro Ile Leu Glu Ala Thr Ala Gly Asp Glu Leu
Val340 345 350Lys Leu Pro Val Lys Leu Ala Ala Tyr Pro Pro Pro Glu
Phe Gln Trp355 360 365Tyr Lys Asp Gly Lys Ala Leu Ser Gly Arg His
Ser Pro His Ala Leu370 375 380Val Leu Lys Glu Val Thr Glu Ala Ser
Thr Gly Thr Tyr Thr Leu Ala385 390 395 400Leu Trp Asn Ser Ala Ala
Gly Leu Arg Arg Asn Ile Ser Leu Glu Leu405 410 415Val Val Asn Val
Pro Pro Gln Ile His Glu Lys Glu Ala Ser Ser Pro420 425 430Ser Ile
Tyr Ser Arg His Ser Arg Gln Ala Leu Thr Cys Thr Ala Tyr435 440
445Gly Val Pro Leu Pro Leu Ser Ile Gln Trp His Trp Arg Pro Trp
Thr450 455 460Pro Cys Lys Met Phe Ala Gln Arg Ser Leu Arg Arg Arg
Gln Gln Gln465 470 475 480Asp Leu Met Pro Gln Cys Arg Asp Trp Arg
Ala Val Thr Thr Gln Asp485 490 495Ala Val Asn Pro Ile Glu Ser Leu
Asp Thr Trp Thr Glu Phe Val Glu500 505 510Gly Lys Asn Lys Thr Val
Ser Lys Leu Val Ile Gln Asn Ala Asn Val515 520 525Ser Ala Met Tyr
Lys Cys Val Val Ser Asn Lys Val Gly Gln Asp Glu530 535 540Arg Leu
Ile Tyr Phe Tyr Val Thr Thr Ile Pro Asp Gly Phe Thr Ile545 550 555
560Glu Ser Lys Pro Ser Glu Glu Leu Leu Glu Gly Gln Pro Val Leu
Leu565 570 575Ser Cys Gln Ala Asp Ser Tyr Lys Tyr Glu His Leu Arg
Trp Tyr Arg580 585 590Leu Asn Leu Ser Thr Leu His Asp Ala His Gly
Asn Pro Leu Leu Leu595 600 605Asp Cys Lys Asn Val His Leu Phe Ala
Thr Pro Leu Ala Ala Ser Leu610 615 620Glu Glu Val Ala Pro Gly Ala
Arg His Ala Thr Leu Ser Leu Ser Ile625 630 635 640Pro Arg Val Ala
Pro Glu His Glu Gly His Tyr Val Cys Glu Val Gln645 650 655Asp Arg
Arg Ser His Asp Lys His Cys His Lys Lys Tyr Leu Ser Val660 665
670Gln Ala Leu Glu Ala Pro Arg Leu Thr Gln Asn Leu Thr Asp Leu
Leu675 680 685Val Asn Val Ser Asp Ser Leu Glu Met Gln Cys Leu Val
Ala Gly Ala690 695 700His Ala Pro Ser Ile Val Trp Tyr Lys Asp Glu
Arg Leu Leu Glu Glu705 710 715 720Lys Ser Gly Val Asp Leu Ala Asp
Ser Asn Gln Lys
Leu Ser Ile Gln725 730 735Arg Val Arg Glu Glu Asp Ala Gly Arg Tyr
Leu Cys Ser Val Cys Asn740 745 750Ala Lys Gly Cys Val Asn Ser Ser
Ala Ser Val Ala Val Glu Gly Ser755 760 765Glu Asp Lys Gly Ser Met
Glu Gly Cys Val Ser Gly Asp Thr Ile Val770 775 780Met Thr Ser Gly
Gly Pro Arg Thr Val Ala Glu Leu Glu Gly Lys Pro785 790 795 800Phe
Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe805 810
815Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu
Gly820 825 830His Cys Leu Arg Leu Thr His Asp His Arg Val Leu Val
Met Asp Gly835 840 845Gly Leu Glu Trp Arg Ala Ala Gly Glu Leu Glu
Arg Gly Asp Arg Leu850 855 860Val Met Asp Asp Ala Ala Gly Glu Phe
Pro Ala Leu Ala Thr Phe Arg865 870 875 880Gly Leu Arg Gly Ala Gly
Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr885 890 895Gly Ala Ser Ala
Phe Thr Ala Asn Gly Phe Ile Val His Ala Cys Gly900 905 910Glu Gln
Pro Gly Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly915 920
925Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu
Val930 935 940Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro His
Thr Ser Leu945 950 955 960Ala Gly Trp Glu Pro Ser Asn Val Pro Ala
Leu Trp Gln Leu Gln965 970 975177951PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 177Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu
Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys
Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu
Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val
Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu
Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val
Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala
Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe
Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala
Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135
140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val
Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro
Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp
Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile
Thr Gly Asn Glu Leu Tyr Asp Ile195 200 205Gln Leu Leu Pro Arg Lys
Ser Leu Glu Leu Leu Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys
Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe225 230 235 240Asp
Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro245 250
255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu
Thr260 265 270Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val
Cys Lys Ala275 280 285Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr
Glu Val Ile Val His290 295 300Glu Asn Pro Phe Ile Ser Val Glu Trp
Leu Lys Gly Pro Ile Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu
Leu Val Lys Leu Pro Val Lys Leu Ala Ala325 330 335Tyr Pro Pro Pro
Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser340 345 350Gly Arg
His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala355 360
365Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly
Leu370 375 380Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro
Pro Gln Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile
Tyr Ser Arg His Ser Arg405 410 415Gln Ala Leu Thr Cys Thr Ala Tyr
Gly Val Pro Leu Pro Leu Ser Ile420 425 430Gln Trp His Trp Arg Pro
Trp Thr Pro Cys Lys Met Phe Ala Gln Arg435 440 445Ser Leu Arg Arg
Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp450 455 460Trp Arg
Ala Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475
480Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser
Lys485 490 495Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys
Cys Val Val500 505 510Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile
Tyr Phe Tyr Val Thr515 520 525Thr Ile Pro Asp Gly Phe Thr Ile Glu
Ser Lys Pro Ser Glu Glu Leu530 535 540Leu Glu Gly Gln Pro Val Leu
Leu Ser Cys Gln Ala Asp Ser Tyr Lys545 550 555 560Tyr Glu His Leu
Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp565 570 575Ala His
Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe580 585
590Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala
Arg595 600 605His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro
Glu His Glu610 615 620Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg
Ser His Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val
Gln Ala Leu Glu Ala Pro Arg Leu645 650 655Thr Gln Asn Leu Thr Asp
Leu Leu Val Asn Val Ser Asp Ser Leu Glu660 665 670Met Gln Cys Leu
Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr675 680 685Lys Asp
Glu Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp690 695
700Ser Asn Gln Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala
Gly705 710 715 720Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys
Val Asn Ser Ser725 730 735Ala Ser Val Ala Val Glu Gly Ser Glu Asp
Lys Gly Ser Met Glu Gly740 745 750Cys Val Ser Gly Asp Thr Ile Val
Met Thr Ser Gly Gly Pro Arg Thr755 760 765Val Ala Glu Leu Glu Gly
Lys Pro Phe Thr Ala Leu Ile Arg Gly Ser770 775 780Gly Tyr Pro Cys
Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp Val785 790 795 800Tyr
Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr His Asp805 810
815His Arg Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg Ala Ala
Gly820 825 830Glu Leu Glu Arg Gly Asp Arg Leu Val Met Asp Asp Ala
Ala Gly Glu835 840 845Phe Pro Ala Leu Ala Thr Phe Arg Gly Leu Arg
Gly Ala Gly Arg Gln850 855 860Asp Val Tyr Asp Ala Thr Val Tyr Gly
Ala Ser Ala Phe Thr Ala Asn865 870 875 880Gly Phe Ile Val His Ala
Cys Gly Glu Gln Pro Gly Thr Gly Leu Asn885 890 895Ser Gly Leu Thr
Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr900 905 910Ala Tyr
Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys915 920
925Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn
Val930 935 940Pro Ala Leu Trp Gln Leu Gln945 950178752PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 178Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu
Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys
Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu
Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val
Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu
Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val
Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala
Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe
Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala
Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135
140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val
Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro
Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp
Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile
Thr Gly Asn Glu Leu Tyr Asp Ile195 200 205Gln Leu Leu Pro Arg Lys
Ser Leu Glu Leu Leu Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys
Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe225 230 235 240Asp
Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro245 250
255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu
Thr260 265 270Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val
Cys Lys Ala275 280 285Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr
Glu Val Ile Val His290 295 300Glu Asn Pro Phe Ile Ser Val Glu Trp
Leu Lys Gly Pro Ile Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu
Leu Val Lys Leu Pro Val Lys Leu Ala Ala325 330 335Tyr Pro Pro Pro
Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser340 345 350Gly Arg
His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala355 360
365Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly
Leu370 375 380Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro
Pro Gln Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile
Tyr Ser Arg His Ser Arg405 410 415Gln Ala Leu Thr Cys Thr Ala Tyr
Gly Val Pro Leu Pro Leu Ser Ile420 425 430Gln Trp His Trp Arg Pro
Trp Thr Pro Cys Lys Met Phe Ala Gln Arg435 440 445Ser Leu Arg Arg
Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp450 455 460Trp Arg
Ala Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475
480Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser
Lys485 490 495Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys
Cys Val Val500 505 510Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile
Tyr Phe Tyr Val Thr515 520 525Thr Ile Pro Asp Gly Phe Thr Ile Glu
Ser Lys Pro Ser Glu Glu Leu530 535 540Leu Glu Gly Gln Pro Val Leu
Leu Ser Cys Gln Ala Asp Ser Tyr Lys545 550 555 560Tyr Glu His Leu
Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp565 570 575Ala His
Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe580 585
590Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala
Arg595 600 605His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro
Glu His Glu610 615 620Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg
Ser His Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val
Gln Ala Leu Glu Ala Pro Arg Leu645 650 655Thr Gln Asn Leu Thr Asp
Leu Leu Val Asn Val Ser Asp Ser Leu Glu660 665 670Met Gln Cys Leu
Val Ala Gly Ala His Ala Pro Ser Ile Val Trp Tyr675 680 685Lys Asp
Glu Arg Leu Leu Glu Glu Lys Ser Gly Val Asp Leu Ala Asp690 695
700Ser Asn Gln Lys Leu Ser Ile Gln Arg Val Arg Glu Glu Asp Ala
Gly705 710 715 720Arg Tyr Leu Cys Ser Val Cys Asn Ala Lys Gly Cys
Val Asn Ser Ser725 730 735Ala Ser Val Ala Val Glu Gly Ser Glu Asp
Lys Gly Ser Met Glu Xaa740 745 750179753PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 179Tyr Ser Met Thr Pro Pro Thr Leu Asn Ile Thr Glu
Glu Ser His Val1 5 10 15Ile Asp Thr Gly Asp Ser Leu Ser Ile Ser Cys
Arg Gly Gln His Pro20 25 30Leu Glu Trp Ala Trp Pro Gly Ala Gln Glu
Ala Pro Ala Thr Gly Asp35 40 45Lys Asp Ser Glu Asp Thr Gly Val Val
Arg Asp Cys Glu Gly Thr Asp50 55 60Ala Arg Pro Tyr Cys Lys Val Leu
Leu Leu His Glu Val His Ala Asn65 70 75 80Asp Thr Gly Ser Tyr Val
Cys Tyr Tyr Lys Tyr Ile Lys Ala Arg Ile85 90 95Glu Gly Thr Thr Ala
Ala Ser Ser Tyr Val Phe Val Arg Asp Phe Glu100 105 110Gln Pro Phe
Ile Asn Lys Pro Asp Thr Leu Leu Val Asn Arg Lys Asp115 120 125Ala
Met Trp Val Pro Cys Leu Val Ser Ile Pro Gly Leu Asn Val Thr130 135
140Leu Arg Ser Gln Ser Ser Val Leu Trp Pro Asp Gly Gln Glu Val
Val145 150 155 160Trp Asp Asp Arg Arg Gly Met Leu Val Ser Thr Pro
Leu Leu His Asp165 170 175Ala Leu Tyr Leu Gln Cys Glu Thr Thr Trp
Gly Asp Gln Asp Phe Leu180 185 190Ser Asn Pro Phe Leu Val His Ile
Thr Gly Asn Glu Leu Tyr Asp Ile195 200 205Gln Leu Leu Pro Arg Lys
Ser Leu Glu Leu Leu Val Gly Glu Lys Leu210 215 220Val Leu Asn Cys
Thr Val Trp Ala Glu Phe Asn Ser Gly Val Thr Phe225 230 235 240Asp
Trp Asp Tyr Pro Gly Lys Gln Ala Glu Arg Gly Lys Trp Val Pro245 250
255Glu Arg Arg Ser Gln Gln Thr His Thr Glu Leu Ser Ser Ile Leu
Thr260 265 270Ile His Asn Val Ser Gln His Asp Leu Gly Ser Tyr Val
Cys Lys Ala275 280 285Asn Asn Gly Ile Gln Arg Phe Arg Glu Ser Thr
Glu Val Ile Val His290 295 300Glu Asn Pro Phe Ile Ser Val Glu Trp
Leu Lys Gly Pro Ile Leu Glu305 310 315 320Ala Thr Ala Gly Asp Glu
Leu Val Lys Leu Pro Val Lys Leu Ala Ala325 330 335Tyr Pro Pro Pro
Glu Phe Gln Trp Tyr Lys Asp Gly Lys Ala Leu Ser340 345 350Gly Arg
His Ser Pro His Ala Leu Val Leu Lys Glu Val Thr Glu Ala355 360
365Ser Thr Gly Thr Tyr Thr Leu Ala Leu Trp Asn Ser Ala Ala Gly
Leu370 375 380Arg Arg Asn Ile Ser Leu Glu Leu Val Val Asn Val Pro
Pro Gln Ile385 390 395 400His Glu Lys Glu Ala Ser Ser Pro Ser Ile
Tyr Ser Arg His Ser Arg405 410 415Gln Ala Leu Thr Cys Thr Ala Tyr
Gly Val Pro Leu Pro Leu Ser Ile420 425 430Gln Trp His Trp Arg Pro
Trp Thr Pro Cys Lys Met Phe Ala Gln Arg435 440 445Ser Leu Arg Arg
Arg Gln Gln Gln Asp Leu Met Pro Gln Cys Arg Asp450 455 460Trp Arg
Ala Val Thr Thr Gln Asp Ala Val Asn Pro Ile Glu Ser Leu465 470 475
480Asp Thr Trp Thr Glu Phe Val Glu Gly Lys Asn Lys Thr Val Ser
Lys485 490 495Leu Val Ile Gln Asn Ala Asn Val Ser Ala Met Tyr Lys
Cys Val Val500 505 510Ser Asn Lys Val Gly Gln Asp Glu Arg Leu Ile
Tyr Phe Tyr Val Thr515 520 525Thr Ile Pro Asp Gly Phe Thr Ile Glu
Ser Lys Pro Ser Glu Glu Leu530 535 540Leu Glu Gly Gln Pro Val Leu
Leu Ser Cys Gln Ala Asp Ser Tyr Lys545 550 555 560Tyr Glu His Leu
Arg Trp Tyr Arg Leu Asn Leu Ser Thr Leu His Asp565 570 575Ala His
Gly Asn Pro Leu Leu Leu Asp Cys Lys Asn Val His Leu Phe580 585
590Ala Thr Pro Leu Ala Ala Ser Leu Glu Glu Val Ala Pro Gly Ala
Arg595 600 605His Ala Thr Leu Ser Leu Ser Ile Pro Arg Val Ala Pro
Glu His Glu610 615 620Gly His Tyr Val Cys Glu Val Gln Asp Arg Arg
Ser His Asp Lys His625 630 635 640Cys His Lys Lys Tyr Leu Ser Val
Gln Ala Leu Glu Ala Pro Arg Leu645 650 655Thr Gln Asn Leu Thr Asp
Leu Leu Val Asn Val Ser
Asp Ser Leu Glu660 665 670Met Gln Cys Leu Val Ala Gly Ala His Ala
Pro Ser Ile Val Trp Tyr675 680 685Lys Asp Glu Arg Leu Leu Glu Glu
Lys Ser Gly Val Asp Leu Ala Asp690 695 700Ser Asn Gln Lys Leu Ser
Ile Gln Arg Val Arg Glu Glu Asp Ala Gly705 710 715 720Arg Tyr Leu
Cys Ser Val Cys Asn Ala Lys Gly Cys Val Asn Ser Ser725 730 735Ala
Ser Val Ala Val Glu Gly Ser Glu Asp Lys Gly Ser Met Glu Gly740 745
750Xaa1802567DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 180aagcttgaat tcccaccatg cgaccctccg
ggacggccgg ggcagcgctc ctggcgctgc 60tggctgcgct ctgcccggcg agtcgggctc
tggaggaaaa gaaagtttgc caaggcacga 120gtaacaagct cacgcagttg
ggcacttttg aagatcattt tctcagcctc cagaggatgt 180tcaataactg
tgaggtggtc cttgggaatt tggaaattac ctatgtgcag aggaattatg
240atctttcctt cttaaagacc atccaggagg tggctggtta tgtcctcatt
gccctcaaca 300cagtggagcg aatccctttg gaaaacctgc agatcatcag
aggaaatatg tactacgaaa 360attcctatgc cttagcagtc ttatctaact
atgatgcaaa taaaaccgga ctgaaggagc 420tgcccatgag aaatttacag
gaaatcctgc atggcgccgt gcggttcagc aacaaccctg 480ccctgtgcaa
cgtggagagc atccagtggc gggacatagt cagcagtgac tttctcagca
540acatgtcgat ggacttccag aaccacctgg gcagctgcca aaagtgtgat
ccaagctgtc 600ccaatgggag ctgctggggt gcaggagagg agaactgcca
gaaactgacc aaaatcatct 660gtgcccagca gtgctccggg cgctgccgtg
gcaagtcccc cagtgactgc tgccacaacc 720agtgtgctgc aggctgcaca
ggcccccggg agagcgactg cctggtctgc cgcaaattcc 780gagacgaagc
cacgtgcaag gacacctgcc ccccactcat gctctacaac cccaccacgt
840accagatgga tgtgaacccc gagggcaaat acagctttgg tgccacctgc
gtgaagaagt 900gtccccgtaa ttatgtggtg acagatcacg gctcgtgcgt
ccgagcctgt ggggccgaca 960gctatgagat ggaggaagac ggcgtccgca
agtgtaagaa gtgcgaaggg ccttgccgca 1020aagtgtgtaa cggaataggt
attggtgaat ttaaagactc actctccata aatgctacga 1080atattaaaca
cttcaaaaac tgcacctcca tcagtggcga tctccacatc ctgccggtgg
1140catttagggg tgactccttc acacatactc ctcctctgga tccacaggaa
ctggatattc 1200tgaaaaccgt aaaggaaatc acagggtttt tgctgattca
ggcttggcct gaaaacagga 1260cggacctcca tgcctttgag aacctagaaa
tcatacgcgg caggaccaag caacatggtc 1320agttttctct tgcagtcgtc
agcctgaaca taacatcctt gggattacgc tccctcaagg 1380agataagtga
tggagatgtg ataatttcag gaaacaaaaa tttgtgctat gcaaatacaa
1440taaactggaa aaaactgttt gggacctccg gtcagaaaac caaaattata
agcaacagag 1500gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc
cttgtgctcc cccgagggct 1560gctggggtcc ggagcccagg gactgcgtct
cttgccggaa tgtcagccga ggcagggaat 1620gcgtggacaa gtgcaagctc
ctggagggtg agccaaggga gtttgtggag aactctgagt 1680gcatacagtg
ccacccagag tgcctgcctc aggccatgaa catcacctgc acaggacggg
1740gaccagacaa ctgtatccag tgtgcccact acattgacgg cccccactgc
gtcaagacct 1800gcccggcagg agtcatggga gaaaacaaca ccctggtctg
gaagtacgca gacgccggcc 1860atgtgtgcca cctgtgccat ccaaactgca
cctacggatg cactgggcca ggtcttgaag 1920gctgtccaac gaatgggcct
aagatcccgt ccgggtgcgt atccggtgac accattgtaa 1980tgactagtgg
cgggccccgc actgtggctg aactggaggg caaaccgttc accgcactga
2040ttcgcggctc tggctaccca tgcccctcag gtttcttccg cacctgtgaa
cgtgacgtat 2100atgatctgcg tacacgtgag ggtcattgct tacgtttgac
ccatgatcac cgtgttctgg 2160tgatggatgg tggcctggaa tggcgtgccg
cgggtgaact ggaacgcggc gaccgcctgg 2220tgatggatga tgcagctggc
gagtttccgg cactggcaac cttccgtggc ctgcgtggcg 2280ctggccgcca
ggatgtttat gacgctactg tttacggtgc tagcgcattc actgctaatg
2340gcttcattgt acacgcatgt ggcgagcagc ccgggaccgg tctgaactca
ggcctcacga 2400caaatcctgg tgtatccgct tggcaggtca acacagctta
tactgcggga caattggtca 2460catataacgg caagacgtat aaatgtttgc
agccccacac ctccttggca ggatgggaac 2520catccaacgt tcctgccttg
tggcagcttc aatgactcga gcggccg 2567181845PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 181Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu
Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu
Lys Lys Val Cys Gln20 25 30Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly
Thr Phe Glu Asp His Phe35 40 45Leu Ser Leu Gln Arg Met Phe Asn Asn
Cys Glu Val Val Leu Gly Asn50 55 60Leu Glu Ile Thr Tyr Val Gln Arg
Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile Gln Glu Val Ala
Gly Tyr Val Leu Ile Ala Leu Asn Thr Val85 90 95Glu Arg Ile Pro Leu
Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr100 105 110Tyr Glu Asn
Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys
Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu130 135
140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val
Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe
Leu Ser Asn Met165 170 175Ser Met Asp Phe Gln Asn His Leu Gly Ser
Cys Gln Lys Cys Asp Pro180 185 190Ser Cys Pro Asn Gly Ser Cys Trp
Gly Ala Gly Glu Glu Asn Cys Gln195 200 205Lys Leu Thr Lys Ile Ile
Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg210 215 220Gly Lys Ser Pro
Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230 235 240Thr
Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp245 250
255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn
Pro260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr
Ser Phe Gly275 280 285Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr
Val Val Thr Asp His290 295 300Gly Ser Cys Val Arg Ala Cys Gly Ala
Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val Arg Lys Cys
Lys Lys Cys Glu Gly Pro Cys Arg Lys Val325 330 335Cys Asn Gly Ile
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn340 345 350Ala Thr
Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360
365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His
Thr370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr
Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp
Pro Glu Asn Arg Thr Asp405 410 415Leu His Ala Phe Glu Asn Leu Glu
Ile Ile Arg Gly Arg Thr Lys Gln420 425 430His Gly Gln Phe Ser Leu
Ala Val Val Ser Leu Asn Ile Thr Ser Leu435 440 445Gly Leu Arg Ser
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser450 455 460Gly Asn
Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470 475
480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly
Glu485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu
Cys Ser Pro500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys
Val Ser Cys Arg Asn515 520 525Val Ser Arg Gly Arg Glu Cys Val Asp
Lys Cys Asn Leu Leu Glu Gly530 535 540Glu Pro Arg Glu Phe Val Glu
Asn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu Pro
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro565 570 575Asp Asn
Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val580 585
590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val
Trp595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His
Pro Asn Cys610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly
Cys Pro Thr Asn Gly625 630 635 640Pro Lys Ile Pro Ser Gly Cys Val
Ser Gly Asp Thr Ile Val Met Thr645 650 655Ser Gly Gly Pro Arg Thr
Val Ala Glu Leu Glu Gly Lys Pro Phe Thr660 665 670Ala Leu Ile Arg
Gly Ser Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg675 680 685Thr Cys
Glu Arg Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys690 695
700Leu Arg Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly
Leu705 710 715 720Glu Trp Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp
Arg Leu Val Met725 730 735Asp Asp Ala Ala Gly Glu Phe Pro Ala Leu
Ala Thr Phe Arg Gly Leu740 745 750Arg Gly Ala Gly Arg Gln Asp Val
Tyr Asp Ala Thr Val Tyr Gly Ala755 760 765Ser Ala Phe Thr Ala Asn
Gly Phe Ile Val His Ala Cys Gly Glu Gln770 775 780Pro Gly Thr Gly
Leu Asn Ser Gly Leu Thr Thr Asn Pro Gly Val Ser785 790 795 800Ala
Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr805 810
815Asn Gly Lys Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala
Gly820 825 830Trp Glu Pro Ser Asn Val Pro Ala Leu Trp Gln Leu
Gln835 840 845182821PRTArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 182Leu Glu Glu Lys Lys Val
Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr Phe Glu
Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn20 25 30Asn Cys Glu Val
Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg35 40 45Asn Tyr Asp
Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr50 55 60Val Leu
Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu65 70 75
80Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala85
90 95Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu
Pro100 105 110Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg
Phe Ser Asn115 120 125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln
Trp Arg Asp Ile Val130 135 140Ser Ser Asp Phe Leu Ser Asn Met Ser
Met Asp Phe Gln Asn His Leu145 150 155 160Gly Ser Cys Gln Lys Cys
Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp165 170 175Gly Ala Gly Glu
Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala180 185 190Gln Gln
Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys195 200
205His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp
Cys210 215 220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys
Asp Thr Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr
Tyr Gln Met Asp Val Asn245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly
Ala Thr Cys Val Lys Lys Cys Pro260 265 270Arg Asn Tyr Val Val Thr
Asp His Gly Ser Cys Val Arg Ala Cys Gly275 280 285Ala Asp Ser Tyr
Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys290 295 300Cys Glu
Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu305 310 315
320Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe
Lys325 330 335Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro
Val Ala Phe340 345 350Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu
Asp Pro Gln Glu Leu355 360 365Asp Ile Leu Lys Thr Val Lys Glu Ile
Thr Gly Phe Leu Leu Ile Gln370 375 380Ala Trp Pro Glu Asn Arg Thr
Asp Leu His Ala Phe Glu Asn Leu Glu385 390 395 400Ile Ile Arg Gly
Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val405 410 415Val Ser
Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile420 425
430Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
Ala435 440 445Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly
Gln Lys Thr450 455 460Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys
Lys Ala Thr Gly Gln465 470 475 480Val Cys His Ala Leu Cys Ser Pro
Glu Gly Cys Trp Gly Pro Glu Pro485 490 495Arg Asp Cys Val Ser Cys
Arg Asn Val Ser Arg Gly Arg Glu Cys Val500 505 510Asp Lys Cys Asn
Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn515 520 525Ser Glu
Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn530 535
540Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
His545 550 555 560Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
Ala Gly Val Met565 570 575Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr
Ala Asp Ala Gly His Val580 585 590Cys His Leu Cys His Pro Asn Cys
Thr Tyr Gly Cys Thr Gly Pro Gly595 600 605Leu Glu Gly Cys Pro Thr
Asn Gly Pro Lys Ile Pro Ser Gly Cys Val610 615 620Ser Gly Asp Thr
Ile Val Met Thr Ser Gly Gly Pro Arg Thr Val Ala625 630 635 640Glu
Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr645 650
655Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr
Asp660 665 670Leu Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr His
Asp His Arg675 680 685Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg
Ala Ala Gly Glu Leu690 695 700Glu Arg Gly Asp Arg Leu Val Met Asp
Asp Ala Ala Gly Glu Phe Pro705 710 715 720Ala Leu Ala Thr Phe Arg
Gly Leu Arg Gly Ala Gly Arg Gln Asp Val725 730 735Tyr Asp Ala Thr
Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn Gly Phe740 745 750Ile Val
His Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly755 760
765Leu Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala
Tyr770 775 780Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr
Lys Cys Leu785 790 795 800Gln Pro His Thr Ser Leu Ala Gly Trp Glu
Pro Ser Asn Val Pro Ala805 810 815Leu Trp Gln Leu
Gln820183622PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 183Leu Glu Glu Lys Lys Val Cys Gln Gly
Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly Thr Phe Glu Asp His Phe
Leu Ser Leu Gln Arg Met Phe Asn20 25 30Asn Cys Glu Val Val Leu Gly
Asn Leu Glu Ile Thr Tyr Val Gln Arg35 40 45Asn Tyr Asp Leu Ser Phe
Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr50 55 60Val Leu Ile Ala Leu
Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu65 70 75 80Gln Ile Ile
Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala85 90 95Val Leu
Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro100 105
110Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser
Asn115 120 125Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg
Asp Ile Val130 135 140Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp
Phe Gln Asn His Leu145 150 155 160Gly Ser Cys Gln Lys Cys Asp Pro
Ser Cys Pro Asn Gly Ser Cys Trp165 170 175Gly Ala Gly Glu Glu Asn
Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala180 185 190Gln Gln Cys Ser
Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys195 200 205His Asn
Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys210 215
220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr
Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln
Met Asp Val Asn245 250 255Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr
Cys Val Lys Lys Cys Pro260 265 270Arg Asn Tyr Val Val Thr Asp His
Gly Ser Cys Val Arg Ala Cys Gly275 280 285Ala Asp Ser Tyr Glu Met
Glu Glu Asp Gly Val Arg Lys Cys Lys Lys290 295 300Cys Glu Gly Pro
Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu305 310 315 320Phe
Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys325 330
335Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala
Phe340 345 350Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro
Gln Glu Leu355 360 365Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly
Phe Leu Leu Ile Gln370 375 380Ala Trp Pro Glu Asn Arg Thr Asp Leu
His Ala
Phe Glu Asn Leu Glu385 390 395 400Ile Ile Arg Gly Arg Thr Lys Gln
His Gly Gln Phe Ser Leu Ala Val405 410 415Val Ser Leu Asn Ile Thr
Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile420 425 430Ser Asp Gly Asp
Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala435 440 445Asn Thr
Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr450 455
460Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly
Gln465 470 475 480Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
Gly Pro Glu Pro485 490 495Arg Asp Cys Val Ser Cys Arg Asn Val Ser
Arg Gly Arg Glu Cys Val500 505 510Asp Lys Cys Asn Leu Leu Glu Gly
Glu Pro Arg Glu Phe Val Glu Asn515 520 525Ser Glu Cys Ile Gln Cys
His Pro Glu Cys Leu Pro Gln Ala Met Asn530 535 540Ile Thr Cys Thr
Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His545 550 555 560Tyr
Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met565 570
575Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
Val580 585 590Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr
Gly Pro Gly595 600 605Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile
Pro Ser Xaa610 615 620184623PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 184Leu Glu Glu
Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln1 5 10 15Leu Gly
Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn20 25 30Asn
Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg35 40
45Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr50
55 60Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn
Leu65 70 75 80Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr
Ala Leu Ala85 90 95Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu
Lys Glu Leu Pro100 105 110Met Arg Asn Leu Gln Glu Ile Leu His Gly
Ala Val Arg Phe Ser Asn115 120 125Asn Pro Ala Leu Cys Asn Val Glu
Ser Ile Gln Trp Arg Asp Ile Val130 135 140Ser Ser Asp Phe Leu Ser
Asn Met Ser Met Asp Phe Gln Asn His Leu145 150 155 160Gly Ser Cys
Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp165 170 175Gly
Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala180 185
190Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys
Cys195 200 205His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu
Ser Asp Cys210 215 220Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr
Cys Lys Asp Thr Cys225 230 235 240Pro Pro Leu Met Leu Tyr Asn Pro
Thr Thr Tyr Gln Met Asp Val Asn245 250 255Pro Glu Gly Lys Tyr Ser
Phe Gly Ala Thr Cys Val Lys Lys Cys Pro260 265 270Arg Asn Tyr Val
Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly275 280 285Ala Asp
Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys290 295
300Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
Glu305 310 315 320Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
Lys His Phe Lys325 330 335Asn Cys Thr Ser Ile Ser Gly Asp Leu His
Ile Leu Pro Val Ala Phe340 345 350Arg Gly Asp Ser Phe Thr His Thr
Pro Pro Leu Asp Pro Gln Glu Leu355 360 365Asp Ile Leu Lys Thr Val
Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln370 375 380Ala Trp Pro Glu
Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu385 390 395 400Ile
Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val405 410
415Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu
Ile420 425 430Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu
Cys Tyr Ala435 440 445Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr
Ser Gly Gln Lys Thr450 455 460Lys Ile Ile Ser Asn Arg Gly Glu Asn
Ser Cys Lys Ala Thr Gly Gln465 470 475 480Val Cys His Ala Leu Cys
Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro485 490 495Arg Asp Cys Val
Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val500 505 510Asp Lys
Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn515 520
525Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met
Asn530 535 540Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln
Cys Ala His545 550 555 560Tyr Ile Asp Gly Pro His Cys Val Lys Thr
Cys Pro Ala Gly Val Met565 570 575Gly Glu Asn Asn Thr Leu Val Trp
Lys Tyr Ala Asp Ala Gly His Val580 585 590Cys His Leu Cys His Pro
Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly595 600 605Leu Glu Gly Cys
Pro Thr Asn Gly Pro Lys Ile Pro Ser Gly Xaa610 615
6201852588DNAArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 185aagcttgaat tcccaccatg gagctggcgg
ccttgtgccg ctgggggctc ctcctcgccc 60tcttgccccc cggagccgcg agcacccaag
tgtgcaccgg cacagacatg aagctgcggc 120tccctgccag tcccgagacc
cacctggaca tgctccgcca cctctaccag ggctgccagg 180tggtgcaggg
aaacctggaa ctcacctacc tgcccaccaa tgccagcctg tccttcctgc
240aggatatcca ggaggtgcag ggctacgtgc tcatcgctca caaccaagtg
aggcaggtcc 300cactgcagag gctgcggatt gtgcgaggca cccagctctt
tgaggacaac tatgccctgg 360ccgtgctaga caatggagac ccgctgaaca
ataccacccc tgtcacaggg gcctccccag 420gaggcctgcg ggagctgcag
cttcgaagcc tcacagagat cttgaaagga ggggtcttga 480tccagcggaa
cccccagctc tgctaccagg acacgatttt gtggaaggac atcttccaca
540agaacaacca gctggctctc acactgatag acaccaaccg ctctcgggcc
tgccacccct 600gttctccgat gtgtaagggc tcccgctgct ggggagagag
ttctgaggat tgtcagagcc 660tgacgcgcac tgtctgtgcc ggtggctgtg
cccgctgcaa ggggccactg cccactgact 720gctgccatga gcagtgtgct
gccggctgca cgggtcccaa gcactctgac tgcctggcct 780gcctccactt
caaccacagt ggcatctgtg agctgcactg cccagccctg gtcacctaca
840acacagacac gtttgagtcc atgcccaatc ccgagggccg gtatacattc
ggcgccagct 900gtgtgactgc ctgtccctac aactaccttt ctacggacgt
gggatcctgc accctcgtct 960gccccctgca caaccaagag gtgacagcag
aggatggaac acagcggtgt gagaagtgca 1020gcaagccctg tgcccgagtg
tgctatggtc tgggcatgga gcacttgcga gaggtgaggg 1080cagttaccag
tgccaatatc caggagtttg ctggctgcaa gaagatcttt gggagcctgg
1140catttctgcc ggagagcttt gatggggacc cagcctccaa cactgccccg
ctccagccag 1200agcagctcca agtgtttgag actctggaag agatcacagg
ttacctatac atctcagcat 1260ggccggacag cctgcctgac ctcagcgtct
tccagaacct gcaagtaatc cggggacgaa 1320ttctgcacaa tggcgcctac
tcgctgaccc tgcaagggct gggcatcagc tggctggggc 1380tgcgctcact
gagggaactg ggcagtggac tggccctcat ccaccataac acccacctct
1440gcttcgtgca cacggtgccc tgggaccagc tctttcggaa cccgcaccaa
gctctgctcc 1500acactgccaa ccggccagag gacgagtgtg tgggcgaggg
cctggcctgc caccagctgt 1560gcgcccgagg gcactgctgg ggtccaggtc
ccacccagtg tgtcaactgc agccagttcc 1620ttcggggcca ggagtgcgtg
gaggaatgcc gagtactgca ggggctcccc agggagtatg 1680tgaatgccag
gcactgtttg ccgtgccacc ctgagtgtca gccccagaat ggctcagtga
1740cctgttttgg accggaggct gaccagtgtg tggcctgtgc ccactataag
gaccctccct 1800tctgcgtggc ccgctgcccc agcggtgtga aacctgacct
ctcctacatg cccatctgga 1860agtttccaga tgaggagggc gcatgccagc
cttgccccat caactgcacc cactcctgtg 1920tggacctgga tgacaagggc
tgccccgccg agcagagagc cagccctctg acggggtgcg 1980tatccggtga
caccattgta atgactagtg gcgggccccg cactgtggct gaactggagg
2040gcaaaccgtt caccgcactg attcgcggct ctggctaccc atgcccctca
ggtttcttcc 2100gcacctgtga acgtgacgta tatgatctgc gtacacgtga
gggtcattgc ttacgtttga 2160cccatgatca ccgtgttctg gtgatggatg
gtggcctgga atggcgtgcc gcgggtgaac 2220tggaacgcgg cgaccgcctg
gtgatggatg atgcagctgg cgagtttccg gcactggcaa 2280ccttccgtgg
cctgcgtggc gctggccgcc aggatgttta tgacgctact gtttacggtg
2340ctagcgcatt cactgctaat ggcttcattg tacacgcatg tggcgagcag
cccgggaccg 2400gtctgaactc aggcctcacg acaaatcctg gtgtatccgc
ttggcaggtc aacacagctt 2460atactgcggg acaattggtc acatataacg
gcaagacgta taaatgtttg cagccccaca 2520cctccttggc aggatgggaa
ccatccaacg ttcctgcctt gtggcagctt caatgactcg 2580agcggccg
2588186852PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 186Met Glu Leu Ala Ala Leu Cys Arg Trp
Gly Leu Leu Leu Ala Leu Leu1 5 10 15Pro Pro Gly Ala Ala Ser Thr Gln
Val Cys Thr Gly Thr Asp Met Lys20 25 30Leu Arg Leu Pro Ala Ser Pro
Glu Thr His Leu Asp Met Leu Arg His35 40 45Leu Tyr Gln Gly Cys Gln
Val Val Gln Gly Asn Leu Glu Leu Thr Tyr50 55 60Leu Pro Thr Asn Ala
Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val65 70 75 80Gln Gly Tyr
Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu85 90 95Gln Arg
Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr100 105
110Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr
Pro115 120 125Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln
Leu Arg Ser130 135 140Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile
Gln Arg Asn Pro Gln145 150 155 160Leu Cys Tyr Gln Asp Thr Ile Leu
Trp Lys Asp Ile Phe His Lys Asn165 170 175Asn Gln Leu Ala Leu Thr
Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys180 185 190His Pro Cys Ser
Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser195 200 205Ser Glu
Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys210 215
220Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln
Cys225 230 235 240Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys
Leu Ala Cys Leu245 250 255His Phe Asn His Ser Gly Ile Cys Glu Leu
His Cys Pro Ala Leu Val260 265 270Thr Tyr Asn Thr Asp Thr Phe Glu
Ser Met Pro Asn Pro Glu Gly Arg275 280 285Tyr Thr Phe Gly Ala Ser
Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu290 295 300Ser Thr Asp Val
Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln305 310 315 320Glu
Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys325 330
335Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg
Glu340 345 350Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala
Gly Cys Lys355 360 365Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu
Ser Phe Asp Gly Asp370 375 380Pro Ala Ser Asn Thr Ala Pro Leu Gln
Pro Glu Gln Leu Gln Val Phe385 390 395 400Glu Thr Leu Glu Glu Ile
Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro405 410 415Asp Ser Leu Pro
Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg420 425 430Gly Arg
Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu435 440
445Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser
Gly450 455 460Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val
His Thr Val465 470 475 480Pro Trp Asp Gln Leu Phe Arg Asn Pro His
Gln Ala Leu Leu His Thr485 490 495Ala Asn Arg Pro Glu Asp Glu Cys
Val Gly Glu Gly Leu Ala Cys His500 505 510Gln Leu Cys Ala Arg Gly
His Cys Trp Gly Pro Gly Pro Thr Gln Cys515 520 525Val Asn Cys Ser
Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys530 535 540Arg Val
Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys545 550 555
560Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr
Cys565 570 575Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His
Tyr Lys Asp580 585 590Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly
Val Lys Pro Asp Leu595 600 605Ser Tyr Met Pro Ile Trp Lys Phe Pro
Asp Glu Glu Gly Ala Cys Gln610 615 620Pro Cys Pro Ile Asn Cys Thr
His Ser Cys Val Asp Leu Asp Asp Lys625 630 635 640Gly Cys Pro Ala
Glu Gln Arg Ala Ser Pro Leu Thr Gly Cys Val Ser645 650 655Gly Asp
Thr Ile Val Met Thr Ser Gly Gly Pro Arg Thr Val Ala Glu660 665
670Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr
Pro675 680 685Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp Val
Tyr Asp Leu690 695 700Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr
His Asp His Arg Val705 710 715 720Leu Val Met Asp Gly Gly Leu Glu
Trp Arg Ala Ala Gly Glu Leu Glu725 730 735Arg Gly Asp Arg Leu Val
Met Asp Asp Ala Ala Gly Glu Phe Pro Ala740 745 750Leu Ala Thr Phe
Arg Gly Leu Arg Gly Ala Gly Arg Gln Asp Val Tyr755 760 765Asp Ala
Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile770 775
780Val His Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly
Leu785 790 795 800Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn
Thr Ala Tyr Thr805 810 815Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys
Thr Tyr Lys Cys Leu Gln820 825 830Pro His Thr Ser Leu Ala Gly Trp
Glu Pro Ser Asn Val Pro Ala Leu835 840 845Trp Gln Leu
Gln850187830PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 187Thr Gln Val Cys Thr Gly Thr Asp Met
Lys Leu Arg Leu Pro Ala Ser1 5 10 15Pro Glu Thr His Leu Asp Met Leu
Arg His Leu Tyr Gln Gly Cys Gln20 25 30Val Val Gln Gly Asn Leu Glu
Leu Thr Tyr Leu Pro Thr Asn Ala Ser35 40 45Leu Ser Phe Leu Gln Asp
Ile Gln Glu Val Gln Gly Tyr Val Leu Ile50 55 60Ala His Asn Gln Val
Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val65 70 75 80Arg Gly Thr
Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp85 90 95Asn Gly
Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro100 105
110Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu
Lys115 120 125Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr
Gln Asp Thr130 135 140Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn
Gln Leu Ala Leu Thr145 150 155 160Leu Ile Asp Thr Asn Arg Ser Arg
Ala Cys His Pro Cys Ser Pro Met165 170 175Cys Lys Gly Ser Arg Cys
Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser180 185 190Leu Thr Arg Thr
Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro195 200 205Leu Pro
Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly210 215
220Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser
Gly225 230 235 240Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr
Asn Thr Asp Thr245 250 255Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
Tyr Thr Phe Gly Ala Ser260 265 270Cys Val Thr Ala Cys Pro Tyr Asn
Tyr Leu Ser Thr Asp Val Gly Ser275 280 285Cys Thr Leu Val Cys Pro
Leu His Asn Gln Glu Val Thr Ala Glu Asp290 295 300Gly Thr Gln Arg
Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys305 310 315 320Tyr
Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser325 330
335Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser
Leu340 345 350Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser
Asn Thr Ala355 360 365Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu
Thr Leu Glu Glu Ile370 375 380Thr Gly Tyr Leu Tyr Ile Ser Ala Trp
Pro Asp Ser Leu Pro Asp Leu385 390 395 400Ser Val Phe Gln Asn Leu
Gln Val Ile Arg Gly Arg Ile Leu His Asn405 410 415Gly Ala Tyr Ser
Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu
Gly420 425 430Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu
Ile His His435 440 445Asn Thr His Leu Cys Phe Val His Thr Val Pro
Trp Asp Gln Leu Phe450 455 460Arg Asn Pro His Gln Ala Leu Leu His
Thr Ala Asn Arg Pro Glu Asp465 470 475 480Glu Cys Val Gly Glu Gly
Leu Ala Cys His Gln Leu Cys Ala Arg Gly485 490 495His Cys Trp Gly
Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe500 505 510Leu Arg
Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu515 520
525Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro
Glu530 535 540Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro
Glu Ala Asp545 550 555 560Gln Cys Val Ala Cys Ala His Tyr Lys Asp
Pro Pro Phe Cys Val Ala565 570 575Arg Cys Pro Ser Gly Val Lys Pro
Asp Leu Ser Tyr Met Pro Ile Trp580 585 590Lys Phe Pro Asp Glu Glu
Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys595 600 605Thr His Ser Cys
Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln610 615 620Arg Ala
Ser Pro Leu Thr Gly Cys Val Ser Gly Asp Thr Ile Val Met625 630 635
640Thr Ser Gly Gly Pro Arg Thr Val Ala Glu Leu Glu Gly Lys Pro
Phe645 650 655Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys Pro Ser
Gly Phe Phe660 665 670Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg
Thr Arg Glu Gly His675 680 685Cys Leu Arg Leu Thr His Asp His Arg
Val Leu Val Met Asp Gly Gly690 695 700Leu Glu Trp Arg Ala Ala Gly
Glu Leu Glu Arg Gly Asp Arg Leu Val705 710 715 720Met Asp Asp Ala
Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly725 730 735Leu Arg
Gly Ala Gly Arg Gln Asp Val Tyr Asp Ala Thr Val Tyr Gly740 745
750Ala Ser Ala Phe Thr Ala Asn Gly Phe Ile Val His Ala Cys Gly
Glu755 760 765Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu Thr Thr Asn
Pro Gly Val770 775 780Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala
Gly Gln Leu Val Thr785 790 795 800Tyr Asn Gly Lys Thr Tyr Lys Cys
Leu Gln Pro His Thr Ser Leu Ala805 810 815Gly Trp Glu Pro Ser Asn
Val Pro Ala Leu Trp Gln Leu Gln820 825 830188631PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 188Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg
Leu Pro Ala Ser1 5 10 15Pro Glu Thr His Leu Asp Met Leu Arg His Leu
Tyr Gln Gly Cys Gln20 25 30Val Val Gln Gly Asn Leu Glu Leu Thr Tyr
Leu Pro Thr Asn Ala Ser35 40 45Leu Ser Phe Leu Gln Asp Ile Gln Glu
Val Gln Gly Tyr Val Leu Ile50 55 60Ala His Asn Gln Val Arg Gln Val
Pro Leu Gln Arg Leu Arg Ile Val65 70 75 80Arg Gly Thr Gln Leu Phe
Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp85 90 95Asn Gly Asp Pro Leu
Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro100 105 110Gly Gly Leu
Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys115 120 125Gly
Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr130 135
140Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu
Thr145 150 155 160Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro
Cys Ser Pro Met165 170 175Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser
Ser Glu Asp Cys Gln Ser180 185 190Leu Thr Arg Thr Val Cys Ala Gly
Gly Cys Ala Arg Cys Lys Gly Pro195 200 205Leu Pro Thr Asp Cys Cys
His Glu Gln Cys Ala Ala Gly Cys Thr Gly210 215 220Pro Lys His Ser
Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly225 230 235 240Ile
Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr245 250
255Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala
Ser260 265 270Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp
Val Gly Ser275 280 285Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu
Val Thr Ala Glu Asp290 295 300Gly Thr Gln Arg Cys Glu Lys Cys Ser
Lys Pro Cys Ala Arg Val Cys305 310 315 320Tyr Gly Leu Gly Met Glu
His Leu Arg Glu Val Arg Ala Val Thr Ser325 330 335Ala Asn Ile Gln
Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu340 345 350Ala Phe
Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala355 360
365Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu
Ile370 375 380Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu
Pro Asp Leu385 390 395 400Ser Val Phe Gln Asn Leu Gln Val Ile Arg
Gly Arg Ile Leu His Asn405 410 415Gly Ala Tyr Ser Leu Thr Leu Gln
Gly Leu Gly Ile Ser Trp Leu Gly420 425 430Leu Arg Ser Leu Arg Glu
Leu Gly Ser Gly Leu Ala Leu Ile His His435 440 445Asn Thr His Leu
Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe450 455 460Arg Asn
Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp465 470 475
480Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg
Gly485 490 495His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys
Ser Gln Phe500 505 510Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg
Val Leu Gln Gly Leu515 520 525Pro Arg Glu Tyr Val Asn Ala Arg His
Cys Leu Pro Cys His Pro Glu530 535 540Cys Gln Pro Gln Asn Gly Ser
Val Thr Cys Phe Gly Pro Glu Ala Asp545 550 555 560Gln Cys Val Ala
Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala565 570 575Arg Cys
Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp580 585
590Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn
Cys595 600 605Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro
Ala Glu Gln610 615 620Arg Ala Ser Pro Leu Thr Xaa625
630189632PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 189Thr Gln Val Cys Thr Gly Thr Asp Met
Lys Leu Arg Leu Pro Ala Ser1 5 10 15Pro Glu Thr His Leu Asp Met Leu
Arg His Leu Tyr Gln Gly Cys Gln20 25 30Val Val Gln Gly Asn Leu Glu
Leu Thr Tyr Leu Pro Thr Asn Ala Ser35 40 45Leu Ser Phe Leu Gln Asp
Ile Gln Glu Val Gln Gly Tyr Val Leu Ile50 55 60Ala His Asn Gln Val
Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val65 70 75 80Arg Gly Thr
Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp85 90 95Asn Gly
Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro100 105
110Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu
Lys115 120 125Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr
Gln Asp Thr130 135 140Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn
Gln Leu Ala Leu Thr145 150 155 160Leu Ile Asp Thr Asn Arg Ser Arg
Ala Cys His Pro Cys Ser Pro Met165 170 175Cys Lys Gly Ser Arg Cys
Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser180 185 190Leu Thr Arg Thr
Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro195 200 205Leu Pro
Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly210 215
220Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser
Gly225 230 235 240Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr
Asn Thr Asp Thr245 250 255Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
Tyr Thr Phe Gly Ala Ser260 265 270Cys Val Thr Ala Cys Pro Tyr Asn
Tyr Leu Ser Thr Asp Val Gly Ser275 280 285Cys Thr Leu Val Cys Pro
Leu His Asn Gln Glu Val Thr Ala Glu Asp290 295 300Gly Thr Gln Arg
Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys305 310 315 320Tyr
Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser325 330
335Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser
Leu340 345 350Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser
Asn Thr Ala355 360 365Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu
Thr Leu Glu Glu Ile370 375 380Thr Gly Tyr Leu Tyr Ile Ser Ala Trp
Pro Asp Ser Leu Pro Asp Leu385 390 395 400Ser Val Phe Gln Asn Leu
Gln Val Ile Arg Gly Arg Ile Leu His Asn405 410 415Gly Ala Tyr Ser
Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly420 425 430Leu Arg
Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His435 440
445Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu
Phe450 455 460Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg
Pro Glu Asp465 470 475 480Glu Cys Val Gly Glu Gly Leu Ala Cys His
Gln Leu Cys Ala Arg Gly485 490 495His Cys Trp Gly Pro Gly Pro Thr
Gln Cys Val Asn Cys Ser Gln Phe500 505 510Leu Arg Gly Gln Glu Cys
Val Glu Glu Cys Arg Val Leu Gln Gly Leu515 520 525Pro Arg Glu Tyr
Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu530 535 540Cys Gln
Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp545 550 555
560Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val
Ala565 570 575Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met
Pro Ile Trp580 585 590Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro
Cys Pro Ile Asn Cys595 600 605Thr His Ser Cys Val Asp Leu Asp Asp
Lys Gly Cys Pro Ala Glu Gln610 615 620Arg Ala Ser Pro Leu Thr Gly
Xaa625 6301902561DNAArtificial Sequenceartificial construct
relating to Homo Sapiens immunoglobulin 190aagcttgaat tcccaccatg
agggcgaacg acgctctgca ggtgctgggc ttgcttttca 60gcctggcccg gggctccgag
gtgggcaact ctcaggcagt gtgtcctggg actctgaatg 120gcctgagtgt
gaccggcgat gctgagaacc aataccagac actgtacaag ctctacgaga
180ggtgtgaggt ggtgatgggg aaccttgaga ttgtgctcac gggacacaat
gccgacctct 240ccttcctgca gtggattcga gaagtgacag gctatgtcct
cgtggccatg aatgaatttt 300ctactctacc attgcccaac ctccgcgtgg
tgcgagggac ccaggtctac gatgggaagt 360ttgccatctt cgtcatgttg
aactataaca ccaactccag ccacgctctg cgccagctcc 420gcttgactca
gctcaccgag attctgtcag ggggtgttta tattgagaag aacgataagc
480tctgtcacat ggacacaatt gactggaggg acatcgtgag ggaccgagat
gctgagatag 540tggtgaagga caatggcaga agctgtcccc cctgtcatga
ggtttgcaag gggcgatgct 600ggggtcctgg atcagaagac tgccagacat
tgaccaagac catctgtgct cctcagtgta 660atggtcactg ctttggtccc
aaccccaacc agtgctgcca tgatgagtgt gccgggggct 720gctcaggccc
tcaggacaca gactgctttg cctgccggca cttcaatgac agtggagcct
780gtgtacctcg ctgtccacag cctcttgtct acaacaagct aactttccag
ctggaaccca 840atccccacac caagtatcag tatggaggag tttgtgtagc
cagctgtccc cataactttg 900tggtggatca aacatcctgt gtcagggcct
gtcctcctga caagatggaa gtagataaaa 960atgggctcaa gatgtgtgag
ccttgtgggg gactatgtcc caaagcctgt gagggaacag 1020gctctgggag
ccgcttccag actgtggatt cgagcaacat tgatggattt gtgaactgca
1080ccaagatcct gggcaacctg gactttctga tcaccggcct caatggagac
ccctggcaca 1140agatccctgc cctggaccca gagaagctca atgtcttccg
gacagtacgg gagatcacag 1200gttacctgaa catccagtcc tggccgcccc
acatgcacaa cttcagtgtt ttttccaatt 1260tgacaaccat tggaggcaga
agcctctaca accggggctt ctcattgttg atcatgaaga 1320acttgaatgt
cacatctctg ggcttccgat ccctgaagga aattagtgct gggcgtatct
1380atataagtgc caataggcag ctctgctacc accactcttt gaactggacc
aaggtgcttc 1440gggggcctac ggaagagcga ctagacatca agcataatcg
gccgcgcaga gactgcgtgg 1500cagagggcaa agtgtgtgac ccactgtgct
cctctggggg atgctgggtc ccaggccctg 1560gtcagtgctt gtcctgtcga
aattatagcc gaggaggtgt ctgtgtgacc cactgcaact 1620ttctgaatgg
ggagccgcga gaatttgccc atgaggccga atgcttctcc tgccacccgg
1680aatgccaacc catggagggc actgccacat gcaatggctc gggctctgat
acttgtgctc 1740aatgtgccca ttttcgagat ggtccccact gtgtgagcag
ctgcccccat ggagtcctag 1800gtgccaaggg tccaatctac aagtacccag
atgttcagaa tgaatgtcgg ccctgccatg 1860agaactgcac ccaggggtgt
aaaggaccag agcttcaaga ctgtttagga caaacactgg 1920tgctgatcgg
caaaacccat ctgacagggt gcgtatccgg tgacaccatt gtaatgacta
1980gtggcgggcc ccgcactgtg gctgaactgg agggcaaacc gttcaccgca
ctgattcgcg 2040gctctggcta cccatgcccc tcaggtttct tccgcacctg
tgaacgtgac gtatatgatc 2100tgcgtacacg tgagggtcat tgcttacgtt
tgacccatga tcaccgtgtt ctggtgatgg 2160atggtggcct ggaatggcgt
gccgcgggtg aactggaacg cggcgaccgc ctggtgatgg 2220atgatgcagc
tggcgagttt ccggcactgg caaccttccg tggcctgcgt ggcgctggcc
2280gccaggatgt ttatgacgct actgtttacg gtgctagcgc attcactgct
aatggcttca 2340ttgtacacgc atgtggcgag cagcccggga ccggtctgaa
ctcaggcctc acgacaaatc 2400ctggtgtatc cgcttggcag gtcaacacag
cttatactgc gggacaattg gtcacatata 2460acggcaagac gtataaatgt
ttgcagcccc acacctcctt ggcaggatgg gaaccatcca 2520acgttcctgc
cttgtggcag cttcaatgac tcgagcggcc g 2561191843PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 191Met Arg Ala Asn Asp Ala Leu Gln Val Leu Gly Leu
Leu Phe Ser Leu1 5 10 15Ala Arg Gly Ser Glu Val Gly Asn Ser Gln Ala
Val Cys Pro Gly Thr20 25 30Leu Asn Gly Leu Ser Val Thr Gly Asp Ala
Glu Asn Gln Tyr Gln Thr35 40 45Leu Tyr Lys Leu Tyr Glu Arg Cys Glu
Val Val Met Gly Asn Leu Glu50 55 60Ile Val Leu Thr Gly His Asn Ala
Asp Leu Ser Phe Leu Gln Trp Ile65 70 75 80Arg Glu Val Thr Gly Tyr
Val Leu Val Ala Met Asn Glu Phe Ser Thr85 90 95Leu Pro Leu Pro Asn
Leu Arg Val Val Arg Gly Thr Gln Val Tyr Asp100 105 110Gly Lys Phe
Ala Ile Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser115 120 125His
Ala Leu Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser130 135
140Gly Gly Val Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp
Thr145 150 155 160Ile Asp Trp Arg Asp Ile Val Arg Asp Arg Asp Ala
Glu Ile Val Val165 170 175Lys Asp Asn Gly Arg Ser Cys Pro Pro Cys
His Glu Val Cys Lys Gly180 185 190Arg Cys Trp Gly Pro Gly Ser Glu
Asp Cys Gln Thr Leu Thr Lys Thr195 200 205Ile Cys Ala Pro Gln Cys
Asn Gly His Cys Phe Gly Pro Asn Pro Asn210 215 220Gln Cys Cys His
Asp Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp225 230 235 240Thr
Asp Cys Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val245 250
255Pro Arg Cys Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln
Leu260 265 270Glu Pro Asn Pro His Thr Lys Tyr Gln Tyr Gly Gly Val
Cys Val Ala275 280 285Ser Cys Pro His Asn Phe Val Val Asp Gln Thr
Ser Cys Val Arg Ala290 295 300Cys Pro Pro Asp Lys Met Glu Val Asp
Lys Asn Gly Leu Lys Met Cys305 310 315 320Glu Pro Cys Gly Gly Leu
Cys Pro Lys Ala Cys Glu Gly Thr Gly Ser325 330 335Gly Ser Arg Phe
Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val340 345 350Asn Cys
Thr Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu355 360
365Asn Gly Asp Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys
Leu370 375 380Asn Val Phe Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu
Asn Ile Gln385 390 395 400Ser Trp Pro Pro His Met His Asn Phe Ser
Val Phe Ser Asn Leu Thr405 410 415Thr Ile Gly Gly Arg Ser Leu Tyr
Asn Arg Gly Phe Ser Leu Leu Ile420 425 430Met Lys Asn Leu Asn Val
Thr Ser Leu Gly Phe Arg Ser Leu Lys Glu435 440 445Ile Ser Ala Gly
Arg Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr450 455 460His His
Ser Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu465
470 475 480Arg Leu Asp Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val
Ala Glu485 490 495Gly Lys Val Cys Asp Pro Leu Cys Ser Ser Gly Gly
Cys Trp Gly Pro500 505 510Gly Pro Gly Gln Cys Leu Ser Cys Arg Asn
Tyr Ser Arg Gly Gly Val515 520 525Cys Val Thr His Cys Asn Phe Leu
Asn Gly Glu Pro Arg Glu Phe Ala530 535 540His Glu Ala Glu Cys Phe
Ser Cys His Pro Glu Cys Gln Pro Met Glu545 550 555 560Gly Thr Ala
Thr Cys Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys565 570 575Ala
His Phe Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly580 585
590Val Leu Gly Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln
Asn595 600 605Glu Cys Arg Pro Cys His Glu Asn Cys Thr Gln Gly Cys
Lys Gly Pro610 615 620Glu Leu Gln Asp Cys Leu Gly Gln Thr Leu Val
Leu Ile Gly Lys Thr625 630 635 640His Leu Thr Gly Cys Val Ser Gly
Asp Thr Ile Val Met Thr Ser Gly645 650 655Gly Pro Arg Thr Val Ala
Glu Leu Glu Gly Lys Pro Phe Thr Ala Leu660 665 670Ile Arg Gly Ser
Gly Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys675 680 685Glu Arg
Asp Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg690 695
700Leu Thr His Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu
Trp705 710 715 720Arg Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu
Val Met Asp Asp725 730 735Ala Ala Gly Glu Phe Pro Ala Leu Ala Thr
Phe Arg Gly Leu Arg Gly740 745 750Ala Gly Arg Gln Asp Val Tyr Asp
Ala Thr Val Tyr Gly Ala Ser Ala755 760 765Phe Thr Ala Asn Gly Phe
Ile Val His Ala Cys Gly Glu Gln Pro Gly770 775 780Thr Gly Leu Asn
Ser Gly Leu Thr Thr Asn Pro Gly Val Ser Ala Trp785 790 795 800Gln
Val Asn Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly805 810
815Lys Thr Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp
Glu820 825 830Pro Ser Asn Val Pro Ala Leu Trp Gln Leu Gln835
840192824PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 192Ser Glu Val Gly Asn Ser Gln Ala Val
Cys Pro Gly Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala Glu
Asn Gln Tyr Gln Thr Leu Tyr Lys20 25 30Leu Tyr Glu Arg Cys Glu Val
Val Met Gly Asn Leu Glu Ile Val Leu35 40 45Thr Gly His Asn Ala Asp
Leu Ser Phe Leu Gln Trp Ile Arg Glu Val50 55 60Thr Gly Tyr Val Leu
Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn Leu
Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe85 90 95Ala Ile
Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu100 105
110Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly
Val115 120 125Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr
Ile Asp Trp130 135 140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile
Val Val Lys Asp Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His
Glu Val Cys Lys Gly Arg Cys Trp165 170 175Gly Pro Gly Ser Glu Asp
Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala180 185 190Pro Gln Cys Asn
Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys195 200 205His Asp
Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys210 215
220Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg
Cys225 230 235 240Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln
Leu Glu Pro Asn245 250 255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val
Cys Val Ala Ser Cys Pro260 265 270His Asn Phe Val Val Asp Gln Thr
Ser Cys Val Arg Ala Cys Pro Pro275 280 285Asp Lys Met Glu Val Asp
Lys Asn Gly Leu Lys Met Cys Glu Pro Cys290 295 300Gly Gly Leu Cys
Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe
Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr325 330
335Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly
Asp340 345 350Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu
Asn Val Phe355 360 365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn
Ile Gln Ser Trp Pro370 375 380Pro His Met His Asn Phe Ser Val Phe
Ser Asn Leu Thr Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn
Arg Gly Phe Ser Leu Leu Ile Met Lys Asn405 410 415Leu Asn Val Thr
Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala420 425 430Gly Arg
Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser435 440
445Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu
Asp450 455 460Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu
Gly Lys Val465 470 475 480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys
Trp Gly Pro Gly Pro Gly485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr
Ser Arg Gly Gly Val Cys Val Thr500 505 510His Cys Asn Phe Leu Asn
Gly Glu Pro Arg Glu Phe Ala His Glu Ala515 520 525Glu Cys Phe Ser
Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala530 535 540Thr Cys
Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555
560Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu
Gly565 570 575Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn
Glu Cys Arg580 585 590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys
Gly Pro Glu Leu Gln595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu
Ile Gly Lys Thr His Leu Thr610 615 620Gly Cys Val Ser Gly Asp Thr
Ile Val Met Thr Ser Gly Gly Pro Arg625 630 635 640Thr Val Ala Glu
Leu Glu Gly Lys Pro Phe Thr Ala Leu Ile Arg Gly645 650 655Ser Gly
Tyr Pro Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu Arg Asp660 665
670Val Tyr Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg Leu Thr
His675 680 685Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu Trp
Arg Ala Ala690 695 700Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met
Asp Asp Ala Ala Gly705 710 715 720Glu Phe Pro Ala Leu Ala Thr Phe
Arg Gly Leu Arg Gly Ala Gly Arg725 730 735Gln Asp Val Tyr Asp Ala
Thr Val Tyr Gly Ala Ser Ala Phe Thr Ala740 745 750Asn Gly Phe Ile
Val His Ala Cys Gly Glu Gln Pro Gly Thr Gly Leu755 760 765Asn Ser
Gly Leu Thr Thr Asn Pro Gly Val Ser Ala Trp Gln Val Asn770 775
780Thr Ala Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr
Tyr785 790 795 800Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp
Glu Pro Ser Asn805 810 815Val Pro Ala Leu Trp Gln Leu
Gln820193625PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 193Ser Glu Val Gly Asn Ser Gln Ala Val
Cys Pro Gly Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala Glu
Asn Gln Tyr Gln Thr Leu Tyr Lys20 25 30Leu Tyr Glu Arg Cys Glu Val
Val Met Gly Asn Leu Glu Ile Val Leu35 40 45Thr Gly His Asn Ala Asp
Leu Ser Phe Leu Gln Trp Ile Arg Glu Val50 55 60Thr Gly Tyr Val Leu
Val Ala Met Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn Leu
Arg Val Val Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe85 90 95Ala Ile
Phe Val Met Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu100 105
110Arg Gln Leu Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly
Val115 120 125Tyr Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr
Ile Asp Trp130 135 140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile
Val Val Lys Asp Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His
Glu Val Cys Lys Gly Arg Cys Trp165 170 175Gly Pro Gly Ser Glu Asp
Cys Gln Thr Leu Thr Lys Thr Ile Cys Ala180 185 190Pro Gln Cys Asn
Gly His Cys Phe Gly Pro Asn Pro Asn Gln Cys Cys195 200 205His Asp
Glu Cys Ala Gly Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys210 215
220Phe Ala Cys Arg His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg
Cys225 230 235 240Pro Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln
Leu Glu Pro Asn245 250 255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val
Cys Val Ala Ser Cys Pro260 265 270His Asn Phe Val Val Asp Gln Thr
Ser Cys Val Arg Ala Cys Pro Pro275 280 285Asp Lys Met Glu Val Asp
Lys Asn Gly Leu Lys Met Cys Glu Pro Cys290 295 300Gly Gly Leu Cys
Pro Lys Ala Cys Glu Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe
Gln Thr Val Asp Ser Ser Asn Ile Asp Gly Phe Val Asn Cys Thr325 330
335Lys Ile Leu Gly Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly
Asp340 345 350Pro Trp His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu
Asn Val Phe355 360 365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn
Ile Gln Ser Trp Pro370 375 380Pro His Met His Asn Phe Ser Val Phe
Ser Asn Leu Thr Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn
Arg Gly Phe Ser Leu Leu Ile Met Lys Asn405 410 415Leu Asn Val Thr
Ser Leu Gly Phe Arg Ser Leu Lys Glu Ile Ser Ala420 425 430Gly Arg
Ile Tyr Ile Ser Ala Asn Arg Gln Leu Cys Tyr His His Ser435 440
445Leu Asn Trp Thr Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu
Asp450 455 460Ile Lys His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu
Gly Lys Val465 470 475 480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys
Trp Gly Pro Gly Pro Gly485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr
Ser Arg Gly Gly Val Cys Val Thr500 505 510His Cys Asn Phe Leu Asn
Gly Glu Pro Arg Glu Phe Ala His Glu Ala515 520 525Glu Cys Phe Ser
Cys His Pro Glu Cys Gln Pro Met Glu Gly Thr Ala530 535 540Thr Cys
Asn Gly Ser Gly Ser Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555
560Arg Asp Gly Pro His Cys Val Ser Ser Cys Pro His Gly Val Leu
Gly565 570 575Ala Lys Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn
Glu Cys Arg580 585 590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys
Gly Pro Glu Leu Gln595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu
Ile Gly Lys Thr His Leu Thr610 615 620Xaa625194626PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 194Ser Glu Val Gly Asn Ser Gln Ala Val Cys Pro Gly
Thr Leu Asn Gly1 5 10 15Leu Ser Val Thr Gly Asp Ala Glu Asn Gln Tyr
Gln Thr Leu Tyr Lys20 25 30Leu Tyr Glu Arg Cys Glu Val Val Met Gly
Asn Leu Glu Ile Val Leu35 40 45Thr Gly His Asn Ala Asp Leu Ser Phe
Leu Gln Trp Ile Arg Glu Val50 55 60Thr Gly Tyr Val Leu Val Ala Met
Asn Glu Phe Ser Thr Leu Pro Leu65 70 75 80Pro Asn Leu Arg Val Val
Arg Gly Thr Gln Val Tyr Asp Gly Lys Phe85 90 95Ala Ile Phe Val Met
Leu Asn Tyr Asn Thr Asn Ser Ser His Ala Leu100 105 110Arg Gln Leu
Arg Leu Thr Gln Leu Thr Glu Ile Leu Ser Gly Gly Val115 120 125Tyr
Ile Glu Lys Asn Asp Lys Leu Cys His Met Asp Thr Ile Asp Trp130 135
140Arg Asp Ile Val Arg Asp Arg Asp Ala Glu Ile Val Val Lys Asp
Asn145 150 155 160Gly Arg Ser Cys Pro Pro Cys His Glu Val Cys Lys
Gly Arg Cys Trp165 170 175Gly Pro Gly Ser Glu Asp Cys Gln Thr Leu
Thr Lys Thr Ile Cys Ala180 185 190Pro Gln Cys Asn Gly His Cys Phe
Gly Pro Asn Pro Asn Gln Cys Cys195 200 205His Asp Glu Cys Ala Gly
Gly Cys Ser Gly Pro Gln Asp Thr Asp Cys210 215 220Phe Ala Cys Arg
His Phe Asn Asp Ser Gly Ala Cys Val Pro Arg Cys225 230 235 240Pro
Gln Pro Leu Val Tyr Asn Lys Leu Thr Phe Gln Leu Glu Pro Asn245 250
255Pro His Thr Lys Tyr Gln Tyr Gly Gly Val Cys Val Ala Ser Cys
Pro260 265 270His Asn Phe Val Val Asp Gln Thr Ser Cys Val Arg Ala
Cys Pro Pro275 280 285Asp Lys Met Glu Val Asp Lys Asn Gly Leu Lys
Met Cys Glu Pro Cys290 295 300Gly Gly Leu Cys Pro Lys Ala Cys Glu
Gly Thr Gly Ser Gly Ser Arg305 310 315 320Phe Gln Thr Val Asp Ser
Ser Asn Ile Asp Gly Phe Val Asn Cys Thr325 330 335Lys Ile Leu Gly
Asn Leu Asp Phe Leu Ile Thr Gly Leu Asn Gly Asp340 345 350Pro Trp
His Lys Ile Pro Ala Leu Asp Pro Glu Lys Leu Asn Val Phe355 360
365Arg Thr Val Arg Glu Ile Thr Gly Tyr Leu Asn Ile Gln Ser Trp
Pro370 375 380Pro His Met His Asn Phe Ser Val Phe Ser Asn Leu Thr
Thr Ile Gly385 390 395 400Gly Arg Ser Leu Tyr Asn Arg Gly Phe Ser
Leu Leu Ile Met Lys Asn405 410 415Leu Asn Val Thr Ser Leu Gly Phe
Arg Ser Leu Lys Glu Ile Ser Ala420 425 430Gly Arg Ile Tyr Ile Ser
Ala Asn Arg Gln Leu Cys Tyr His His Ser435 440 445Leu Asn Trp Thr
Lys Val Leu Arg Gly Pro Thr Glu Glu Arg Leu Asp450 455 460Ile Lys
His Asn Arg Pro Arg Arg Asp Cys Val Ala Glu Gly Lys Val465 470 475
480Cys Asp Pro Leu Cys Ser Ser Gly Gly Cys Trp Gly Pro Gly Pro
Gly485 490 495Gln Cys Leu Ser Cys Arg Asn Tyr Ser Arg Gly Gly Val
Cys Val Thr500 505 510His Cys Asn Phe Leu Asn Gly Glu Pro Arg Glu
Phe Ala His Glu Ala515 520 525Glu Cys Phe Ser Cys His Pro Glu Cys
Gln Pro Met Glu Gly Thr Ala530 535 540Thr Cys Asn Gly Ser Gly Ser
Asp Thr Cys Ala Gln Cys Ala His Phe545 550 555 560Arg Asp Gly Pro
His Cys Val Ser Ser Cys Pro His Gly Val Leu Gly565 570 575Ala Lys
Gly Pro Ile Tyr Lys Tyr Pro Asp Val Gln Asn Glu Cys Arg580 585
590Pro Cys His Glu Asn Cys Thr Gln Gly Cys Lys Gly Pro Glu Leu
Gln595 600 605Asp Cys Leu Gly Gln Thr Leu Val Leu Ile Gly Lys Thr
His Leu Thr610 615 620Gly Xaa6251952585DNAArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 195aagcttgaat tcccaccatg aagccggcga caggactttg
ggtctgggtg agccttctcg 60tggcggcggg gaccgtccag cccagcgatt ctcagtcagt
gtgtgcagga acggagaata 120aactgagctc tctctctgac ctggaacagc
agtaccgagc cttgcgcaag tactatgaaa 180actgtgaggt tgtcatgggc
aacctggaga taaccagcat tgagcacaac cgggacctct 240ccttcctgcg
gtctgttcga gaagtcacag gctacgtgtt agtggctctt aatcagtttc
300gttacctgcc tctggagaat ttacgcatta ttcgtgggac aaaactttat
gaggatcgat 360atgccttggc aatattttta aactacagaa aagatggaaa
ctttggactt caagaacttg 420gattaaagaa cttgacagaa atcctaaatg
gtggagtcta tgtagaccag aacaaattcc 480tttgttatgc agacaccatt
cattggcaag atattgttcg gaacccatgg ccttccaact 540tgactcttgt
gtcaacaaat ggtagttcag gatgtggacg ttgccataag tcctgtactg
600gccgttgctg gggacccaca gaaaatcatt gccagacttt gacaaggacg
gtgtgtgcag 660aacaatgtga cggcagatgc tacggacctt acgtcagtga
ctgctgccat cgagaatgtg 720ctggaggctg ctcaggacct aaggacacag
actgctttgc ctgcatgaat ttcaatgaca 780gtggagcatg tgttactcag
tgtccccaaa cctttgtcta caatccaacc acctttcaac 840tggagcacaa
tttcaatgca aagtacacat atggagcatt ctgtgtcaag aaatgtccac
900ataactttgt
ggtagattcc agttcttgtg tgcgtgcctg ccctagttcc aagatggaag
960tagaagaaaa tgggattaaa atgtgtaaac cttgcactga catttgccca
aaggcttgtg 1020atggcattgg cacaggatca ttgatgtcag ctcagactgt
ggattccagt aacattgaca 1080aattcataaa ctgtaccaag atcaatggga
atttgatctt tctagtcact ggtattcatg 1140gggaccctta caatgcaatt
gaagccatag acccagagaa actgaacgtc tttcggacag 1200tcagagagat
aacaggtttc ctgaacatac agtcatggcc accaaacatg actgacttca
1260gtgttttttc taacctggtg accattggtg gaagagtact ctatagtggc
ctgtccttgc 1320ttatcctcaa gcaacagggc atcacctctc tacagttcca
gtccctgaag gaaatcagcg 1380caggaaacat ctatattact gacaacagca
acctgtgtta ttatcatacc attaactgga 1440caacactctt cagcacaatc
aaccagagaa tagtaatccg ggacaacaga aaagctgaaa 1500attgtactgc
tgaaggaatg gtgtgcaacc atctgtgttc cagtgatggc tgttggggac
1560ctgggccaga ccaatgtctg tcgtgtcgcc gcttcagtag aggaaggatc
tgcatagagt 1620cttgtaacct ctatgatggt gaatttcggg agtttgagaa
tggctccatc tgtgtggagt 1680gtgaccccca gtgtgagaag atggaagatg
gcctcctcac atgccatgga ccgggtcctg 1740acaactgtac aaagtgctct
cattttaaag atggcccaaa ctgtgtggaa aaatgtccag 1800atggcttaca
gggggcaaac agtttcattt tcaagtatgc tgatccagat cgggagtgcc
1860acccatgcca tccaaactgc acccaagggt gtaacggtcc cactagtcat
gactgcattt 1920actacccatg gacgggccat tccactttac cacaacatgc
tagaactccc gggtgcgtat 1980ccggtgacac cattgtaatg actagtggcg
ggccccgcac tgtggctgaa ctggagggca 2040aaccgttcac cgcactgatt
cgcggctctg gctacccatg cccctcaggt ttcttccgca 2100cctgtgaacg
tgacgtatat gatctgcgta cacgtgaggg tcattgctta cgtttgaccc
2160atgatcaccg tgttctggtg atggatggtg gcctggaatg gcgtgccgcg
ggtgaactgg 2220aacgcggcga ccgcctggtg atggatgatg cagctggcga
gtttccggca ctggcaacct 2280tccgtggcct gcgtggcgct ggccgccagg
atgtttatga cgctactgtt tacggtgcta 2340gcgcattcac tgctaatggc
ttcattgtac acgcatgtgg cgagcagccc gggaccggtc 2400tgaactcagg
cctcacgaca aatcctggtg tatccgcttg gcaggtcaac acagcttata
2460ctgcgggaca attggtcaca tataacggca agacgtataa atgtttgcag
ccccacacct 2520ccttggcagg atgggaacca tccaacgttc ctgccttgtg
gcagcttcaa tgactcgagc 2580ggccg 2585196851PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 196Met Lys Pro Ala Thr Gly Leu Trp Val Trp Val Ser
Leu Leu Val Ala1 5 10 15Ala Gly Thr Val Gln Pro Ser Asp Ser Gln Ser
Val Cys Ala Gly Thr20 25 30Glu Asn Lys Leu Ser Ser Leu Ser Asp Leu
Glu Gln Gln Tyr Arg Ala35 40 45Leu Arg Lys Tyr Tyr Glu Asn Cys Glu
Val Val Met Gly Asn Leu Glu50 55 60Ile Thr Ser Ile Glu His Asn Arg
Asp Leu Ser Phe Leu Arg Ser Val65 70 75 80Arg Glu Val Thr Gly Tyr
Val Leu Val Ala Leu Asn Gln Phe Arg Tyr85 90 95Leu Pro Leu Glu Asn
Leu Arg Ile Ile Arg Gly Thr Lys Leu Tyr Glu100 105 110Asp Arg Tyr
Ala Leu Ala Ile Phe Leu Asn Tyr Arg Lys Asp Gly Asn115 120 125Phe
Gly Leu Gln Glu Leu Gly Leu Lys Asn Leu Thr Glu Ile Leu Asn130 135
140Gly Gly Val Tyr Val Asp Gln Asn Lys Phe Leu Cys Tyr Ala Asp
Thr145 150 155 160Ile His Trp Gln Asp Ile Val Arg Asn Pro Trp Pro
Ser Asn Leu Thr165 170 175Leu Val Ser Thr Asn Gly Ser Ser Gly Cys
Gly Arg Cys His Lys Ser180 185 190Cys Thr Gly Arg Cys Trp Gly Pro
Thr Glu Asn His Cys Gln Thr Leu195 200 205Thr Arg Thr Val Cys Ala
Glu Gln Cys Asp Gly Arg Cys Tyr Gly Pro210 215 220Tyr Val Ser Asp
Cys Cys His Arg Glu Cys Ala Gly Gly Cys Ser Gly225 230 235 240Pro
Lys Asp Thr Asp Cys Phe Ala Cys Met Asn Phe Asn Asp Ser Gly245 250
255Ala Cys Val Thr Gln Cys Pro Gln Thr Phe Val Tyr Asn Pro Thr
Thr260 265 270Phe Gln Leu Glu His Asn Phe Asn Ala Lys Tyr Thr Tyr
Gly Ala Phe275 280 285Cys Val Lys Lys Cys Pro His Asn Phe Val Val
Asp Ser Ser Ser Cys290 295 300Val Arg Ala Cys Pro Ser Ser Lys Met
Glu Val Glu Glu Asn Gly Ile305 310 315 320Lys Met Cys Lys Pro Cys
Thr Asp Ile Cys Pro Lys Ala Cys Asp Gly325 330 335Ile Gly Thr Gly
Ser Leu Met Ser Ala Gln Thr Val Asp Ser Ser Asn340 345 350Ile Asp
Lys Phe Ile Asn Cys Thr Lys Ile Asn Gly Asn Leu Ile Phe355 360
365Leu Val Thr Gly Ile His Gly Asp Pro Tyr Asn Ala Ile Glu Ala
Ile370 375 380Asp Pro Glu Lys Leu Asn Val Phe Arg Thr Val Arg Glu
Ile Thr Gly385 390 395 400Phe Leu Asn Ile Gln Ser Trp Pro Pro Asn
Met Thr Asp Phe Ser Val405 410 415Phe Ser Asn Leu Val Thr Ile Gly
Gly Arg Val Leu Tyr Ser Gly Leu420 425 430Ser Leu Leu Ile Leu Lys
Gln Gln Gly Ile Thr Ser Leu Gln Phe Gln435 440 445Ser Leu Lys Glu
Ile Ser Ala Gly Asn Ile Tyr Ile Thr Asp Asn Ser450 455 460Asn Leu
Cys Tyr Tyr His Thr Ile Asn Trp Thr Thr Leu Phe Ser Thr465 470 475
480Ile Asn Gln Arg Ile Val Ile Arg Asp Asn Arg Lys Ala Glu Asn
Cys485 490 495Thr Ala Glu Gly Met Val Cys Asn His Leu Cys Ser Ser
Asp Gly Cys500 505 510Trp Gly Pro Gly Pro Asp Gln Cys Leu Ser Cys
Arg Arg Phe Ser Arg515 520 525Gly Arg Ile Cys Ile Glu Ser Cys Asn
Leu Tyr Asp Gly Glu Phe Arg530 535 540Glu Phe Glu Asn Gly Ser Ile
Cys Val Glu Cys Asp Pro Gln Cys Glu545 550 555 560Lys Met Glu Asp
Gly Leu Leu Thr Cys His Gly Pro Gly Pro Asp Asn565 570 575Cys Thr
Lys Cys Ser His Phe Lys Asp Gly Pro Asn Cys Val Glu Lys580 585
590Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe Ile Phe Lys Tyr
Ala595 600 605Asp Pro Asp Arg Glu Cys His Pro Cys His Pro Asn Cys
Thr Gln Gly610 615 620Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr
Tyr Pro Trp Thr Gly625 630 635 640His Ser Thr Leu Pro Gln His Ala
Arg Thr Pro Gly Cys Val Ser Gly645 650 655Asp Thr Ile Val Met Thr
Ser Gly Gly Pro Arg Thr Val Ala Glu Leu660 665 670Glu Gly Lys Pro
Phe Thr Ala Leu Ile Arg Gly Ser Gly Tyr Pro Cys675 680 685Pro Ser
Gly Phe Phe Arg Thr Cys Glu Arg Asp Val Tyr Asp Leu Arg690 695
700Thr Arg Glu Gly His Cys Leu Arg Leu Thr His Asp His Arg Val
Leu705 710 715 720Val Met Asp Gly Gly Leu Glu Trp Arg Ala Ala Gly
Glu Leu Glu Arg725 730 735Gly Asp Arg Leu Val Met Asp Asp Ala Ala
Gly Glu Phe Pro Ala Leu740 745 750Ala Thr Phe Arg Gly Leu Arg Gly
Ala Gly Arg Gln Asp Val Tyr Asp755 760 765Ala Thr Val Tyr Gly Ala
Ser Ala Phe Thr Ala Asn Gly Phe Ile Val770 775 780His Ala Cys Gly
Glu Gln Pro Gly Thr Gly Leu Asn Ser Gly Leu Thr785 790 795 800Thr
Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala805 810
815Gly Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln
Pro820 825 830His Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro
Ala Leu Trp835 840 845Gln Leu Gln850197826PRTArtificial
Sequenceartificial construct relating to Homo Sapiens
immunoglobulin 197Gln Ser Val Cys Ala Gly Thr Glu Asn Lys Leu Ser
Ser Leu Ser Asp1 5 10 15Leu Glu Gln Gln Tyr Arg Ala Leu Arg Lys Tyr
Tyr Glu Asn Cys Glu20 25 30Val Val Met Gly Asn Leu Glu Ile Thr Ser
Ile Glu His Asn Arg Asp35 40 45Leu Ser Phe Leu Arg Ser Val Arg Glu
Val Thr Gly Tyr Val Leu Val50 55 60Ala Leu Asn Gln Phe Arg Tyr Leu
Pro Leu Glu Asn Leu Arg Ile Ile65 70 75 80Arg Gly Thr Lys Leu Tyr
Glu Asp Arg Tyr Ala Leu Ala Ile Phe Leu85 90 95Asn Tyr Arg Lys Asp
Gly Asn Phe Gly Leu Gln Glu Leu Gly Leu Lys100 105 110Asn Leu Thr
Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln Asn Lys115 120 125Phe
Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile Val Arg Asn130 135
140Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr Asn Gly Ser Ser
Gly145 150 155 160Cys Gly Arg Cys His Lys Ser Cys Thr Gly Arg Cys
Trp Gly Pro Thr165 170 175Glu Asn His Cys Gln Thr Leu Thr Arg Thr
Val Cys Ala Glu Gln Cys180 185 190Asp Gly Arg Cys Tyr Gly Pro Tyr
Val Ser Asp Cys Cys His Arg Glu195 200 205Cys Ala Gly Gly Cys Ser
Gly Pro Lys Asp Thr Asp Cys Phe Ala Cys210 215 220Met Asn Phe Asn
Asp Ser Gly Ala Cys Val Thr Gln Cys Pro Gln Thr225 230 235 240Phe
Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His Asn Phe Asn Ala245 250
255Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys Lys Cys Pro His Asn
Phe260 265 270Val Val Asp Ser Ser Ser Cys Val Arg Ala Cys Pro Ser
Ser Lys Met275 280 285Glu Val Glu Glu Asn Gly Ile Lys Met Cys Lys
Pro Cys Thr Asp Ile290 295 300Cys Pro Lys Ala Cys Asp Gly Ile Gly
Thr Gly Ser Leu Met Ser Ala305 310 315 320Gln Thr Val Asp Ser Ser
Asn Ile Asp Lys Phe Ile Asn Cys Thr Lys325 330 335Ile Asn Gly Asn
Leu Ile Phe Leu Val Thr Gly Ile His Gly Asp Pro340 345 350Tyr Asn
Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn Val Phe Arg355 360
365Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile Gln Ser Trp Pro
Pro370 375 380Asn Met Thr Asp Phe Ser Val Phe Ser Asn Leu Val Thr
Ile Gly Gly385 390 395 400Arg Val Leu Tyr Ser Gly Leu Ser Leu Leu
Ile Leu Lys Gln Gln Gly405 410 415Ile Thr Ser Leu Gln Phe Gln Ser
Leu Lys Glu Ile Ser Ala Gly Asn420 425 430Ile Tyr Ile Thr Asp Asn
Ser Asn Leu Cys Tyr Tyr His Thr Ile Asn435 440 445Trp Thr Thr Leu
Phe Ser Thr Ile Asn Gln Arg Ile Val Ile Arg Asp450 455 460Asn Arg
Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val Cys Asn His465 470 475
480Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro Gly Pro Asp Gln Cys
Leu485 490 495Ser Cys Arg Arg Phe Ser Arg Gly Arg Ile Cys Ile Glu
Ser Cys Asn500 505 510Leu Tyr Asp Gly Glu Phe Arg Glu Phe Glu Asn
Gly Ser Ile Cys Val515 520 525Glu Cys Asp Pro Gln Cys Glu Lys Met
Glu Asp Gly Leu Leu Thr Cys530 535 540His Gly Pro Gly Pro Asp Asn
Cys Thr Lys Cys Ser His Phe Lys Asp545 550 555 560Gly Pro Asn Cys
Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn565 570 575Ser Phe
Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys His Pro Cys580 585
590His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp
Cys595 600 605Ile Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln
His Ala Arg610 615 620Thr Pro Gly Cys Val Ser Gly Asp Thr Ile Val
Met Thr Ser Gly Gly625 630 635 640Pro Arg Thr Val Ala Glu Leu Glu
Gly Lys Pro Phe Thr Ala Leu Ile645 650 655Arg Gly Ser Gly Tyr Pro
Cys Pro Ser Gly Phe Phe Arg Thr Cys Glu660 665 670Arg Asp Val Tyr
Asp Leu Arg Thr Arg Glu Gly His Cys Leu Arg Leu675 680 685Thr His
Asp His Arg Val Leu Val Met Asp Gly Gly Leu Glu Trp Arg690 695
700Ala Ala Gly Glu Leu Glu Arg Gly Asp Arg Leu Val Met Asp Asp
Ala705 710 715 720Ala Gly Glu Phe Pro Ala Leu Ala Thr Phe Arg Gly
Leu Arg Gly Ala725 730 735Gly Arg Gln Asp Val Tyr Asp Ala Thr Val
Tyr Gly Ala Ser Ala Phe740 745 750Thr Ala Asn Gly Phe Ile Val His
Ala Cys Gly Glu Gln Pro Gly Thr755 760 765Gly Leu Asn Ser Gly Leu
Thr Thr Asn Pro Gly Val Ser Ala Trp Gln770 775 780Val Asn Thr Ala
Tyr Thr Ala Gly Gln Leu Val Thr Tyr Asn Gly Lys785 790 795 800Thr
Tyr Lys Cys Leu Gln Pro His Thr Ser Leu Ala Gly Trp Glu Pro805 810
815Ser Asn Val Pro Ala Leu Trp Gln Leu Gln820
825198627PRTArtificial Sequenceartificial construct relating to
Homo Sapiens immunoglobulin 198Gln Ser Val Cys Ala Gly Thr Glu Asn
Lys Leu Ser Ser Leu Ser Asp1 5 10 15Leu Glu Gln Gln Tyr Arg Ala Leu
Arg Lys Tyr Tyr Glu Asn Cys Glu20 25 30Val Val Met Gly Asn Leu Glu
Ile Thr Ser Ile Glu His Asn Arg Asp35 40 45Leu Ser Phe Leu Arg Ser
Val Arg Glu Val Thr Gly Tyr Val Leu Val50 55 60Ala Leu Asn Gln Phe
Arg Tyr Leu Pro Leu Glu Asn Leu Arg Ile Ile65 70 75 80Arg Gly Thr
Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala Ile Phe Leu85 90 95Asn Tyr
Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu Leu Gly Leu Lys100 105
110Asn Leu Thr Glu Ile Leu Asn Gly Gly Val Tyr Val Asp Gln Asn
Lys115 120 125Phe Leu Cys Tyr Ala Asp Thr Ile His Trp Gln Asp Ile
Val Arg Asn130 135 140Pro Trp Pro Ser Asn Leu Thr Leu Val Ser Thr
Asn Gly Ser Ser Gly145 150 155 160Cys Gly Arg Cys His Lys Ser Cys
Thr Gly Arg Cys Trp Gly Pro Thr165 170 175Glu Asn His Cys Gln Thr
Leu Thr Arg Thr Val Cys Ala Glu Gln Cys180 185 190Asp Gly Arg Cys
Tyr Gly Pro Tyr Val Ser Asp Cys Cys His Arg Glu195 200 205Cys Ala
Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys Phe Ala Cys210 215
220Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr Gln Cys Pro Gln
Thr225 230 235 240Phe Val Tyr Asn Pro Thr Thr Phe Gln Leu Glu His
Asn Phe Asn Ala245 250 255Lys Tyr Thr Tyr Gly Ala Phe Cys Val Lys
Lys Cys Pro His Asn Phe260 265 270Val Val Asp Ser Ser Ser Cys Val
Arg Ala Cys Pro Ser Ser Lys Met275 280 285Glu Val Glu Glu Asn Gly
Ile Lys Met Cys Lys Pro Cys Thr Asp Ile290 295 300Cys Pro Lys Ala
Cys Asp Gly Ile Gly Thr Gly Ser Leu Met Ser Ala305 310 315 320Gln
Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile Asn Cys Thr Lys325 330
335Ile Asn Gly Asn Leu Ile Phe Leu Val Thr Gly Ile His Gly Asp
Pro340 345 350Tyr Asn Ala Ile Glu Ala Ile Asp Pro Glu Lys Leu Asn
Val Phe Arg355 360 365Thr Val Arg Glu Ile Thr Gly Phe Leu Asn Ile
Gln Ser Trp Pro Pro370 375 380Asn Met Thr Asp Phe Ser Val Phe Ser
Asn Leu Val Thr Ile Gly Gly385 390 395 400Arg Val Leu Tyr Ser Gly
Leu Ser Leu Leu Ile Leu Lys Gln Gln Gly405 410 415Ile Thr Ser Leu
Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala Gly Asn420 425 430Ile Tyr
Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His Thr Ile Asn435 440
445Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg Ile Val Ile Arg
Asp450 455 460Asn Arg Lys Ala Glu Asn Cys Thr Ala Glu Gly Met Val
Cys Asn His465 470 475 480Leu Cys Ser Ser Asp Gly Cys Trp Gly Pro
Gly Pro Asp Gln Cys Leu485 490 495Ser Cys Arg Arg Phe Ser Arg Gly
Arg Ile Cys Ile Glu Ser Cys Asn500 505 510Leu Tyr Asp Gly Glu Phe
Arg Glu Phe Glu Asn Gly Ser Ile Cys Val515 520 525Glu Cys Asp Pro
Gln Cys Glu Lys Met Glu Asp Gly Leu Leu Thr Cys530 535 540His Gly
Pro Gly Pro Asp Asn Cys Thr Lys Cys Ser His Phe Lys Asp545 550 555
560Gly Pro Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala
Asn565 570 575Ser Phe Ile Phe Lys Tyr Ala Asp Pro Asp Arg Glu Cys
His Pro Cys580 585 590His Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro
Thr Ser His Asp Cys595 600 605Ile Tyr Tyr Pro Trp Thr Gly His Ser
Thr Leu Pro Gln His Ala Arg610 615 620Thr Pro
Xaa625199628PRTArtificial Sequenceartificial
construct relating to Homo Sapiens immunoglobulin 199Gln Ser Val
Cys Ala Gly Thr Glu Asn Lys Leu Ser Ser Leu Ser Asp1 5 10 15Leu Glu
Gln Gln Tyr Arg Ala Leu Arg Lys Tyr Tyr Glu Asn Cys Glu20 25 30Val
Val Met Gly Asn Leu Glu Ile Thr Ser Ile Glu His Asn Arg Asp35 40
45Leu Ser Phe Leu Arg Ser Val Arg Glu Val Thr Gly Tyr Val Leu Val50
55 60Ala Leu Asn Gln Phe Arg Tyr Leu Pro Leu Glu Asn Leu Arg Ile
Ile65 70 75 80Arg Gly Thr Lys Leu Tyr Glu Asp Arg Tyr Ala Leu Ala
Ile Phe Leu85 90 95Asn Tyr Arg Lys Asp Gly Asn Phe Gly Leu Gln Glu
Leu Gly Leu Lys100 105 110Asn Leu Thr Glu Ile Leu Asn Gly Gly Val
Tyr Val Asp Gln Asn Lys115 120 125Phe Leu Cys Tyr Ala Asp Thr Ile
His Trp Gln Asp Ile Val Arg Asn130 135 140Pro Trp Pro Ser Asn Leu
Thr Leu Val Ser Thr Asn Gly Ser Ser Gly145 150 155 160Cys Gly Arg
Cys His Lys Ser Cys Thr Gly Arg Cys Trp Gly Pro Thr165 170 175Glu
Asn His Cys Gln Thr Leu Thr Arg Thr Val Cys Ala Glu Gln Cys180 185
190Asp Gly Arg Cys Tyr Gly Pro Tyr Val Ser Asp Cys Cys His Arg
Glu195 200 205Cys Ala Gly Gly Cys Ser Gly Pro Lys Asp Thr Asp Cys
Phe Ala Cys210 215 220Met Asn Phe Asn Asp Ser Gly Ala Cys Val Thr
Gln Cys Pro Gln Thr225 230 235 240Phe Val Tyr Asn Pro Thr Thr Phe
Gln Leu Glu His Asn Phe Asn Ala245 250 255Lys Tyr Thr Tyr Gly Ala
Phe Cys Val Lys Lys Cys Pro His Asn Phe260 265 270Val Val Asp Ser
Ser Ser Cys Val Arg Ala Cys Pro Ser Ser Lys Met275 280 285Glu Val
Glu Glu Asn Gly Ile Lys Met Cys Lys Pro Cys Thr Asp Ile290 295
300Cys Pro Lys Ala Cys Asp Gly Ile Gly Thr Gly Ser Leu Met Ser
Ala305 310 315 320Gln Thr Val Asp Ser Ser Asn Ile Asp Lys Phe Ile
Asn Cys Thr Lys325 330 335Ile Asn Gly Asn Leu Ile Phe Leu Val Thr
Gly Ile His Gly Asp Pro340 345 350Tyr Asn Ala Ile Glu Ala Ile Asp
Pro Glu Lys Leu Asn Val Phe Arg355 360 365Thr Val Arg Glu Ile Thr
Gly Phe Leu Asn Ile Gln Ser Trp Pro Pro370 375 380Asn Met Thr Asp
Phe Ser Val Phe Ser Asn Leu Val Thr Ile Gly Gly385 390 395 400Arg
Val Leu Tyr Ser Gly Leu Ser Leu Leu Ile Leu Lys Gln Gln Gly405 410
415Ile Thr Ser Leu Gln Phe Gln Ser Leu Lys Glu Ile Ser Ala Gly
Asn420 425 430Ile Tyr Ile Thr Asp Asn Ser Asn Leu Cys Tyr Tyr His
Thr Ile Asn435 440 445Trp Thr Thr Leu Phe Ser Thr Ile Asn Gln Arg
Ile Val Ile Arg Asp450 455 460Asn Arg Lys Ala Glu Asn Cys Thr Ala
Glu Gly Met Val Cys Asn His465 470 475 480Leu Cys Ser Ser Asp Gly
Cys Trp Gly Pro Gly Pro Asp Gln Cys Leu485 490 495Ser Cys Arg Arg
Phe Ser Arg Gly Arg Ile Cys Ile Glu Ser Cys Asn500 505 510Leu Tyr
Asp Gly Glu Phe Arg Glu Phe Glu Asn Gly Ser Ile Cys Val515 520
525Glu Cys Asp Pro Gln Cys Glu Lys Met Glu Asp Gly Leu Leu Thr
Cys530 535 540His Gly Pro Gly Pro Asp Asn Cys Thr Lys Cys Ser His
Phe Lys Asp545 550 555 560Gly Pro Asn Cys Val Glu Lys Cys Pro Asp
Gly Leu Gln Gly Ala Asn565 570 575Ser Phe Ile Phe Lys Tyr Ala Asp
Pro Asp Arg Glu Cys His Pro Cys580 585 590His Pro Asn Cys Thr Gln
Gly Cys Asn Gly Pro Thr Ser His Asp Cys595 600 605Ile Tyr Tyr Pro
Trp Thr Gly His Ser Thr Leu Pro Gln His Ala Arg610 615 620Thr Pro
Gly Xaa625
* * * * *