U.S. patent application number 15/359100 was filed with the patent office on 2017-03-16 for compositions and methods for conjugating activatable antibodies.
The applicant listed for this patent is CytomX Therapeutics, Inc.. Invention is credited to Luc Roland Desnoyers, Andrei William Konradi, Tony W. Liang, Henry Bernard Lowman, Shweta Singh.
Application Number | 20170072067 15/359100 |
Document ID | / |
Family ID | 51023194 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170072067 |
Kind Code |
A1 |
Lowman; Henry Bernard ; et
al. |
March 16, 2017 |
COMPOSITIONS AND METHODS FOR CONJUGATING ACTIVATABLE ANTIBODIES
Abstract
The invention relates generally to compositions and methods for
conjugating antibodies and activatable antibodies, and methods of
partially reducing antibodies and/or activatable antibodies prior
to conjugation, e.g., thiol-based conjugation, with an agent, e.g.,
a therapeutic and/or diagnostic agent.
Inventors: |
Lowman; Henry Bernard; (El
Granada, CA) ; Desnoyers; Luc Roland; (San Francisco,
CA) ; Liang; Tony W.; (San Mateo, CA) ;
Konradi; Andrei William; (Burlingame, CA) ; Singh;
Shweta; (Fremont, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CytomX Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
51023194 |
Appl. No.: |
15/359100 |
Filed: |
November 22, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14296207 |
Jun 4, 2014 |
9517276 |
|
|
15359100 |
|
|
|
|
61830913 |
Jun 4, 2013 |
|
|
|
61919935 |
Dec 23, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/18 20130101;
A61P 35/00 20180101; A61K 47/6803 20170801; C07K 16/2863 20130101;
A61K 47/6851 20170801; A61P 43/00 20180101; A61P 29/00 20180101;
A61K 39/3955 20130101; A61K 49/00 20130101; A61P 37/06 20180101;
A61K 2039/505 20130101; C07K 2319/00 20130101; C07K 2317/21
20130101; A61K 47/6889 20170801; C07K 2317/76 20130101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 16/18 20060101 C07K016/18 |
Claims
1. A method of partially reducing and conjugating an agent to an
activatable antibody resulting in selectivity in the placement of
the agent, the method comprising partially reducing at least one
disulfide bond in the activatable antibody with a reducing agent
without disturbing any intrachain disulfide bonds in the
activatable antibody, and conjugating the agent to at least one
thiol, wherein the activatable antibody comprises an antibody or an
antigen binding fragment thereof (AB) that specifically binds to a
target, wherein the target is Jagged 1 or Jagged 2, a masking
moiety (MM) that inhibits the binding of the AB of the activatable
antibody in an uncleaved state to the target, and a cleavable
moiety (CM) coupled to the AB, wherein the CM is a polypeptide that
functions as a substrate for a protease.
2. The method of claim 1, wherein the at least one disulfide bond
is an interchain disulfide bond.
3. The method of claim 1, wherein the at least one disulfide bond
is a disulfide bond between the activatable antibody and a second
molecule.
4. The method of claim 3, wherein the second molecule is cysteine
or glutathione.
5. The method of claim 1, wherein the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM or wherein the
activatable antibody in the uncleaved state comprises a spacer that
is joined directly to the MM and has the structural arrangement
from N-terminus to C-terminus of spacer-MM-CM-AB.
6. The method of claim 1, wherein the reducing agent is TCEP.
7. The method of claim 1, wherein the agent is a toxin or fragment
thereof.
8. The method of claim 7, wherein the agent is a microtubule
inhibitor or a nucleic acid damaging agent.
9. The method of claim 7, wherein the agent is a dolastatin or a
derivative thereof, an auristatin or a derivative thereof, a
maytansinoid or a derivative thereof, a duocarmycin or a derivative
thereof, or a calicheamicin or a derivative thereof.
10. The method of claim 1, wherein the agent is auristatin E or a
derivative thereof.
11. The method of claim 1, wherein the agent is monomethyl
auristatin E (MMAE).
12. The method of claim 1, wherein the agent is monomethyl
auristatin D (MMAD).
13. The method of claim 1, wherein the agent is DM1 or DM4.
14. The method of claim 1, wherein the agent is conjugated to the
AB via a linker.
15. The method of claim 14, wherein the linker is a cleavable
linker.
16. The method of claim 1, wherein the agent is a detectable
moiety.
17. The method of claim 16, wherein the detectable moiety is a
diagnostic agent.
18. The method of claim 1, wherein the antigen binding fragment
thereof is selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single domain heavy
chain antibody, and a single domain light chain antibody.
19. The method of claim 1, wherein the MM has an equilibrium
dissociation constant for binding to the AB that is greater than
the equilibrium dissociation constant of the AB to the target.
20. The method of claim 1, wherein the MM does not interfere or
compete with the AB for binding to the target when the activatable
antibody is in a cleaved state.
21. The method of claim 1, wherein the MM is a polypeptide of no
more than 40 amino acids in length.
22. The method of claim 1, wherein the MM polypeptide sequence is
different from that of the target, and wherein the MM polypeptide
sequence is no more than 50/o identical to any natural binding
partner of the AB.
23. The method of claim 1, wherein the CM is a polypeptide of up to
15 amino acids in length.
24. The method of claim 1, wherein the protease is co-localized
with the target in a tissue, and wherein the protease cleaves the
CM in the activatable antibody when the activatable antibody is
exposed to the protease.
25. The method of claim 1, wherein the activatable antibody
comprises a linking peptide between the MM and the CM, or wherein
the activatable antibody comprises a linking peptide between the CM
and the AB.
26. The method of claim 1, wherein the activatable antibody
comprises a first linking peptide (LP1) and a second linking
peptide (LP2), and wherein the activatable antibody in an uncleaved
state has the structural arrangement from N-terminus to C-terminus
as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
27. The method of claim 26, wherein the two linking peptides need
not be identical to each other.
28. The method of claim 26, wherein each of LP1 and LP2 is a
peptide of about 1 to 20 amino acids in length.
29. The method of claim 26, wherein at least one of LP1 or LP2
comprises an amino acid sequence selected from the group consisting
of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n (SEQ ID NO: 21),
(GGGS).sub.n (SEQ ID NO: 22), GGSG (SEQ ID NO: 23), GGSGG (SEQ ID
NO: 24), GSGSG (SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ
ID NO: 27), and GSSSG (SEQ ID NO: 28), where n is an integer of at
least one.
30. The method of claim 1, wherein the CM is a substrate for an
enzyme selected from the group consisting of those shown in Table
3.
31. The method of claim 1, wherein the CM is a substrate for one or
more enzymes selected from the group consisting of: a urokinase
plasminogen activator (uPA), a legumain, a matriptase, a matrix
metalloprotease (MMP), MMP-9, and MMP-14.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/296,207, filed Jun. 4, 2014, which claims the benefit
of U.S. Provisional Application No. 61/830,913, filed Jun. 4, 2013
and U.S. Provisional Application No. 61/919,935, filed Dec. 23,
2013. The contents of each of which are hereby incorporated by
reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The contents of the text file named
"CYTM023D01USSeqList.txt", which was created on Nov. 22, 2016 and
is 160 KB in size, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The invention relates generally to compositions and methods
for conjugating antibodies and activatable antibodies, and methods
of partially reducing antibodies and/or activatable antibodies
prior to conjugation, e.g., thiol-based conjugation, with an agent,
e.g., a therapeutic and/or diagnostic agent.
BACKGROUND OF THE INVENTION
[0004] Antibody-based therapies have proven effective treatments
for some diseases but in some cases, toxicities due to broad target
expression have limited their therapeutic effectiveness. In
addition, antibody-based therapeutics have exhibited other
limitations such as rapid clearance from the circulation following
administration. Conjugating agents to antibodies has been used to
further advance the use of antibody-based therapies. Molecules such
as toxins, radionuclides and drugs including anti-cancer drugs have
been conjugated to certain antibodies to generate immunotoxins,
radioimmunoconjugates, and/or antibody-drug conjugates (ADCs).
[0005] In the realm of small molecule therapeutics, strategies have
been developed to provide prodrugs of an active chemical entity.
Such prodrugs are administered in a relatively inactive (or
significantly less active) form. Once administered, the prodrug is
metabolized in vivo into the active compound. Such prodrug
strategies can provide for increased selectivity of the drug for
its intended target and for a reduction of adverse effects.
[0006] Accordingly, there is a continued need in the field of
antibody-based therapeutics for antibodies that mimic the desirable
characteristics of the small molecule prodrug, as well as a need
for improved methods of conjugating agents to these antibodies
without negatively impacting their ability to mimic the desirable
characteristics of the small molecule prodrug.
SUMMARY OF THE INVENTION
[0007] The present invention provides conjugates that include an
activatable antibody and methods of making these activatable
antibody conjugates. Also provided are activatable antibodies
having points of conjugation for receiving a drug or label. The
conjugates can be used therapeutically, diagnostically (e.g., in
vitro or in vivo), for in vivo imaging, and for any of a variety of
other therapeutic, diagnostic and/or prophylactic uses.
[0008] Generally, the compositions and methods provided herein
include an activatable antibody that includes an antibody or
antibody fragment (AB) that specifically binds a target, where the
AB is coupled to a masking moiety (MM) that decreases the ability
of the AB to bind its target. In some embodiments, the activatable
antibody further includes a cleavable moiety (CM) that is a
substrate for a protease. The compositions and methods provided
herein enable the attachment of one or more agents to one or more
cysteine residues in the AB without compromising the activity
(e.g., the masking, activating or binding activity) of the
activatable antibody. In some embodiments, the compositions and
methods provided herein enable the attachment of one or more agents
to one or more cysteine residues in the AB without reducing or
otherwise disturbing one or more disulfide bonds within the MM. The
compositions and methods provided herein produce an activatable
antibody that is conjugated to one or more agents, e.g., any of a
variety of therapeutic, diagnostic and/or prophylactic agents, and,
in some embodiments, without any of the agent(s) being conjugated
to the MM of the activatable antibody. The compositions and methods
provided herein produce conjugated activatable antibodies in which
the MM retains the ability to effectively and efficiently mask the
AB of the activatable antibody in an uncleaved state. The
compositions and methods provided herein produce conjugated
activatable antibodies in which the activatable antibody is still
activated, i.e., cleaved, in the presence of a protease that can
cleave the CM.
[0009] The activatable antibodies have at least one point of
conjugation for an agent, but in the methods and compositions
provided herein less than all possible points of conjugation are
available for conjugation to an agent. In some embodiments, the one
or more points of conjugation are sulfur atoms involved in
disulfide bonds. In some embodiments, the one or more points of
conjugation are sulfur atoms involved in interchain disulfide
bonds. In some embodiments, the one or more points of conjugation
are sulfur atoms involved in interchain sulfide bonds, but not
sulfur atoms involved in intrachain disulfide bonds. In some
embodiments, the one or more points of conjugation are sulfur atoms
of cysteine or other amino acid residues containing a sulfur atom.
Such residues may occur naturally in the antibody structure or may
be incorporated into the antibody by site-directed mutagenesis,
chemical conversion, or mis-incorporation of non-natural amino
acids.
[0010] Also provided are methods of preparing a conjugate of an
activatable antibody having one or more interchain disulfide bonds
in the AB and one or more intrachain disulfide bonds in the MM, and
a drug reactive with free thiols is provided. The method generally
includes partially reducing interchain disulfide bonds in the
activatable antibody with a reducing agent, such as, for example,
TCEP; and conjugating the drug reactive with free thiols to the
partially reduced activatable antibody. As used herein, the term
partial reduction refers to situations where an activatable
antibody is contacted with a reducing agent and less than all
disulfide bonds, e.g., less than all possible sites of conjugation
are reduced. In some embodiments, less than 99%, 98%/0, 97%, 96%,
95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%/0, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10% or less than 5% of all possible sites of
conjugation are reduced.
[0011] In some embodiments, a method of reducing and conjugating an
agent, e.g., a drug, to an activatable antibody resulting in
selectivity in the placement of the agent is provided. The method
generally includes partially reducing the activatable antibody with
a reducing agent such that any conjugation sites in the masking
moiety or other non-AB portion of the activatable antibody are not
reduced, and conjugating the agent to interchain thiols in the AB.
The conjugation site(s) are selected so as to allow desired
placement of an agent to allow conjugation to occur at a desired
site. The reducing agent is, for example, TCEP. The reduction
reaction conditions such as, for example, the ratio of reducing
agent to activatable antibody, the length of incubation, the
temperature during the incubation, the pH of the reducing reaction
solution, etc., are determined by identifying the conditions that
produce a conjugated activatable antibody in which the MM retains
the ability to effectively and efficiently mask the AB of the
activatable antibody in an uncleaved state. The ratio of reduction
agent to activatable antibody will vary depending on the
activatable antibody. In some embodiments, the ratio of reducing
agent to activatable antibody will be in a range from about 20:1 to
1:1, from about 10:1 to 1:1, from about 9:1 to 1:1, from about 8:1
to 1:1, from about 7:1 to 1:1, from about 6:1 to 1:1, from about
5:1 to 1:1, from about 4:1 to 1:1, from about 3:1 to 1:1, from
about 2:1 to 1:1, from about 20:1 to 1:1.5, from about 10:1 to
1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5, from about
7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to 1:1.5,
from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about 2:1 to
1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In
some embodiments, the ratio is in a range of from about 5:1 to 1:1.
In some embodiments, the ratio is in a range of from about 5:1 to
1.5:1. In some embodiments, the ratio is in a range of from about
4:1 to 1:1. In some embodiments, the ratio is in a range from about
4:1 to 1.5:1. In some embodiments, the ratio is in a range from
about 8:1 to about 1:1. In some embodiments, the ratio is in a
range of from about 2.5:1 to 1:1.
[0012] In some embodiments, a method of reducing interchain
disulfide bonds in the AB of an activatable antibody and
conjugating an agent, e.g., a thiol-containing agent such as a
drug, to the resulting interchain thiols to selectively locate
agent(s) on the AB is provided. The method generally includes
partially reducing the AB with a reducing agent to form at least
two interchain thiols without forming all possible interchain
thiols in the activatable antibody; and conjugating the agent to
the interchain thiols of the partially reduced AB. For example, the
AB of the activatable antibody is partially reduced for about 1
hour at about 37.degree. C. at a desired ratio of reducing
agent:activatable antibody. In some embodiments, the ratio of
reducing agent to activatable antibody will be in a range from
about 20:1 to 1:1, from about 10:1 to 1:1, from about 9:1 to 1:1,
from about 8:1 to 1:1, from about 7:1 to 1:1, from about 6:1 to
1:1, from about 5:1 to 1:1, from about 4:1 to 1:1, from about 3:1
to 1:1, from about 2:1 to 1:1, from about 20:1 to 1:1.5, from about
10:1 to 1:1.5, from about 9:1 to 1:1.5, from about 8:1 to 1:1.5,
from about 7:1 to 1:1.5, from about 6:1 to 1:1.5, from about 5:1 to
1:1.5, from about 4:1 to 1:1.5, from about 3:1 to 1:1.5, from about
2:1 to 1:1.5, from about 1.5:1 to 1:1.5, or from about 1:1 to
1:1.5. In some embodiments, the ratio is in a range of from about
5:1 to 1:1. In some embodiments, the ratio is in a range of from
about 5:1 to 1.5:1. In some embodiments, the ratio is in a range of
from about 4:1 to 1:1. In some embodiments, the ratio is in a range
from about 4:1 to 1.5:1. In some embodiments, the ratio is in a
range from about 8:1 to about 1:1. In some embodiments, the ratio
is in a range of from about 2.5:1 to 1:1.
[0013] The thiol-containing reagent can be, for example, cysteine
or N-acetyl cysteine. The reducing agent can be, for example, TCEP.
In some embodiments, the reduced activatable antibody can be
purified prior to conjugation, using for example, column
chromatography, dialysis, or diafiltration. In some embodiments,
the reduced antibody is not purified after partial reduction and
prior to conjugation.
[0014] In some embodiments, the activatable antibody includes an
antibody or antigen-binding fragment thereof (AB) that specifically
binds a target, wherein the AB is coupled to a masking moiety (MM),
such that coupling of the MM to the AB decreases the ability of the
antibody or antigen-binding fragment thereof to bind the target. In
some embodiments, the MM is coupled to the AB via a cleavable
moiety (CM) that includes a substrate for a protease, for example,
a protease that is co-localized with the target at a treatment site
in a subject. The activatable antibodies provided herein are stable
in circulation, activated at intended sites of therapy and/or
diagnosis but not in normal, e.g., healthy, tissue, and, when
activated, exhibit binding to the target that is at least
comparable to the corresponding, unmodified antibody.
[0015] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM.
[0016] In some embodiments, the activatable antibody includes an
antibody or antigen-binding fragment thereof (AB) that specifically
binds the target. In some embodiments, the antibody or
immunologically active fragment thereof that binds the target is a
monoclonal antibody, domain antibody, single chain, Fab fragment, a
F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single domain heavy
chain antibody, and a single domain light chain antibody. In some
embodiments, such an antibody or immunologically active fragment
thereof that binds the target is a mouse, chimeric, humanized or
fully human monoclonal antibody. In some embodiments, the antigen
binding fragment thereof is a Fab fragment, a F(ab').sub.2
fragment, a scFv, or a scAb.
[0017] In some embodiments, the antibody or an antigen binding
fragment thereof (AB) specifically binds to a target selected from
those shown Table 1. In some embodiments, the AB specifically binds
to Epidermal Growth Factor Receptor (EGFR). In some embodiments,
the AB specifically binds to Jagged 1 and/or Jagged 2. In some
embodiments, the AB specifically binds to interleukin 6 receptor
(IL-6R).
[0018] In some embodiments, the antibody or an antigen binding
fragment thereof (AB) is or is derived from an antibody selected
from those shown in Table 2.
[0019] In some embodiments, the AB has an equilibrium dissociation
constant of about 100 nM or less for binding to the target.
[0020] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target.
[0021] In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is no more than the equilibrium
dissociation constant of the AB to the target.
[0022] In some embodiments, the MM does not interfere or compete
with the AB of the activatable antibody in a cleaved state for
binding to the target.
[0023] In some embodiments, the MM is a polypeptide of about 2 to
40 amino acids in length, for example, no more than 40 amino acids
long.
[0024] In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 500 identical to any natural binding partner of the
AB.
[0025] In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 25% identical to any natural binding partner of the
AB. In some embodiments, the MM polypeptide sequence is different
from that of the target, and the MM polypeptide sequence is no more
than 10% identical to any natural binding partner of the AB.
[0026] In some embodiments, the coupling of the MM to the AB
decreases the ability of the AB to bind the target such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards the target is at least 20 times greater than the K.sub.d of
the AB when not coupled to the MM towards the target. In some
embodiments, the coupling of the MM to the AB decreases the ability
of the AB to bind the target such that the dissociation constant
(K.sub.d) of the AB when coupled to the MM towards the target is at
least 40 times greater than the K.sub.d of the AB when not coupled
to the MM towards the target. In some embodiments, the coupling of
the MM to the AB decreases the ability of the AB to bind the target
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards the target is at least 50 times greater
than the K.sub.d of the AB when not coupled to the MM towards the
target. In some embodiments, the coupling of the MM to the AB
decreases the ability of the AB to bind the target such that the
K.sub.d of the AB when coupled to the MM towards the target is at
least 100 times greater than the K.sub.d of the AB when not coupled
to the MM towards the target. In some embodiments, the coupling of
the MM to the AB decreases the ability of the AB to bind the target
such that the K.sub.d of the AB when coupled to the MM towards the
target is at least 1000 times greater than the K.sub.d of the AB
when not coupled to the MM towards the target. In some embodiments,
the coupling of the MM to the AB decreases the ability of the AB to
bind the target such that the K.sub.d of the AB when coupled to the
MM towards the target is at least 10,000 times greater than the
K.sub.d of the AB when not coupled to the MM towards the
target.
[0027] In some embodiments, in the presence of the target, the MM
decreases the ability of the AB to bind the target by at least 90%
when the CM is uncleaved, as compared to when the CM is cleaved
when assayed in vitro using a target displacement assay such as,
for example, the assay described in PCT Publication Nos. WO
2009/025846 and WO 2010/081173.
[0028] In some embodiments, the protease is co-localized with the
target in a tissue, and the protease cleaves the CM in the
activatable antibody when the activatable antibody is exposed to
the protease. In some embodiments, the protease is not active or is
significantly less active in tissues that do not significantly
express the target. In some embodiments, the protease is not active
or is significantly less active in healthy, e.g., non-diseased
tissues.
[0029] In some embodiments, the CM is a polypeptide of up to 15
amino acids in length.
[0030] In some embodiments, the CM is a substrate for a protease
selected from the group consisting of those shown in Table 3. In
some embodiments, the CM is a substrate for a protease selected
from the group consisting of uPA (urokinase plasminogen activator),
legumain and MT-SP1 (matriptase). In some embodiments, the protease
comprises uPA. In some embodiments, the protease comprises
legumain. In some embodiments, the protease comprises MT-SP1.
[0031] In some embodiments, the CM is a substrate for at least two
proteases. In some embodiments, each protease is selected from the
group consisting of those shown in Table 3. In some embodiments,
the CM is a substrate for at least two proteases, and one of the
proteases is selected from the group consisting of uPA, legumain
and MT-SP1 and the other protease is selected from the group
consisting of those shown in Table 3. In some embodiments, the CM
is a substrate for at least two proteases selected from the group
consisting of uPA, legumain and MT-SP1.
[0032] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to the target is decreased such that it occurs
with an equilibrium dissociation constant that is at least 20-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, and whereas the AB of the activatable
antibody in the cleaved state binds the target.
[0033] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to the target is decreased such that it occurs
with an equilibrium dissociation constant that is at least 40-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, and whereas the AB of the activatable
antibody in the cleaved state binds the target.
[0034] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to the target is decreased such that it occurs
with an equilibrium dissociation constant that is at least 50-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, and whereas the AB of the activatable
antibody in the cleaved state binds the target.
[0035] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to the target is decreased such that it occurs
with an equilibrium dissociation constant that is at least 100-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, and whereas the AB of the activatable
antibody in the cleaved state binds the target.
[0036] In some embodiments, the CM is positioned in the activatable
antibody such that in the uncleaved state, binding of the
activatable antibody to the target is decreased such that it occurs
with an equilibrium dissociation constant that is at least 200-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, and whereas the AB of the activatable
antibody in the cleaved state binds the target.
[0037] In some embodiments, the activatable antibody includes a
linking peptide between the MM and the CM.
[0038] In some embodiments, the activatable antibody includes a
linking peptide between the CM and the AB.
[0039] In some embodiments, the activatable antibody includes a
first linking peptide (LP1) and a second linking peptide (LP2), and
the activatable antibody in the uncleaved state has the structural
arrangement from N-terminus to C-terminus as follows:
MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In some embodiments, the two
linking peptides need not be identical to each other.
[0040] In some embodiments, each of LP1 and LP2 is a peptide of
about 1 to 20 amino acids in length.
[0041] In some embodiments, at least one of LP1 or LP2 includes an
amino acid sequence selected from the group consisting of
(GS).sub.n, (GGS).sub.n, (GSGGS).sub.n (SEQ ID NO: 21) and
(GGGS).sub.n (SEQ ID NO: 22), where n is an integer of at least
one. In some embodiments, at least one of LP1 or LP2 includes an
amino acid sequence selected from the group consisting of GGSG (SEQ
ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG (SEQ ID NO: 25), GSGGG
(SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and GSSSG (SEQ ID NO:
28).
[0042] In some embodiments, the activatable antibody is exposed to
and cleaved by a protease such that, in the activated or cleaved
state, the activated antibody includes a light chain amino acid
sequence that includes at least a portion of LP2 and/or CM sequence
after the protease has cleaved the CM.
[0043] In some embodiments, the activatable antibody also includes
a signal peptide. In some embodiments, the signal peptide is
conjugated to the activatable antibody via a spacer. In some
embodiments, the spacer is conjugated to the activatable antibody
in the absence of a signal peptide. In some embodiments, the spacer
is joined directly to the MM of the activatable antibody.
[0044] In some embodiments, the activatable antibody in an
uncleaved state comprises a spacer that is joined directly to the
MM and has the structural arrangement from N-terminus to C-terminus
of spacer-MM-CM-AB. In some embodiments, the spacer includes at
least the amino acid sequence QGQSGQ (SEQ ID NO: 11).
[0045] In some embodiments, the AB of the activatable antibody
naturally contains one or more disulfide bonds. In some
embodiments, the AB can be engineered to include one or more
disulfide bonds.
[0046] In some embodiments, the agent conjugated to the activatable
antibody is a therapeutic agent. In some embodiments, the agent
conjugated to the activatable antibody is a diagnostic agent. In
some embodiments, the agent conjugated to the activatable antibody
is a prophylactic agent.
[0047] In some embodiments, the agent is an antineoplastic agent.
In some embodiments, the agent is a toxin or fragment thereof. As
used herein, a fragment of a toxin is a fragment that retains toxic
activity. In some embodiments, the agent is an agent selected from
the group listed in Table 4. In some embodiments, the agent is a
microtubule inhibitor. In some embodiments, the agent is a
dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is
monomethyl auristatin D (MMAD). In some embodiments, the agent is a
maytansinoid or maytansinoid derivative. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a nucleic
acid damaging agent. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof.
[0048] In some embodiments, the agent is conjugated to the AB via a
linker. In some embodiments, the linker is a thiol-containing
linker. In some embodiments, the linker is a cleavable linker. In
some embodiments, the linker is selected from the group consisting
of the linkers shown in Tables 5 and 6.
[0049] In some embodiments, the activatable antibody also includes
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent. In some embodiments, the detectable moiety is a
conjugatable detection reagent. In some embodiments, the detectable
moiety is, for example, a fluorophore, for example, a fluorescein
derivative such as fluorescein isothiocyanate (FITC).
[0050] In some embodiments, the activatable antibody and/or
conjugated activatable antibody is monospecific. In some
embodiments, the activatable antibody and/or conjugated activatable
antibody is multispecific, e.g., by way of non-limiting example,
bispecific or trifunctional. In some embodiments, the activatable
antibody and/or conjugated activatable antibody is formulated as
part of a pro-Bispecific T Cell Engager (BITE) molecule. In some
embodiments, the activatable antibody and/or conjugated activatable
antibody is formulated as part of a pro-Chimeric Antigen Receptor
(CAR) modified T cell or other engineered receptor.
[0051] In some embodiments, the serum half-life of the activatable
antibody is longer than that of the corresponding antibody; e.g.,
the pK of the activatable antibody is longer than that of the
corresponding antibody. In some embodiments, the serum half-life of
the activatable antibody is similar to that of the corresponding
antibody. In some embodiments, the serum half-life of the
activatable antibody is at least 15 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 12 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 11 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 10 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 9 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 8 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 7 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 6 days when administered to an
organism. In some embodiments, the serum half-life of the
conjugated activatable antibody is at least 5 days when
administered to an organism. In some embodiments, the serum
half-life of the conjugated activatable antibody is at least 4 days
when administered to an organism. In some embodiments, the serum
half-life of the conjugated activatable antibody is at least 3 days
when administered to an organism. In some embodiments, the serum
half-life of the conjugated activatable antibody is at least 2 days
when administered to an organism. In some embodiments, the serum
half-life of the conjugated activatable antibody is at least 24
hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
20 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
18 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
16 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
14 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
12 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
10 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
8 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
6 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
4 hours when administered to an organism. In some embodiments, the
serum half-life of the conjugated activatable antibody is at least
3 hours when administered to an organism.
[0052] The invention provides partially reduced activatable
antibodies in which at least one interchain disulfide bond in the
activatable antibody has been reduced with a reducing agent without
disturbing any intrachain disulfide bonds in the activatable
antibody, wherein the activatable antibody includes an antibody or
an antigen binding fragment thereof (AB) that specifically binds to
a target, a masking moiety (MM) that inhibits the binding of the AB
of the activatable antibody in an uncleaved state to the target,
and a cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease. In some
embodiments, one or more intrachain disulfide bond(s) of the
activatable antibody is not disturbed by the reducing agent. In
some embodiments, one or more intrachain disulfide bond(s) of the
MM within the activatable antibody is not disturbed by the reducing
agent. In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP.
[0053] The invention also provides partially reduced activatable
antibodies in which at least one interchain disulfide bond in the
activatable antibody has been reduced with a reducing agent without
disturbing or otherwise compromising the activity and/or efficacy
of the activatable antibody, wherein the activatable antibody
includes an antibody or an antigen binding fragment thereof (AB)
that specifically binds to a target, a masking moiety (MM) that
inhibits the binding of the AB of the activatable antibody in an
uncleaved state to the target, and a cleavable moiety (CM) coupled
to the AB, and the CM is a polypeptide that functions as a
substrate for a protease. The activity and/or efficacy of the
activatable antibody is, by way of nonlimiting example, masking
activity, activation of the activatable antibody, and/or binding
activity of the activated activatable antibody. In some
embodiments, one or more intrachain disulfide bond(s) of the
activatable antibody is not disturbed by the reducing agent. In
some embodiments, one or more intrachain disulfide bond(s) of the
MM within the activatable antibody is not disturbed by the reducing
agent. In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP.
[0054] In some embodiments, the partially reduced activatable
antibody is conjugated to an agent through at least one interchain
thiol. In some embodiments, the agent is selected from the group of
agents listed in Table 4. In some embodiments, the agent is a toxin
or fragment thereof. In some embodiments, the agent is a
microtubule inhibitor. In some embodiments, the agent is a
dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is
monomethyl auristatin D (MMAD). In some embodiments, the agent is a
maytansinoid or a derivative thereof. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a nucleic
acid damaging agent. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is conjugated to the AB via a linker. In some embodiments,
the linker is a cleavable linker. In some embodiments, the agent is
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0055] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50%/o identical to any natural binding partner of
the AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0056] The invention also provides partially reduced activatable
antibodies in which at least one disulfide bond between the
activatable antibody and a second molecule has been reduced with a
reducing agent without disturbing any intrachain disulfide bonds in
the activatable antibody, wherein the activatable antibody includes
an antibody or an antigen binding fragment thereof (AB) that
specifically binds to a target, a masking moiety (MM) that inhibits
the binding of the AB of the activatable antibody in an uncleaved
state to the target, and a cleavable moiety (CM) coupled to the AB,
and the CM is a polypeptide that functions as a substrate for a
protease. In some embodiments, the second molecule is cysteine. In
some embodiments, the second molecule is glutathione.
[0057] The invention also provides partially reduced activatable
antibodies in which at least one disulfide bond between the
activatable antibody and a second molecule has been reduced with a
reducing agent without disturbing or otherwise compromising the
activity and/or efficacy of the activatable antibody, wherein the
activatable antibody includes an antibody or an antigen binding
fragment thereof (AB) that specifically binds to a target, a
masking moiety (MM) that inhibits the binding of the AB of the
activatable antibody in an uncleaved state to the target, and a
cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease. The
activity and/or efficacy of the activatable antibody is, by way of
nonlimiting example, masking activity, activation of the
activatable antibody, and/or binding activity of the activated
activatable antibody. In some embodiments, the second molecule is
cysteine. In some embodiments, the second molecule is
glutathione.
[0058] In some embodiments, the partial reduction method does not
disturb one or more intrachain disulfide bonds of the activatable
antibody. In some embodiments, the method does not disturb one or
more intrachain disulfide bonds of the MM within the activatable
antibody.
[0059] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP.
[0060] In some embodiments, the partially reduced activatable
antibody is conjugated to an agent through at least one thiol. In
some embodiments, the agent is selected from the group of agents
listed in Table 4. In some embodiments, the agent is a toxin or
fragment thereof. In some embodiments, the agent is a microtubule
inhibitor. In some embodiments, the agent is a dolastatin. In some
embodiments, the agent is an auristatin or derivative thereof. In
some embodiments, the agent is auristatin E or a derivative
thereof. In some embodiments, the agent is monomethyl auristatin E
(MMAE). In some embodiments, the agent is monomethyl auristatin D
(MMAD). In some embodiments, the agent is a maytansinoid or a
derivative thereof. In some embodiments, the agent is DM1 or DM4.
In some embodiments, the agent is a nucleic acid damaging agent. In
some embodiments, the agent is a duocarmycin or derivative thereof.
In some embodiments, the agent is a calicheamicin or derivative
thereof. In some embodiments, the agent is conjugated to the AB via
a linker. In some embodiments, the linker is a cleavable linker. In
some embodiments, the agent is a detectable moiety. In some
embodiments, the detectable moiety is a diagnostic agent.
[0061] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0062] The invention provides methods of selectively conjugating an
agent to an activatable antibody. For example, the invention
provides a method of partially reducing and conjugating an agent to
an activatable antibody resulting in selectivity in the placement
of the agent by partially reducing at least one interchain
disulfide bond in the activatable antibody with a reducing agent
without disturbing any intrachain disulfide bonds in the
activatable antibody, and conjugating the agent to at least one
interchain thiol, wherein the activatable antibody includes an
antibody or an antigen binding fragment thereof (AB) that
specifically binds to a target, a masking moiety (MM) that inhibits
the binding of the AB of the activatable antibody in an uncleaved
state to the target, and a cleavable moiety (CM) coupled to the AB,
and the CM is a polypeptide that functions as a substrate for a
protease.
[0063] The invention also provides a method of partially reducing
and conjugating an agent to an activatable antibody resulting in
selectivity in the placement of the agent by partially reducing at
least one interchain disulfide bond in the activatable antibody
with a reducing agent without disturbing or otherwise compromising
the activity and/or efficacy of the activatable antibody, wherein
the activatable antibody includes an antibody or an antigen binding
fragment thereof (AB) that specifically binds to a target, a
masking moiety (MM) that inhibits the binding of the AB of the
activatable antibody in an uncleaved state to the target, and a
cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease. The
activity and/or efficacy of the activatable antibody is, by way of
nonlimiting example, masking activity, activation of the
activatable antibody, and/or binding activity of the activated
activatable antibody.
[0064] In some embodiments, the method does not disturb one or more
intrachain disulfide bonds of the activatable antibody. In some
embodiments, the method does not disturb one or more intrachain
disulfide bonds of the MM within the activatable antibody.
[0065] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP. In some embodiments, the agent is selected
from the group of agents listed in Table 4. In some embodiments,
the agent is a toxin or fragment thereof. In some embodiments, the
agent is a microtubule inhibitor. In some embodiments, the agent is
a dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is
monomethyl auristatin D (MMAD). In some embodiments, the agent is a
maytansinoid or a derivative thereof. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a nucleic
acid damaging agent. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is conjugated to the AB via a linker. In some embodiments,
the linker is a cleavable linker. In some embodiments, the agent is
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0066] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0067] The invention provides a method of partially reducing and
conjugating an agent to an activatable antibody resulting in
selectivity in the placement of the agent by partially reducing at
least one disulfide bond between the activatable antibody and a
second molecule with a reducing agent without disturbing any
intrachain disulfide bonds in the activatable antibody, and
conjugating the agent to at least one thiol, wherein the
activatable antibody includes an antibody or an antigen binding
fragment thereof (AB) that specifically binds to a target, a
masking moiety (MM) that inhibits the binding of the AB of the
activatable antibody in an uncleaved state to the target, and a
cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease. In some
embodiments, the second molecule is cysteine. In some embodiments,
the second molecule is glutathione.
[0068] The invention also provides a method of partially reducing
and conjugating an agent to an activatable antibody resulting in
selectivity in the placement of the agent by partially reducing at
least one disulfide bond between the activatable antibody and a
second molecule with a reducing agent without disturbing or
otherwise compromising the activity and/or efficacy of the
activatable antibody, wherein the activatable antibody includes an
antibody or an antigen binding fragment thereof (AB) that
specifically binds to a target, a masking moiety (MM) that inhibits
the binding of the AB of the activatable antibody in an uncleaved
state to the target, and a cleavable moiety (CM) coupled to the AB,
and the CM is a polypeptide that functions as a substrate for a
protease. The activity and/or efficacy of the activatable antibody
is, by way of nonlimiting example, masking activity, activation of
the activatable antibody, and/or binding activity of the activated
activatable antibody. In some embodiments, the second molecule is
cysteine. In some embodiments, the second molecule is
glutathione.
[0069] In some embodiments, the method does not disturb one or more
intrachain disulfide bonds of the activatable antibody. In some
embodiments, the method does not disturb one or more intrachain
disulfide bonds of the MM within the activatable antibody.
[0070] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP. In some embodiments, the agent is selected
from the group of agents listed in Table 4. In some embodiments,
the agent is a toxin or fragment thereof. In some embodiments, the
agent is a microtubule inhibitor. In some embodiments, the agent is
a dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is
monomethyl auristatin D (MMAD). In some embodiments, the agent is a
maytansinoid or a derivative thereof. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a nucleic
acid damaging agent. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is conjugated to the AB via a linker. In some embodiments,
the linker is a cleavable linker. In some embodiments, the agent is
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0071] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0072] The invention also provides a method of partially reducing
an activatable antibody resulting in selectivity in the placement
of one or more potential conjugation sites in the activatable
antibody by partially reducing at least one interchain disulfide
bond in the activatable antibody with a reducing agent without
disturbing any intrachain disulfide bonds in the activatable
antibody, wherein the activatable antibody includes an antibody or
an antigen binding fragment thereof (AB) that specifically binds to
a target, a masking moiety (MM) that inhibits the binding of the AB
of the activatable antibody in an uncleaved state to the target,
and a cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease.
[0073] The invention also provides a method of partially reducing
an activatable antibody resulting in selectivity in the placement
of one or more potential conjugation sites in the activatable
antibody by partially reducing at least one interchain disulfide
bond in the activatable antibody with a reducing agent without
disturbing or otherwise compromising the activity and/or efficacy
of the activatable antibody, wherein the activatable antibody
includes an antibody or an antigen binding fragment thereof (AB)
that specifically binds to a target, a masking moiety (MM) that
inhibits the binding of the AB of the activatable antibody in an
uncleaved state to the target, and a cleavable moiety (CM) coupled
to the AB, and the CM is a polypeptide that functions as a
substrate for a protease. The activity and/or efficacy of the
activatable antibody is, by way of nonlimiting example, masking
activity, activation of the activatable antibody, and/or binding
activity of the activated activatable antibody.
[0074] In some embodiments, the method does not disturb one or more
intrachain disulfide bonds of the activatable antibody. In some
embodiments, the method does not disturb one or more intrachain
disulfide bonds of the MM within the activatable antibody.
[0075] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP.
[0076] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0077] The invention also provides a method of partially reducing
an activatable antibody resulting in selectivity in the placement
of one or more potential conjugation sites in the activatable
antibody by partially reducing at least one disulfide bond between
the activatable antibody and a second molecule with a reducing
agent without disturbing any intrachain disulfide bonds in the
activatable antibody, wherein the activatable antibody includes an
antibody or an antigen binding fragment thereof (AB) that
specifically binds to a target, a masking moiety (MM) that inhibits
the binding of the AB of the activatable antibody in an uncleaved
state to the target, and a cleavable moiety (CM) coupled to the AB,
and the CM is a polypeptide that functions as a substrate for a
protease. In some embodiments, the second molecule is cysteine. In
some embodiments, the second molecule is glutathione.
[0078] The invention also provides a method of partially reducing
an activatable antibody resulting in selectivity in the placement
of one or more potential conjugation sites in the activatable
antibody by partially reducing at least one disulfide bond between
the activatable antibody and a second molecule with a reducing
agent without disturbing or otherwise compromising the activity
and/or efficacy of the activatable antibody, wherein the
activatable antibody includes an antibody or an antigen binding
fragment thereof (AB) that specifically binds to a target, a
masking moiety (MM) that inhibits the binding of the AB of the
activatable antibody in an uncleaved state to the target, and a
cleavable moiety (CM) coupled to the AB, and the CM is a
polypeptide that functions as a substrate for a protease. The
activity and/or efficacy of the activatable antibody is, by way of
nonlimiting example, masking activity, activation of the
activatable antibody, and/or binding activity of the activated
activatable antibody. In some embodiments, the second molecule is
cysteine. In some embodiments, the second molecule is
glutathione.
[0079] In some embodiments, the method does not disturb one or more
intrachain disulfide bonds of the activatable antibody. In some
embodiments, the method does not disturb one or more intrachain
disulfide bonds of the MM within the activatable antibody.
[0080] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
reducing agent is TCEP. In some embodiments, the agent is selected
from the group of agents listed in Table 4. In some embodiments,
the agent is a toxin or fragment thereof. In some embodiments, the
agent is a microtubule inhibitor. In some embodiments, the agent is
a dolastatin. In some embodiments, the agent is an auristatin or
derivative thereof. In some embodiments, the agent is auristatin E
or a derivative thereof. In some embodiments, the agent is
monomethyl auristatin E (MMAE). In some embodiments, the agent is
monomethyl auristatin D (MMAD). In some embodiments, the agent is a
maytansinoid or a derivative thereof. In some embodiments, the
agent is DM1 or DM4. In some embodiments, the agent is a nucleic
acid damaging agent. In some embodiments, the agent is a
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is conjugated to the AB via a linker. In some embodiments,
the linker is a cleavable linker. In some embodiments, the agent is
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0081] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the AB is or is
derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n(SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0082] The invention also provides conjugated activatable
antibodies that include an activatable antibody linked to
monomethyl auristatin D (MMAD) payload, wherein the activatable
antibody includes an antibody or an antigen binding fragment
thereof (AB) that specifically binds to a target, a masking moiety
(MM) that inhibits the binding of the AB of the activatable
antibody in an uncleaved state to the target, and a cleavable
moiety (CM) coupled to the AB, and the CM is a polypeptide that
functions as a substrate for a protease.
[0083] In some embodiments, the MMAD-conjugated activatable
antibody can be conjugated using any of several methods for
attaching agents to ABs: (a) attachment to the carbohydrate
moieties of the AB, or (b) attachment to sulfhydryl groups of the
AB, or (c) attachment to amino groups of the AB, or (d) attachment
to carboxylate groups of the AB.
[0084] In some embodiments, the MMAD payload is conjugated to the
AB via a linker. In some embodiments, the MMAD payload is
conjugated to a cysteine in the AB via a linker. In some
embodiments, the MMAD payload is conjugated to a lysine in the AB
via a linker. In some embodiments, the MMAD payload is conjugated
to another residue of the AB via a linker, such as those residues
disclosed herein. In some embodiments, the linker is a
thiol-containing linker. In some embodiments, the linker is a
cleavable linker. In some embodiments, the linker is a
non-cleavable linker. In some embodiments, the linker is selected
from the group consisting of the linkers shown in Tables 5 and 6.
In some embodiments, the activatable antibody and the MMAD payload
are linked via a maleimide caproyl-valine-citrulline linker. In
some embodiments, the activatable antibody and the MMAD payload are
linked via a maleimide PEG-valine-citrulline linker. In some
embodiments, the activatable antibody and the MMAD payload are
linked via a maleimide
caproyl-valine-citrulline-para-aminobenzyloxycarbonyl linker. In
some embodiments, the activatable antibody and the MMAD payload are
linked via a maleimide
PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some
embodiments, the MMAD payload is conjugated to the AB using the
partial reduction and conjugation technology disclosed herein.
[0085] In some embodiments, the target is selected from the group
of targets listed in Table 1. In some embodiments, the target is
EGFR. In some embodiments, the target is a Jagged protein, e.g.,
Jagged 1 and/or Jagged 2. In some embodiments, the target is
interleukin 6 receptor (IL-6R). In some embodiments, the AB is or
is derived from an antibody selected from the group of antibodies
listed in Table 2. In some embodiments, the antigen binding
fragment thereof is selected from the group consisting of a Fab
fragment, a F(ab').sub.2 fragment, a scFv, a scAb, a dAb, a single
domain heavy chain antibody, and a single domain light chain
antibody. In some embodiments, the AB has an equilibrium
dissociation constant of about 100 nM or less for binding to the
target. In some embodiments, the MM has an equilibrium dissociation
constant for binding to the AB that is greater than the equilibrium
dissociation constant of the AB to the target. In some embodiments,
the MM does not interfere or compete with the AB of the activatable
antibody in a cleaved state for binding to the target. In some
embodiments, the MM is a polypeptide of no more than 40 amino acids
in length. In some embodiments, the MM polypeptide sequence is
different from that of the target, and the MM polypeptide sequence
is no more than 50% identical to any natural binding partner of the
AB. In some embodiments, the MM does not include more than 25%
amino acid sequence identity to the target. In some embodiments,
the MM does not include more than 10% amino acid sequence identity
to the target. In some embodiments, the CM is a polypeptide of up
to 15 amino acids in length. In some embodiments, the protease is
co-localized with the target in a tissue, and the protease cleaves
the CM in the activatable antibody when the activatable antibody is
exposed to the protease. In some embodiments, the activatable
antibody includes a linking peptide between the MM and the CM. In
some embodiments, the activatable antibody includes a linking
peptide between the CM and the AB. In some embodiments, the
activatable antibody includes a first linking peptide (LP1) and a
second linking peptide (LP2), and the activatable antibody in an
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, each of LP1 and LP2 is a peptide
of about 1 to 20 amino acids in length. In some embodiments, at
least one of LP1 or LP2 includes an amino acid sequence selected
from the group consisting of (GS).sub.n, (GGS).sub.n, (GSGGS).sub.n
(SEQ ID NO: 21) and (GGGS).sub.n (SEQ ID NO: 22), where n is an
integer of at least one. In some embodiments, at least one of LP1
or LP2 includes an amino acid sequence selected from the group
consisting of GGSG (SEQ ID NO: 23), GGSGG (SEQ ID NO: 24), GSGSG
(SEQ ID NO: 25), GSGGG (SEQ ID NO: 26), GGGSG (SEQ ID NO: 27), and
GSSSG (SEQ ID NO: 28). In some embodiments, the CM is a substrate
for an enzyme selected from the group consisting of those shown in
Table 3. In some embodiments, the activatable antibody in an
uncleaved state includes a spacer, and the spacer is joined
directly to the MM and has the structural arrangement from
N-terminus to C-terminus of spacer-MM-CM-AB.
[0086] The invention provides methods of treating, preventing
and/or delaying the onset or progression of, or alleviating a
symptom of an indication, e.g., disease or disorder, associated
with expression and/or activity of the target in a subject using a
conjugated activatable antibody that in an activated state binds
the target, particularly a conjugated activatable antibody that
binds and neutralizes or otherwise inhibits at least one biological
activity of the target. Suitable conjugated activatable antibodies
for use in any of the methods and kits of the invention include any
of the conjugated activatable antibodies described herein,
including any partially conjugated activatable antibodies and/or
partially reduced activatable antibodies described herein.
[0087] In some embodiments, the invention provides methods of
treating, preventing and/or delaying the onset or progression of,
or alleviating a symptom of an indication, e.g., disease or
disorder, associated with a detectable level of expression and/or
activity of the target in a subject using a conjugated activatable
antibody that in an activated state binds the target, particularly
a conjugated activatable antibody that binds and neutralizes or
otherwise inhibits at least one biological activity of the target.
In some embodiments, the conjugated activatable antibody in an
activated state binds the target and is internalized. In some
embodiments, the detectable level of expression and/or activity of
the target is found in at least one intended site of therapy and/or
diagnosis. In some embodiments, the detectable level of expression
and/or activity of the target is found in normal, e.g., healthy,
tissue, and the conjugated activatable antibody is activated at the
intended site(s) of therapy and/or diagnosis but not in the normal,
e.g., healthy, tissue. The conjugated activatable antibody is
activated, for example, by a protease that is co-localized with the
target at the intended site(s) of therapy and/or diagnosis. In some
embodiments, the detectable level of expression and/or activity of
the target is found in at least one intended site of therapy and/or
diagnosis and in normal, e.g., healthy, tissue, and the conjugated
activatable antibody is activated at the intended site(s) of
therapy and/or diagnosis but not in the normal, e.g., healthy,
tissue. The conjugated activatable antibody is activated, for
example, by a protease that is co-localized with the target at the
intended site(s) of therapy and/or diagnosis.
[0088] In some embodiments, the indication, e.g., disease or
disorder, associated with expression and/or activity of the target
is a cancer. In some embodiments, the indication, e.g., disease or
disorder, associated with expression and/or activity of the target
is an inflammatory disorder and/or an autoimmune disease.
[0089] The invention also provides methods of inhibiting
angiogenesis in a subject by administering a therapeutically
effective amount of a conjugated activatable antibody described
herein to a subject in need thereof.
[0090] The conjugated activatable antibody can be administered at
any stage of the disease. In some embodiments, a conjugated
activatable antibody can be administered to a patient suffering
cancer of any stage, from early to metastatic. In some embodiments,
a conjugated activatable antibody can be administered to a patient
suffering from an inflammatory disorder and/or autoimmune disease
of any stage, from early onset to an advanced stage. It is to be
understood that the terms subject and patient are used
interchangeably herein.
[0091] The conjugated activatable antibodies are also useful in
other therapeutic indications and treatment regimens. For example,
the conjugated activatable antibodies of the embodiments provided
herein can be used in a treatment regimen that includes neoadjuvant
therapy.
[0092] In some embodiments, a conjugated activatable antibody is
administered in combination with one or more additional agents such
as, by way of non-limiting example, a chemotherapeutic agent, such
as an alkylating agent, an anti-metabolite, an anti-microtubule
agent, a topoisomerase inhibitor, a cytotoxic antibiotic, and any
other nucleic acid damaging agent. In some embodiments, the
additional agent is a taxane, such as paclitaxel (e.g.,
Abraxane.RTM.). In some embodiments, the additional agent is an
anti-metabolite, such as gemcitabine. In some embodiments, the
additional agent is an alkylating agent, such as platinum-based
chemotherapy, such as carboplatin or cisplatin. In some
embodiments, the additional agent is a targeted agent, such as a
kinase inhibitor, e.g., sorafenib or erlotinib. In some
embodiments, the additional agent is a targeted agent, such as
another antibody, e.g., a monoclonal antibody (e.g., bevacizumab),
a bispecific antibody, or a multispecific antibody. In some
embodiments, the additional agent is a proteosome inhibitor, such
as bortezomib or carfilzomib. In some embodiments, the additional
agent is an immune modulating agent, such as lenolidominde or IL-2.
In some embodiments, the additional agent is radiation. In some
embodiments, the additional agent is an agent considered standard
of care by those skilled in the art. In some embodiments, the
additional agent is a chemotherapeutic agent well known to those
skilled in the art. In some embodiments, the conjugated activatable
antibody and the additional agent(s) are formulated in a single
composition. In some embodiments, the conjugated activatable
antibody and the additional agent(s) are administered as two or
more separate compositions. In some embodiments, the conjugated
activatable antibody and the additional agent(s) are administered
simultaneously. In some embodiments, the conjugated activatable
antibody and the additional agent(s) are administered
sequentially.
[0093] In some embodiments, the subject is a mammal. In some
embodiments, the subject is a human. In some embodiments, the
subject is a non-human mammal, such as a non-human primate,
companion animal (e.g., cat, dog, horse), farm animal, work animal,
or zoo animal. In some embodiments, the subject is a rodent. In
some embodiments, the subject is a human. In some embodiments, the
subject is a companion animal. In some embodiments, the subject is
an animal in the care of a veterinarian.
[0094] The conjugated activatable antibody and therapeutic
formulations thereof are administered to a subject suffering from
or susceptible to a disease or disorder associated with expression
and/or activity of the target. A subject suffering from or
susceptible to a disease or disorder associated with expression
and/or activity of the target is identified using any of a variety
of methods known in the art. For example, subjects suffering from
cancer or other neoplastic condition are identified using any of a
variety of clinical and/or laboratory tests such as, physical
examination and blood, urine and stool analysis to evaluate health
status.
[0095] Administration of a conjugated activatable antibody to a
patient suffering from a disease or disorder associated with target
expression and/or activity is considered successful if any of a
variety of laboratory or clinical objectives is achieved. For
example, administration of a conjugated activatable antibody to a
patient suffering from a disease or disorder associated with target
expression and/or activity is considered successful if one or more
of the symptoms associated with the disease or disorder is
alleviated, reduced, inhibited or does not progress to a further,
i.e., worse, state. Administration of a conjugated activatable
antibody to a patient suffering from a disease or disorder
associated with target expression and/or activity is considered
successful if the disease enters remission or does not progress to
a further, i.e., worse, state.
[0096] The invention also provides methods of using conjugated
activatable antibodies that bind the target in a variety of
diagnostic and/or prophylactic indications, as well as kits for use
in these methods. In some embodiments of these methods and/or kits,
the conjugated activatable antibody includes a detectable label. In
some embodiments of these methods and/or kits, the detectable label
includes an imaging agent, a contrasting agent, an enzyme, a
fluorescent label, a chromophore, a dye, one or more metal ions, or
a ligand-based label. In some embodiments of these methods and/or
kits, the imaging agent comprises a radioisotope. In some
embodiments of these methods, the radioisotope is indium or
technetium. In some embodiments of these methods, the radioisotope
is or is derived from iodine. In some embodiments of these methods,
the radioisotope is .sup.125I or .sup.133I. In some embodiments of
these methods and/or kits, the contrasting agent comprises iodine,
gadolinium or iron oxide. In some embodiments of these methods
and/or kits, the enzyme comprises horseradish peroxidase, alkaline
phosphatase, or .beta.-galactosidase. In some embodiments of these
methods and/or kits, the fluorescent label comprises yellow
fluorescent protein (YFP), cyan fluorescent protein (CFP), green
fluorescent protein (GFP), modified red fluorescent protein (mRFP),
red fluorescent protein tdimer2 (RFP tdimer2), HCRED, or a europium
derivative. In some embodiments of these methods and/or kits, the
luminescent label comprises an N-methylacrydium derivative. In some
embodiments of these methods and/or kits, the label comprises an
Alexa Fluor.RTM. label, such as Alex Fluor.RTM. 680 or Alexa
Fluor.RTM. 750. In some embodiments of these methods and/or kits,
the ligand-based label comprises biotin, avidin, streptavidin or
one or more haptens.
[0097] In some embodiments of these methods and/or kits, the
subject is a mammal. In some embodiments of these methods and/or
kits, the subject is a human. In some embodiments, the subject is a
non-human mammal, such as a non-human primate, companion animal
(e.g., cat, dog, horse), farm animal, work animal, or zoo animal.
In some embodiments, the subject is a rodent. In some embodiments,
the subject is a human. In some embodiments, the subject is a
companion animal. In some embodiments, the subject is an animal in
the care of a veterinarian.
[0098] The invention also provides methods of using the conjugated
activatable antibodies (i.e., activatable antibody conjugates) in a
variety of diagnostic and/or prophylactic indications. For example,
the invention provides methods of detecting presence or absence of
a cleaving agent and a target of interest in a subject or a sample
by (i) contacting a subject or sample with a conjugated activatable
antibody and (ii) measuring a level of conjugated activatable
antibody in the subject or sample, wherein a detectable level of
activated conjugated activatable antibody in the subject or sample
indicates that the cleaving agent and the target are present in the
subject or sample and wherein no detectable level of activated
conjugated activatable antibody in the subject or sample indicates
that the cleaving agent, the target or both the cleaving agent and
the target are absent in the subject or sample. In some embodiments
an unconjugated activatable antibody corresponding to the
activatable antibody conjugated in the conjugated activatable
antibody is used to contact the subject or sample.
[0099] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or sample with a conjugated activatable
antibody in the presence of the target, and (ii) measuring a level
of activated conjugated activatable antibody in the subject or
sample, wherein a detectable level of activated conjugated
activatable antibody in the subject or sample indicates that the
cleaving agent is present in the subject or sample and wherein no
detectable level of conjugated activatable antibody in the subject
or sample indicates that the cleaving agent is absent in the
subject or sample. In some embodiments an unconjugated activatable
antibody corresponding to the activatable antibody conjugated in
the conjugated activatable antibody is used to contact the subject
or sample.
[0100] The invention also provides methods of detecting presence or
absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or sample with a conjugated activatable
antibody; and (ii) measuring a level of detectable label in the
subject or sample, wherein a detectable level of the detectable
label in the subject or sample indicates that the cleaving agent is
absent in the subject or sample and wherein no detectable level of
the detectable label in the subject or sample indicates that the
cleaving agent is present in the subject or sample. In some
embodiments an unconjugated activatable antibody corresponding to
the activatable antibody conjugated in the conjugated activatable
antibody is used to contact the subject or sample.
[0101] In some embodiments of these methods, the conjugated
activatable antibody or corresponding unconjugated activatable
antibody includes a detectable label selected from the group
consisting of an imaging agent, a contrasting agent, an enzyme, a
fluorescent label, a chromophore, a dye, one or more metal ions,
and a ligand-based label. In some embodiments of these methods, the
imaging agent comprises a radioisotope. In some embodiments of
these methods, the radioisotope is indium or technetium. In some
embodiments of these methods, the contrasting agent comprises
iodine, gadolinium or iron oxide. In some embodiments of these
methods, the enzyme comprises horseradish peroxidase, alkaline
phosphatase, or 3-galactosidase. In some embodiments of these
methods, the fluorescent label comprises yellow fluorescent protein
(YFP), cyan fluorescent protein (CFP), green fluorescent protein
(GFP), modified red fluorescent protein (mRFP), red fluorescent
protein tdimer2 (RFP tdimer2), HCRED, or a europium derivative. In
some embodiments of these methods, the luminescent label comprises
an N-methylacrydium derivative. In some embodiments of these
methods, the label comprises an Alexa Fluor.RTM. label, such as
Alex Fluor.RTM. 680 or Alexa Fluor.RTM. 750. In some embodiments of
these methods, the ligand-based label comprises biotin, avidin,
streptavidin or one or more haptens.
[0102] In some embodiments of these methods, the subject is a
mammal. In some embodiments of these methods, the subject is a
human. In some embodiments, the subject is a non-human mammal, such
as a non-human primate, companion animal (e.g., cat, dog, horse),
farm animal, work animal, or zoo animal. In some embodiments, the
subject is a rodent. In some embodiments, the subject is a human.
In some embodiments, the subject is a companion animal. In some
embodiments, the subject is an animal in the care of a
veterinarian.
[0103] In some embodiments of these methods, the method is an in
vivo method. In some embodiments of these methods, the method is an
in situ method. In some embodiments of these methods, the method is
an ex vivo method. In some embodiments of these methods, the method
is an in vitro method.
[0104] In some embodiments of the methods, the method is used to
identify or otherwise refine a patient population suitable for
treatment with a conjugated activatable antibody of the disclosure.
For example, patients that test positive for both the target and a
protease that cleaves the substrate in the cleavable moiety (CM) of
the conjugated activatable antibody being tested in these methods
are identified as suitable candidates for treatment with such a
conjugated activatable antibody comprising such a CM. Likewise,
patients that test negative for either or both of the target and
the protease that cleaves the substrate in the CM in the conjugated
activatable antibody or corresponding unconjugated activatable
antibody being tested using these methods might be identified as
suitable candidates for another form of therapy. In some
embodiments, such patients can be tested with other activatable
antibodies and/or conjugated activatable antibodies until a
suitable conjugated activatable antibody for treatment is
identified (e.g., a conjugated activatable antibody comprising a CM
that is cleaved by the patient at the site of disease).
[0105] In some embodiments of the methods, the method is used to
identify or otherwise refine a patient population suitable for
treatment with a conjugated activatable antibody of the disclosure
followed by treatment by administering that conjugated activatable
antibody to a subject in need thereof. For example, patients that
test positive for both the target and a protease that cleaves the
substrate in the cleavable moiety (CM) of the conjugated
activatable antibody or corresponding unconjugated activatable
antibody being tested in these methods are identified as suitable
candidates for treatment with such a conjugated activatable
antibody comprising such a CM, and the patient is then administered
a therapeutically effective amount of the conjugated activatable
antibody. Likewise, patients that test negative for either or both
of the target and the protease that cleaves the substrate in the CM
in the conjugated activatable antibody or corresponding
unconjugated activatable antibody being tested using these methods
might be identified as suitable candidates for another form of
therapy. In some embodiments, such patients can be tested with
other activatable antibodies and/or conjugated activatable
antibodies until a conjugated activatable antibody for treatment is
identified (e.g., a conjugated activatable antibody comprising a CM
that is cleaved by the patient at the site of disease). In some
embodiments, the patient is then administered a therapeutically
effective amount of the conjugated activatable antibody for which
the patient tested positive.
[0106] The invention also provides conjugated activatable
antibodies that in an activated state bind a target, wherein the
conjugated activatable antibody includes an antibody or an antigen
binding fragment thereof (AB) that specifically binds to the
target, wherein the AB is conjugated to monomethyl auristatin D
(MMAD); a masking moiety (MM) that inhibits the binding of the AB
to the target when the activatable antibody is in an uncleaved
state; and a cleavable moiety (CM) coupled to the AB, wherein the
CM is a polypeptide that functions as a substrate for a
protease.
[0107] In some embodiments, the activatable antibody in the
uncleaved state has the structural arrangement from N-terminus to
C-terminus as follows: MM-CM-AB or AB-CM-MM. In some embodiments,
the activatable antibody comprises a linking peptide between the MM
and the CM. In some embodiments, the activatable antibody comprises
a linking peptide between the CM and the AB. In some embodiments,
the activatable antibody comprises a first linking peptide (LP1)
and a second linking peptide (LP2), and wherein the activatable
antibody in the uncleaved state has the structural arrangement from
N-terminus to C-terminus as follows: MM-LP1-CM-LP2-AB or
AB-LP2-CM-LP1-MM. In some embodiments, the two linking peptides
need not be identical to each other. In some embodiments, each of
LP1 and LP2 is a peptide of about 1 to 20 amino acids in
length.
[0108] In some embodiments, he MM has an equilibrium dissociation
constant for binding to the AB which is greater than the
equilibrium dissociation constant of the AB to the target. In some
embodiments, the MM does not interfere or compete with the AB for
binding to the target when the activatable antibody is in a cleaved
state. In some embodiments, the MM is a polypeptide of about no
more than 40 amino acids in length. In some embodiments, the MM
polypeptide sequence is different from that of the target and
wherein the MM polypeptide sequence is no more than 50% identical
to any natural binding partner of the AB.
[0109] In some embodiments, the protease is co-localized with the
target in a tissue, and wherein the protease cleaves the CM in the
activatable antibody when the activatable antibody is exposed to
the protease. In some embodiments, the CM is a polypeptide of up to
15 amino acids in length. In some embodiments, the CM is a
substrate for an enzyme selected from the group consisting of those
shown in Table 3.
[0110] In some embodiments, the antigen binding fragment thereof is
selected from the group consisting of a Fab fragment, a
F(ab').sub.2 fragment, a scFv, a scab, a dAb, a single domain heavy
chain antibody, and a single domain light chain antibody. In some
embodiments, the target for the AB is selected from the group
consisting of the targets listed in Table 1. In some embodiments,
the AB is or is derived from an antibody listed in Table 2.
[0111] In some embodiments, the MMAD is conjugated to the AB via a
linker. In some embodiments, linker is a cleavable linker. In some
embodiments, the linker is a non-cleavable linker. In some
embodiments, the linker is selected from the group consisting of
the linkers shown in Tables 5 and 6. In some embodiments, the
activatable antibody and the MMAD payload are linked via a
maleimide caproyl-valine-citrulline linker. In some embodiments,
the activatable antibody and the MMAD payload are linked via a
maleimide PEG-valine-citrulline linker. In some embodiments, the
activatable antibody and the MMAD payload are linked via a
maleimide caproyl-valine-citrulline-para-aminobenzyloxycarbonyl
linker. In some embodiments, the activatable antibody and the MMAD
payload are linked via a maleimide
PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker. In some
embodiments, the MMAD payload is conjugated to the AB using the
partial reduction and conjugation technology disclosed herein.
[0112] Pharmaceutical compositions according to the invention can
include a conjugated antibody of the invention and a carrier. These
pharmaceutical compositions can be included in kits, such as, for
example, diagnostic kits for use in the methods disclosed
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0113] FIG. 1 is a graph demonstrating that partial reduction of an
activatable anti-EGFR antibody using three different
TCEP-to-activatable antibody ratios (i.e., ratios of 1.5:1, of 2:1,
and of 4:1), and subsequent thiol conjugation of a fluorescent dye
(Alexa 680) to such partially reduced activatable anti-EGFR
antibody with a cleavable moiety (3954-1204-c225v5) using the
methods provided herein successfully conjugates the dye to the
activatable antibody, while maintaining the masking efficiency of
the masking moiety of the activatable anti-EGFR antibody. As used
in these figures, "(1.5)", "(2)" and "(4)" signify the ratios of
TCEP-to-activatable antibody used in the TCEP reduction step.
[0114] FIG. 2 is a graph demonstrating that partial reduction of a
masked anti-EGFR antibody with a noncleavable moiety using three
different TCEP-to-masked antibody ratios (i.e., ratios of 1.5:1, of
2:1, and of 4:1), and subsequent thiol conjugation of Alexa 680 to
such partially reduced masked anti-EGFR antibody with a
noncleavable moiety (3954-NSUB-c225v5) using the methods provided
herein successfully conjugates the dye to the noncleavable masked
antibody, while maintaining the masking efficiency of the masking
moiety of the masked anti-EGFR antibody. As used in these figures,
"(1.5)", "(2)" and "(4)" signify the ratios of TCEP-to-masked
antibody used in the TCEP reduction step.
[0115] FIGS. 3A and 3B are an illustration and a graph
demonstrating the non-reduced analysis by LabChip.RTM. of TCEP
reduction of 3954-1204-c225v5, Alexa 680 thiol-conjugation and
activation by the protease uPA. As shown in these figures using two
different TCEP-to-activatable antibody ratios (i.e., ratios of
1.5:1 and of 4:1), partial reduction, subsequent thiol conjugation
of Alexa 680 to the partially reduced activatable anti-EGFR
antibody 3954-1204-c225v5 and activation by uPA does not disturb or
otherwise negatively affect the activation and/or masking
efficiency of the activatable antibody. As used in these figures,
"1.5" and "4" signify the ratios of TCEP-to-activatable antibody
used in the TCEP reduction step; and "(U)" signifies that the
activatable antibody has been activated, i.e., cleaved, by
incubation with uPA.
[0116] FIGS. 4A and 4B are an illustration and a graph
demonstrating the reduced analysis by LabChip.RTM. of TCEP
reduction of 3954-1204-c225v5, Alexa 680 thiol-conjugation and
activation by the protease uPA. As shown in these figures using two
different TCEP-to-activatable antibody ratios (i.e., ratios of
1.5:1 and of 4:1), partial reduction, subsequent thiol conjugation
of Alexa 680 to the partially reduced activatable anti-EGFR
antibody 3954-1204-c225v5 and activation by uPA does not disturb or
otherwise negatively affect the activation and/or masking
efficiency of the activatable antibody. As used in these figures,
"1.5" and "4" signify the ratios of TCEP-to-activatable antibody
used in the TCEP reduction step; "(U)" signifies that the
activatable antibody has been activated, i.e., cleaved, by
incubation with uPA, and "R" signifies reduced analysis.
[0117] FIGS. 5A and 5B are an illustration and a graph
demonstrating the non-reduced analysis by LabChip.RTM. of TCEP
reduction of 3954-NSUB-c225v5, Alexa 680 thiol-conjugation and
activation by the protease uPA. As shown in these figures using two
different TCEP-to-masked antibody ratios (i.e., ratios of 1.5:1 and
of 4:1), partial reduction, subsequent thiol conjugation of Alexa
680 to the partially reduced masked anti-EGFR antibody
3954-NSUB-c225v5 and activation by uPA does not disturb or
otherwise negatively affect the activation and/or masking
efficiency of the noncleavable masked antibody. As used in these
figures, "1.5" and "4" signify the ratios of TCEP-to-masked
antibody used in the TCEP reduction step; and "(U)" signifies that
the noncleavable masked antibody was not activated, i.e., not
cleaved, by incubation with uPA.
[0118] FIGS. 6A and 6B are an illustration and a graph
demonstrating the reduced analysis by LabChip.RTM. of TCEP
reduction of 3954-NSUB-c225v5, Alexa 680 thiol-conjugation and
activation by the protease uPA. As shown in these figures using two
different TCEP-to-masked antibody ratios (i.e., ratios of 1.5:1 and
of 4:1), partial reduction, subsequent thiol conjugation of Alexa
680 to the partially reduced masked anti-EGFR antibody
3954-NSUB-c225v5 and activation by uPA does not disturb or
otherwise negatively affect the activation and/or masking
efficiency of the noncleavable masked antibody. As used in these
figures, "1.5" and "4" signify the ratios of TCEP-to-masked
antibody used in the TCEP reduction step; "(U)" signifies that the
noncleavable masked antibody was not been activated, i.e., not
cleaved by incubation with uPA, and "R" signifies reduced
analysis.
[0119] FIG. 7 is a table and a photograph depicting reduction of
the activatable anti-Jagged antibody 5342-1204-4D11 at a ratio of
TCEP to activatable antibody equaling 4:1 using a 120-minute
reduction time. For the data shown, reduction was followed by
conjugation to a fluorescent dye, Alexa 680.
[0120] FIG. 8 is a graph depicting varying degrees of Alexa 680
conjugation to an anti-Jagged antibody 4D11 or anti-Jagged
activatable antibody 5342-1204-4D11 using thiol conjugatable Alexa
680 as a surrogate for thiol conjugatable toxin. This figure also
demonstrates that such conjugation can be effected so as to
maintain activation of anti-Jagged activatable antibody by uPA.
[0121] FIG. 9A is a graph demonstrating the binding activities of
anti-Jagged antibody 4D11, antibody conjugate 4D11-vc-MMAD,
activatable antibody conjugate 5342-1204-4D11-vc-MMAD,
uPA-activated activatable antibody conjugate
5342-1204-4D11-vc-MMAD, and Synagis to the pancreatic
adenocarcinoma cell line BxPC3. Cells were incubated with the
respective compositions and then stained with AF-647-labelled anti
human IgG.
[0122] FIG. 9B is a graph demonstrating cytotoxicity activities of
anti-Jagged antibody conjugate 4D11-vc-MMAD, activatable antibody
conjugate 5342-1204-4D1 I-vc-MMAD, uPA-activated activatable
antibody conjugate 5342-1204-4D1 I-vc-MMAD, and rituximab antibody
conjugated to linker payload vc-MMAD on BxPC3 cells. Viability was
measured using Cell Titer Glo reagent and relative Luminescence
units plotted against dose.
[0123] FIG. 10A is a graph demonstrating the binding activities of
anti-Jagged antibody 4D11, antibody conjugate 4D11-vc-MMAE,
activatable antibody 5342-1204-4D11, activatable antibody conjugate
5342-1204-4D11-vc-MMAE, uPA-activated activatable antibody
conjugate 5342-1204-4D11-vc-MMAE, and Synagis conjugated to linker
payload vc-MMAE to the pancreatic adenocarcinoma cell line BxPC3.
Cells were incubated with the respective compositions and then
stained with AF-647-labelled anti human IgG.
[0124] FIG. 10B is a graph demonstrating cytotoxicity activities of
anti-Jagged antibody conjugate 4D11-vc-MMAE, activatable antibody
conjugate 5342-1204-4D11-vc-MMAE, uPA-activated activatable
antibody conjugate 5342-1204-4D11-vc-MMAE, and Synagis conjugated
to linker payload vc-MMAE on BxPC3. Viability was measured using
Cell Titer Glo reagent and relative Luminescence units plotted
against dose.
DETAILED DESCRIPTION OF THE INVENTION
[0125] The present invention provides conjugates that include an
activatable antibody and methods of making these activatable
antibody conjugates. Also provided are activatable antibodies
having points of conjugation for receiving a drug or label. The
conjugates can be used therapeutically, diagnostically (e.g., in
vitro or in vivo), for in vivo imaging, and for any of a variety of
other therapeutic, diagnostic and/or prophylactic uses.
[0126] Generally, the compositions and methods provided herein
include an activatable antibody that includes an antibody or
antibody fragment (AB) that specifically binds a target, where the
AB is coupled to a masking moiety (MM) that decreases the ability
of the AB to bind its target. In some embodiments, the activatable
antibody further includes a cleavable moiety (CM) that is a
substrate for a protease. The compositions and methods provided
herein enable the attachment of one or more agents to one or more
cysteine residues in the AB without compromising the activity
(e.g., the masking, activating or binding activity) of the
activatable antibody. In some embodiments, the compositions and
methods provided herein enable the attachment of one or more agents
to one or more cysteine residues in the AB without reducing or
otherwise disturbing one or more intrachain disulfide bonds within
the activatable antibody. In some embodiments, the compositions and
methods provided herein enable the attachment of one or more agents
to one or more cysteine residues in the AB without reducing or
otherwise disturbing one or more disulfide bonds within the MM.
[0127] The compositions and methods provided herein produce an
activatable antibody that is conjugated to one or more agents,
e.g., any of a variety of therapeutic, diagnostic and/or
prophylactic agents, without any of the agent(s) being conjugated
to the MM of the activatable antibody. The compositions and methods
provided herein produce conjugated activatable antibodies in which
the MM retains the ability to effectively mask the AB of the
activatable antibody. In addition, such a conjugated activatable
antibody retains the ability to be activated, and the activated AB
retains the ability to bind to its target.
[0128] In some embodiments, the compositions and methods provided
herein do not compromise the masking activity and/or masking
efficiency of the MM in the activatable antibody. For example, in
situations where the compositions and methods might be found to
negatively impact the masking activity and/or masking efficiency of
the MM in the activatable antibody, the compositions and methods
decrease or otherwise disturb the masking activity and/or masking
efficiency of the activatable antibody by no more than 50%, no more
than 40%, no more than 30%, no more than 25%, no more than 200, no
more than 15%, no more than 10%, no more than 9%/0, no more than
8%, no more than 7%, no more than 6%, no more than 5%, no more than
4%, no more than 3%, no more than 2% or no more than 1%, as
compared to the level of masking activity and/or masking efficiency
of the activatable antibody prior to conjugation or in the absence
of any conjugation.
[0129] In some embodiments, the compositions and methods provided
herein do not compromise the activating activity and/or activating
efficiency of the activatable antibody. For example, in situations
where the compositions and methods might be found to negatively
impact the activating activity and/or activating efficiency, the
compositions and methods decrease or otherwise disturb the
activating activity and/or activating efficiency of the activatable
antibody by no more than 50%, no more than 40%, no more than 30%,
no more than 25%, no more than 20%, no more than 15%, no more than
10%, no more than 9%, no more than 8%, no more than 7%, no more
than 6%, no more than 5%, no more than 4%, no more than 3%, no more
than 2% or no more than 1%, as compared to the level of activating
activity and/or activating efficiency of the activatable antibody
prior to conjugation or in the absence of any conjugation.
[0130] In some embodiments, the compositions and methods provided
herein do not compromise the binding activity of the activatable
antibody. For example, in situations where the compositions and
methods might be found to negatively impact the binding activity,
the compositions and methods decrease or otherwise disturb the
binding activity of the activatable antibody by no more than 50%,
no more than 40%, no more than 30%, no more than 25%, no more than
20%, no more than 15%, no more than 10%, no more than 9%, no more
than 8%, no more than 7%, no more than 6%, no more than 5%, no more
than 4%, no more than 3%, no more than 2% or no more than 1%, as
compared to the level of binding activity of the activatable
antibody prior to conjugation or in the absence of any
conjugation.
[0131] The compositions and methods provided herein determine the
combination of reagents and reaction conditions to produce the
desired partial reduction followed by conjugation. When reduction
and subsequent conjugation is not controlled properly, activatable
antibodies will be completely reduced, and the masking efficiency
of the activatable antibody is/will be compromised.
[0132] The conjugated activatable antibodies include an antibody or
antigen-binding fragment thereof (AB) that specifically binds a
target, and the AB is coupled to a masking moiety (MM), such that
coupling of the MM to the AB decreases the ability of the antibody
or antigen-binding fragment thereof to bind the target. In some
embodiments, the MM is coupled to the AB via a cleavable moiety
(CM) that includes a substrate for a protease, for example, a
protease that is co-localized with the target at a treatment site
in a subject. Numerous studies have demonstrated the correlation of
aberrant protease levels, e.g., uPA, legumain, MT-SP1, matrix
metalloproteases (MMPs), in solid tumors. (See e.g., Murthy R V, et
al. "Legumain expression in relation to clinicopathologic and
biological variables in colorectal cancer." Clin Cancer Res. 11
(2005): 2293-2299; Nielsen B S, et al. "Urokinase plasminogen
activator is localized in stromal cells in ductal breast cancer."
Lab Invest 81 (2001): 1485-1501; Mook O R, et al. "In situ
localization of gelatinolytic activity in the extracellular matrix
of metastases of colon cancer in rat liver using quenched
fluorogenic DQ-gelatin." J Histochem Cytochem. 51 (2003):
821-829).
[0133] The conjugated activatable antibodies provided herein
include a substrate for a protease, which is useful in leveraging
the protease activity in tumor cells for targeted conjugated
antibody activation at the site of treatment and/or diagnosis. The
substrate selection process is used to identify substrates that
have a number of desirable characteristics. For example, the
selected substrates are systemically stable (i.e., stable in the
systemic circulation of a subject), are generally not susceptible
to cleavage by circulating proteases such as plasmin, thrombin,
tissue plasminogen activator (tPA) or a kallikrein (KLK) such as
KLK-5 and/or KLK-7, are non-toxic, are generally not susceptible to
cleavage at potential sites of toxicity such as the skin by
proteases such as ADAM 9, ADAM 10, ADAM 17 and/or kallikreins, such
as KLK-5 and KLK-7, and are active at an intended site of treatment
and/or diagnosis. In some embodiments, the identified substrates
are selected for proteases that are dysregulated, due to, for
example, being overexpressed or showing excess activity, or being
less susceptible to protease inhibition (due, e.g., to
underexpression of the corresponding inhibitor or reduction in
inhibitor activity) at an intended site of therapy and/or diagnosis
but are not typically expressed at or in normal, healthy or
otherwise non-diseased or damaged tissue, and then the selected
substrates are subsequently counter-screened against proteases
expressed in normal, e.g., non-diseased, tissue.
[0134] As a non-limiting example, the AB is a binding partner for
any target listed in Table 1.
TABLE-US-00001 TABLE 1 Exemplary Targets 1-92-LFA-3 Alpha-4
integrin Alpha-V integrin alpha4beta1 integrin alpha4beta7 integrin
AGR2 Anti-Lewis-Y Apelin J receptor APRIL B7-H4 BAFF BTLA C5
complement C-242 CD2 CD3 CD6 CD9 CD11a CD19 CD20 CD22 CD24 CD25
CD27 CD28 CD30 CD33 CD38 CD40 CD40L CD41 CD44 CD47 CD51 CD52 CD56
CD64 CD70 CD74 CD80 CD81 CD86 CD95 CD117 CD125 CD132 (IL-2RG) CD133
CD137 CD138 CD166 CD172A CD248 CEACAM5 (CEA) CEACAM6 (NCA-90)
CLAUDIN-3 CLAUDIN-4 cMet Collagen Cripto CSFR CSFR-1 CTLA-4 CTGF
CXCL10 CXCL13 CXCR1 CXCR2 CXCR4 CYR61 DL44 DLL4 DPP-4 EGFR
Endothelin B receptor (ETBR) EpCAM EPHA2 ERBB3 F protein of RSV FAP
FGF-2 FGF8 FGFR1 FGFR2 FGFR3 FGFR4 Folate receptor G-CSF G-CSFR GD2
GITR GLUT1 GLUT4 GM-CSF GM-CSFR GP IIb/IIIa receptors Gp130
GPIIB/IIIA GPNMB GRP78 HER2/neu HGF hGH HVEM Hyaluronidase ICOS
IFNalpha IFNbeta IFNgamma IgE IgE Receptor (FceRI) IGF IGF1R IL1B
IL1R IL2 IL11 IL12 IL12p40 IL-12R, IL-12Rbeta1 IL13 IL13R IL15 IL17
IL18 IL21 IL23 IL23R IL27/IL27R (wsx1) IL29 IL-31R IL31/IL31R IL2R
IL4 IL4R IL6, IL6R Insulin Receptor Jagged Ligands Jagged 1 Jagged
2 LAG-3 LIF-R LIGHT MRP4 MUC1 Mucin-16 Na/K ATPase Neutrophil
elastase NGF Nicastrin Notch Receptors Notch 1 Notch 2 Notch 3
Notch 4 NOV OSM-R OX-40 PAR2 PDGF-AA PDGF-BB PDGFRalpha PDGFRbeta
PD-1 PD-L1 PD-L2 Phosphatidyl-serine P1GF PSCA PSMA RAAG12 RAGE
SLC44A4 Sphingosine 1 Phosphate STEAP1 TAPA1 TGFbeta TIGIT TIM-3
TLR2 TLR4 TLR6 TLR7 TLR8 TLR9 TMEM31 TNFalpha TNFR TNFRS12A
TRAIL-R1 TRAIL-R2 Transferrin Transferrin receptor TRK-A TRK-B uPAR
VAP1 VCAM-1 VEGF VEGF-A VEGF-B VEGF-C VEGF-D VEGFR1 VEGFR2 VEGFR3
VISTA WISP-1 WISP-2 WISP-3
[0135] As a nonlimiting example, the AB is or is derived from an
antibody listed in Table
TABLE-US-00002 TABLE 2 Exemplary sources for Abs Antibody Trade
Name (antibody name) Target Avastin .TM. (bevacizumab) VEGF
Lucentis .TM. (ranibizumab) VEGF Erbitux .TM. (cetuximab) EGFR
Vectibix .TM. (panitumumab) EGFR Remicade .TM. (infliximab)
TNF.alpha. Humira .TM. (adalimumab) TNF.alpha. Tysabri .TM.
(natalizumab) Integrin.alpha.4 Simulect .TM. (basiliximab) IL2R
Soliris .TM. (eculizumab) Complement C5 Raptiva .TM. (efalizumab)
CD11a Bexxar .TM. (tositumomab) CD20 Zevalin .TM. (ibritumomab
tiuxetan) CD20 Rituxan .TM. (rituximab) CD20 Ocrelizumab CD20
Arzerra .TM. (ofatumumab) CD20 Obinutuzumab CD20 Zenapax .TM.
(daclizumab) CD25 Adcetris .TM. (brentuximab vedotin) CD30
Myelotarg .TM. (gemtuzumab) CD33 Mylotarg .TM. (gemtuzumab
ozogamicin) CD33 Campath .TM. (alemtuzumab) CD52 ReoPro .TM.
(abiciximab) Glycoprotein receptor IIb/IIIa Xolair .TM.
(omalizumab) IgE Herceptin .TM. (trastuzumab) Her2 Kadcyla .TM.
(trastuzumab emtansine) Her2 Synagis .TM. (palivizumab) F protein
of RSV (ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L
(pertuzumab) Her2-neu (ertumaxomab) CD3/Her2-neu Orencia .TM.
(abatacept) CTLA-4 (tanezumab) NGF (bavituximab) Phosphatidylserine
(zalutumumab) EGFR (mapatumumab) EGFR (matuzumab) EGFR
(nimotuzumab) EGFR ICR62 EGFR mAb 528 EGFR CH806 EGFR MDX-447
EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12 huJ591 PSMA Enbrel .TM.
(etanercept) TNF-R Amevive .TM. (alefacept) 1-92-LFA-3 Antril .TM.,
Kineret .TM. (ankinra) IL-1Ra GC1008 TGFbeta Notch, e.g., Notch 1
Jagged 1 or Jagged 2 (adecatumumab) EpCAM (figitumumab) IGF1R
(tocilizumab) IL-6 receptor Stelara .TM. (ustekinumab) IL-12/IL-23
Prolia .TM. (denosumab) RANKL
[0136] In some embodiments, the AB binds Epidermal Growth Factor
Receptor (EGFR). In some embodiments, the AB that binds EGFR
includes one or more of the heavy chain and/or light chain
sequences shown below.
TABLE-US-00003 C225v5 Antibody Heavy Chain Nucleotide Sequence:
(SEQ ID NO: 1)
CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCT
GCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAA
AGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACC
AGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGC
AAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGC
GTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
AAATGA C225v5 Antibody Heavy Chain Amino Acid Sequence (SEQ ID NO:
2) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT
SRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEATHNHYTQKSLSLSPG
C225v5 Antibody Light Chain Nucleotide Sequence: (SEQ ID NO: 10)
CAGATCTTGCTGACCCAGAGCCCGGTGATTCTGAGCGTGAGCCCGGGCGAACGTGTGAGCTTTA
GCTGCCGCGCGAGCCAGAGCATTGGCACCAACATTCATTGGTATCAGCAGCGCACCAACGGCAG
AGCGGCAGCGGCACCGATTTTACCCTGAGCATTAACAGCGTGGAAAGCGAAGATATTGCGGATT
ATTATTGCCAGCAGAACAACAACTGGCCGACCACCTTTGGCGCGGGCACCAAACTGGAACTGAA
ACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGA
ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG
TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAG
CACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC
GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGT
GTTAG C225v5 Antibody Light Chain Amino Acid Sequence: (SEQ ID NO:
16)
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG
SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSG
TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC* C225v4 Antibody Heavy Chain Nucleotide
Sequence: (SEQ ID NO: 238)
CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCT
GCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAA
AGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACC
AGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGC
AAAGCAACGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGC
GTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAA
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
AAATGA C225v4 Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO:
239)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT
SRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPFPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*
C225v6 Antibody Heavy Chain Nucleotide Sequence: (SEQ ID NO: 240)
CAGGTGCAGCTGAAACAGAGCGGCCCGGGCCTGGTGCAGCCGAGCCAGAGCCTGAGCATTACCT
GCACCGTGAGCGGCTTTAGCCTGACCAACTATGGCGTGCATTGGGTGCGCCAGAGCCCGGGCAA
AGGCCTGGAATGGCTGGGCGTGATTTGGAGCGGCGGCAACACCGATTATAACACCCCGTTTACC
AGCCGCCTGAGCATTAACAAAGATAACAGCAAAAGCCAGGTGTTTTTTAAAATGAACAGCCTGC
AAAGCCAGGATACCGCGATTTATTATTGCGCGCGCGCGCTGACCTATTATGATTATGAATTTGC
GTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGCTAGCACCAAGGGCCCATCGGTCTTC
CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACAC
CTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCC
AGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGG
ACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGA
ACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCC
CGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCA
ACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGC
CAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAACTGACCAAGAA
CCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAG
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATG
CTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
AAATGA] C225v6 Antibody Heavy Chain Amino Acid Sequence (SEQ ID NO:
241)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFT
SRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVF
PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS
SSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
K*
[0137] In some embodiments, the AB binds interleukin 6 receptor
(IL-6R). In some embodiments, the AB that binds IL-6R includes one
or more of the heavy chain and/or light chain sequences shown
below.
TABLE-US-00004 Av1 Antibody Heavy Chain Amino Acid Sequence: (SEQ
ID NO: 242) QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIG
YISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSL
ARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Av1 Antibody
Light Chain Amino Acid Sequence: (SEQ ID NO: 243)
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYY
TSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0138] In some embodiments, the AB binds a Jagged target, e.g.,
Jagged 1, Jagged 2 or both Jagged 1 and Jagged 2. In some
embodiments, the AB that binds a Jagged target includes one or more
of the heavy chain and/or light chain sequences shown below.
TABLE-US-00005 4D11 Light Chain sequence: (SEQ ID NO: 244)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQTVVAPPLFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVYKSENRGEC
4D11 Heavy Chain sequence: (SEQ ID NO: 245)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Heavy
Chain sequence (SEQ ID NO: 246)
EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Light
Chain Sequence (SEQ ID NO: 247)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0139] In some embodiments, the AB that binds a Jagged target
includes one or more of the variable heavy chain and/or variable
light chain sequences shown below.
TABLE-US-00006 Variable Light Chain Amino Sequence Lc4 (SEQ ID NO:
248) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc4 (SEQ ID NO: 249)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFT1SRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc5 (SEQ ID
NO: 250) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc5 (SEQ ID NO: 251)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PYHGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc7 (SEQ ID
NO: 252) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc7 (SEQ ID NO: 253)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PFFGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc8 (SEQ ID
NO: 254) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc8 (SEQ ID NO: 255)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHI
GRTNPFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc13 (SEQ
ID NO: 256) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYL
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc13 (SEQ ID NO: 257)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable Light Chain Amino Sequence Lc16 (SEQ ID NO: 258)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc16 (SEQ ID NO: 259)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PYYGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc19 (SEQ
ID NO: 260) DIQMTOSPSSLSASVGDRVTITCRASQSISSYLNWYQ0KPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc19 (SEQ ID NO: 261)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQNGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PFFGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc21 (SEQ
ID NO: 262) DIQMIQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc21 (SEQ ID NO: 263)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc24 (SEQ
ID NO: 264) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy ChaiN Amino Sequence Hc24 (SEQ ID NO: 265)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable Light Chain Amino Sequence Lc26 (SEQ ID NO: 266)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc26 (SEQ ID NO: 267)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc27 (SEQ
ID NO: 268) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ TKVEIKR Variable
Heavy Chain Amino Sequence Hc27 (SEQ ID NO: 269)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PFYGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc28 (SEQ
ID NO: 270) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYL
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc28 (SEQ ID NO: 271)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PFFGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc30 (SEQ
ID NO: 272) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc30 (SEQ ID NO: 273)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEEMGWQTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAA AFDYWGQGTLVTVSS
Variable Light Chain Amino Sequence Lc31 (SEQ ID NO: 274)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc31 (SEQ ID NO: 275)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc32 (SEQ
ID NO: 276) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc32 (SEQ ID NO: 277)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable Light Chain Amino Sequence Lc37 (SEQ ID NO: 278)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc37 (SEQ ID NO: 279)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTTSRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PHNGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc39 (SEQ
ID NO: 280) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc39 (SEQ ID NO: 281)
EVQLLESGGGLVQPGGSLRISCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable Light Chain Amino Sequence Lc40 (SEQ ID NO: 282)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQSVVAPLTFGQ GTKVEIKR Heavy
Chain Amino Sequence Hc40 (SEQ ID NO: 283)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSP
PFFGQFDYWGQGTLVTVSS Variable Light Chain Amino Sequence Lc47 (SEQ
ID NO: 284) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQ GTKVEIKR
Variable Heavy Chain Amino Sequence Hc47 (SEQ ID NO: 285)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDEMGWQTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGOGTLVTVSS
Variable 4B2 Light Chain (SEQ ID NO: 286)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQ GTKVEIKR
Variable 4B2 Heavy Chain (SEQ ID NO: 287)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEQMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable 4D11 Light Chain (SEQ ID NO: 288)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ GTKVEIKR
Variable 4D11 Heavy Chain (SEQ ID NO: 289)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable 4E7 Light Chain (SEQ ID NO: 290)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQ GTKVEIKR
Variable 4E7 Heavy Chain (SEQ ID NO: 291)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEEMGWQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable 4E11 Light Chain (SEQ ID NO: 292)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQ GTKVEIKR
Variable 4E11 Heavy Chain (SEQ ID NO: 293)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IEPMGQLTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSS Variable 6B7 Light Chain (SEQ ID NO: 294)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQALVAPLTFGQ GTKVEIKR
Variable 6B7 Heavy Chain (SEQ ID NO: 295)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA AAFDYWGQGTLVTVSS
Variable 6F8 Light Chain (SEQ ID NO: 296)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATTYCQQALVAPLTFGQ GTKVEIKR
Variable 6F8 Heavy Chain (SEQ ID NO: 297)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDEMGWQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA
AAFDYWGQGTLVTVSS
[0140] By way of non-limiting example, the CM includes an amino
acid sequence that is a substrate or is derived from a substrate
that is cleaved by one or more of the following enzymes or
proteases listed in Table 3.
TABLE-US-00007 TABLE 3 Exemplary Enzymes/Proteases ADAMS, ADAMTS,
e.g. ADAM8 ADAM9 ADAM10 ADAM12 ADAM15 ADAM17/TACE ADAMDEC1 ADAMTS1
ADAMTS4 ADAMTS5 Aspartate proteases, e.g., BACE Renin Aspartic
cathepsins, e.g., Cathepsin D Cathepsin E Caspases, e.g., Caspase 1
Caspase 2 Caspase 3 Caspase 4 Caspase 5 Caspase 6 Caspase 7 Caspase
8 Caspase 9 Caspase 10 Caspase 14 Cysteine cathepsins, e.g.,
Cathepsin B Cathepsin C Cathepsin K Cathepsin L Cathepsin S
Cathepsin V/L2 Cathepsin X/Z/P Cysteine proteinases, e.g.,
Cruzipain Legumain Otubain-2 KLKs, e.g., KLK4 KLK5 KLK6 KLK7 KLK8
KLK10 KLK11 KLK13 KLK14 Metallo proteinases, e.g., Meprin
Neprilysin PSMA BMP-1 MMPs, e.g., MMP1 MMP2 MMP3 MMP7 MMP8 MMP9
MMP10 MMP11 MMP12 MMP13 MMP14 MMP15 MMP16 MMP17 MMP19 MMP20 MMP23
MMP24 MMP26 MMP27 Serine proteases, e.g., activated protein C
Cathepsin A Cathepsin G Chymase coagulation factor proteases (e.g.,
FVIIa, FIXa, FXa, FXIa, FXIIa) Elastase Granzyme B
Guanidinobenzoatase HtrA1 Human Neutrophil Elastase Lactoferrin
Marapsin NS3/4A PACE4 Plasmin PSA tPA Thrombin Tryptase uPA Type II
Transmembrane Serine Proteases (TTSPs), e.g., DESC1 DPP-4 FAP
Hepsin Matriptase-2 MT-SP1/Matriptase TMPRSS2 TMPRSS3 TMPRSS4
[0141] The conjugated activatable antibodies provided herein
include a masking moiety. In some embodiments, the masking moiety
is an amino acid sequence that is coupled or otherwise attached to
the activatable antibody and is positioned within the activatable
antibody construct such that the masking moiety decreases the
ability of the antibody to specifically bind the target. Suitable
masking moieties are identified using any of a variety of known
techniques. For example, peptide masking moieties are identified
using the methods described in U.S. Pat. No. 8,293,685 by Daugherty
et al., the contents of which are hereby incorporated by reference
in their entirety.
[0142] In some embodiments, the masking moiety is selected for use
with a specific antibody or antibody fragment. For example,
suitable masking moieties for use with antibodies that bind EGFR
include MMs that include the sequence CISPRG (SEQ ID NO: 29). By
way of non-limiting examples, the MM can include a sequence such as
CISPRGCG (SEQ ID NO: 30); CISPRGCPDGPYVMY (SEQ ID NO: 31);
CISPRGCPDGPYVM (SEQ ID NO: 32), CISPRGCEPGTYVPT (SEQ ID NO: 33) and
CISPRGCPGQIWHPP (SEQ ID NO: 34). Other suitable masking moieties
include any of the EGFR-specific masks disclosed in PCT Publication
No. WO 2010/081173, such as, by way of non-limiting example,
GSHCLIPINMGAPSC (SEQ ID NO: 35); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC
(SEQ ID NO: 36); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 37);
ADHVFWGSYGCISPRGCPG (SEQ ID NO: 38); CHHVYWGHCGCISPRGCPG (SEQ ID
NO: 39); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 40); CNHHYHYYCGCISPRGCPG
(SEQ ID NO: 41); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 42);
CPYYTLSYCGCISPRGCPG (SEQ ID NO: 43); CNHVYFGTCGCISPRGCPG (SEQ ID
NO: 44); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 45); CHHFTLTTCGCISPRGCPG
(SEQ ID NO: 46); YNPCATPMCCISPRGCPG (SEQ ID NO: 47),
CNHHYFYTCGCISPRGCG (SEQ ID NO: 48); CNHHYHYYCGCISPRGCG (SEQ ID NO:
49); CNHVYFGTCGCISPRGCG (SEQ ID NO: 50); CHHVYWGHCGCISPRGCG (SEQ ID
NO: 51); CPHFTTTSCGCISPRGCG (SEQ ID NO: 52); CNHFTLTTCGCISPRGCG
(SEQ ID NO: 53); CHHFTLTTCGCISPRGCG (SEQ ID NO: 54);
CPYYTLSYCGCISPRGCG (SEQ ID NO: 55); CPHVSFGSCGCISPRGCG (SEQ ID NO:
56); ADHVFWGSYGCISPRGCG (SEQ ID NO: 57); YNPCATPMCCISPRGCG (SEQ ID
NO: 58); CHHVYWGHCGCISPRGCG (SEQ ID NO: 59);
C(N/P)H(H/V/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID
NO: 60); CISPRGCGQPIPSVK (SEQ ID NO: 61); CISPRGCTQPYHVSR (SEQ ID
NO: 62); and/or CISPRGCNAVSGLGS (SEQ ID NO: 63).
[0143] Suitable masking moieties for use with antibodies that bind
a Jagged target, e.g., Jagged 1 and/or Jagged 2, include, by way of
non-limiting example, masking moieties that include a sequence such
as QGQSGQCNIWLVGGDCRGWQG (SEQ ID NO: 232);
QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 64); PWCMQRQDFLRCPQP (SEQ ID
NO: 65); QLGLPAYMCTFECLR (SEQ ID NO: 66); CNLWVSGGDCGGLQG (SEQ ID
NO: 67); SCSLWTSGSCLPHSP (SEQ ID NO: 68); YCLQLPHYMQAMCGR (SEQ ID
NO: 69); CFLYSCTDVSYWNNT (SEQ ID NO: 70); PWCMQRQDYLRCPQP (SEQ ID
NO: 71); CNLWISGGDCRGLAG (SEQ ID NO: 72); CNLWVSGGDCRGVQG (SEQ ID
NO: 73); CNLWVSGGDCRGLRG (SEQ ID NO: 74); CNLWISGGDCRGLPG (SEQ ID
NO: 75); CNLWVSGGDCRDAPW (SEQ ID NO: 76); CNLWVSGGDCRDLLG (SEQ ID
NO: 77); CNLWVSGGDCRGLQG (SEQ ID NO: 78); CNLWLHGGDCRGWQG (SEQ ID
NO: 79); CNIWLVGGDCRGWQG (SEQ ID NO: 80); CTTWFCGGDCGVMRG (SEQ ID
NO: 81); CNIWGPSVDCGALLG (SEQ ID NO: 82); CNIWVNGGDCRSFEG (SEQ ID
NO: 83); YCLNLPRYMQDMCWA (SEQ ID NO: 84); YCLALPHYMQADCAR (SEQ ID
NO: 85); CFLYSCGDVSYWGSA (SEQ ID NO: 86); CYLYSCTDSAFWNNR (SEQ ID
NO: 87); CYLYSCNDVSYWSNT (SEQ ID NO: 88); CFLYSCTDVSYW (SEQ ID NO:
89); CFLYSCTDVAYWNSA (SEQ ID NO: 90); CFLYSCTDVSYWGDT (SEQ ID NO:
91); CFLYSCTDVSYWGNS (SEQ ID NO: 92); CFLYSCTDVAYWNNT (SEQ ID NO:
93); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 94); CFLYSCTDVAYWSGL (SEQ ID
NO: 95); CYLYSCTDGSYWNST (SEQ ID NO: 96); CFLYSCSDVSYWGNI (SEQ ID
NO: 97); CFLYSCTDVAYW (SEQ ID NO: 98); CFLYSCTDVSYWGST (SEQ ID NO:
99); CFLYSCTDVAYWGDT (SEQ ID NO: 100); GCNIWLNGGDCRGWVDPLQG (SEQ ID
NO: 101); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 102);
GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 103); GCNIWANGGDCRGWIDNIDG (SEQ ID
NO: 104); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 105);
GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 106); GGPALCNIWLNGGDCRGWSG (SEQ ID
NO: 107); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 108);
GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 109); GKSEFCNIWLNGGDCRGWIG (SEQ ID
NO: 110); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 111);
GASQYCNLWINGGDCRGWRG (SEQ ID NO: 112); GCNIWLVGGDCRPWVEGG (SEQ ID
NO: 113); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 114); GCNIWLNGGDCRAWVDTG
(SEQ ID NO: 115); GCNIWIVGGDCRPFINDG (SEQ ID NO: 116);
GCNIWLNGGDCRPVVFGG (SEQ ID NO: 117); GCNIWLSGGDCRMFMNEG (SEQ ID NO:
118); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 119); GCNIWLNGGDCRGWEASG (SEQ
ID NO: 120); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 121);
GCNIWLNGGDCRTFVASG (SEQ ID NO: 122); GCNIWAHGGDCRGFIEPG (SEQ ID NO:
123); GFLENCNIWLNGGDCRTG (SEQ ID NO: 124); GIYENCNIWLNGGDCRMG (SEQ
ID NO: 125); and/or GIPDNCNIWINGGDCRYG (SEQ ID NO: 126).
[0144] Suitable masking moieties for use with antibodies that bind
an interleukin 6 target, e.g., interleukin 6 receptor (IL-6R),
include, by way of non-limiting example, masking moieties that
include a sequence such as QGQSGQYGSCSWNYVHIFMDC (SEQ ID NO: 127);
QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 128); QGQSGQMGVPAGCVWNYAHIFMDC
(SEQ ID NO: 129); YRSCNWNYVSIFLDC (SEQ ID NO: 130);
PGAFDIPFPAHWVPNT (SEQ ID NO: 131); ESSCVWNYVHIYMDC (SEQ ID NO:
132); YPGCKWNYDRIFLDC (SEQ ID NO: 133); YRTCSWNYVGIFLDC (SEQ ID NO:
134); YGSCSWNYVHIFMDC (SEQ ID NO: 135); YGSCSWNYVHIFLDC (SEQ ID NO:
136); YGSCNWNYVHIFLDC (SEQ ID NO: 137); YTSCNWNYVHIFMDC (SEQ ID NO:
138); YPGCKWNYDRIFLDC (SEQ ID NO: 139); WRSCNWNYAHIFLDC (SEQ ID NO:
140); WSNCHWNYVHIFLDC (SEQ ID NO: 141); DRSCTWNYVRISYDC (SEQ ID NO:
142); SGSCKWDYVHIFLDC (SEQ ID NO: 143); SRSCIWNYAHIHLDC (SEQ ID NO:
144); SMSCYWQYERIFLDC (SEQ ID NO: 145); YRSCNWNYVSIFLDC (SEQ ID NO:
146); YGSCSWNYVHIFMDC (SEQ ID NO: 147); SGSCKWDYVHIFLDC (SEQ ID NO:
148); YKSCHWDYVHIFLDC (SEQ ID NO: 149); YGSCTWNYVHIFMEC (SEQ ID NO:
150); FSSCNWNYVHIFLDC (SEQ ID NO: 151); WRSCNWNYAHIFLDC (SEQ ID NO:
152); YGSCQWNYVHIFLDC (SEQ ID NO: 153); YRSCNWNYVHIFLDC (SEQ ID NO:
154); NMSCHWDYVHIFLDC (SEQ ID NO: 155); FGPCTWNYARISWDC (SEQ ID NO:
156); XXsCXWXYvhlfXdC (SEQ ID NO: 157); MGVPAGCVWNYAHIFMDC (SEQ ID
NO: 158); RDTGGQCRWDYVHIFMDC (SEQ ID NO: 159); AGVPAGCTWNYVHIFMEC
(SEQ ID NO: 160); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 161);
DGGPAGCSWNYVHIFMEC (SEQ ID NO: 162); AVGPAGCWWNYVHIFMEC (SEQ ID NO:
163); CTWNYVHIFMDCGEGEGP (SEQ ID NO: 164); GGVPEGCTWNYAHIFMEC (SEQ
ID NO: 165); AEVPAGCWWNYVHIFMEC (SEQ ID NO: 166);
AGVPAGCTWNYVHIFMEC (SEQ ID NO: 167); SGASGGCKWNYVHIFMDC (SEQ ID NO:
168); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 169); TPGCRWNYVHIFMECEAL (SEQ
ID NO: 170); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 171); PGAFDIPFPAHWVPNT
(SEQ ID NO: 172); RGACDIPFPAHWIPNT (SEQ ID NO: 173);
QGDFDIPFPAHWVPIT (SEQ ID NO: 174); XGafDIPFPAHWvPnT (SEQ ID NO:
175); RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 176); SGVGRDRDIPFPAHWVPRT
(SEQ ID NO: 177); WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 178);
WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 179); AGSGNDSDIPFPAHWVPRT (SEQ ID
NO: 180); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 181); and/or
RECGRCGDIPFPAHWVPRT (SEQ ID NO: 182).
[0145] In some embodiments, the masking moiety is selected for use
with any antibody or antibody fragment. For example, in some
embodiments, the masking moiety is a non-binding steric moiety (NB)
or a binding partner (BP) for a non-binding steric moiety, where
the BP recruits or otherwise attracts the NB to the activatable
antibody. For example, in some embodiments, the NB is a soluble,
globular protein. In some embodiments, the NB is a protein that
circulates in the bloodstream. In some embodiments, the NB is
selected from the group consisting of albumin, fibrinogen,
fibronectin, hemoglobin, transferrin, an immunoglobulin domain, and
other serum proteins. In some embodiments, the BP is selected from
the group consisting of an albumin binding peptide, a fibrinogen
binding peptide, a fibronectin binding peptide, a hemoglobin
binding peptide, a transferrin binding peptide, an immunoglobulin
domain binding peptide, and other serum protein binding peptides.
In some embodiments, the activatable antibody has the structural
arrangement from N-terminus to C-terminus as follows in the
uncleaved state: NB-CM-AB, AB-CM-NB, BP-CM-AB or AB-CM-BP. In
embodiments where the activatable antibody includes a BP and the
activatable antibody is in the presence of the corresponding NB,
the activatable antibody has a structural arrangement from
N-terminus to C-terminus as follows in the uncleaved state:
NB:BP-CM-AB or AB-CM-BP:NB, where ":" represents an interaction,
e.g., binding, between the NB and BP. In some embodiments, the
activatable antibody has the structural arrangement from N-terminus
to C-terminus as follows in the uncleaved state: NB-LP1-CM-LP2-AB,
AB-LP2-CM-LP1-NB, BP-LP1-CM-LP2-AB or AB-LP2-CM-LP1-BP. In
embodiments where the activatable antibody includes a BP and the
activatable antibody is in the presence of the corresponding NB,
the activatable antibody has a structural arrangement from
N-terminus to C-terminus as follows in the uncleaved state:
NB:BP-LP1-CM-LP2-AB or AB-LP2-CM-LP1-BP:NB, where ":" represents an
interaction, e.g., binding, between the NB and BP.
[0146] The conjugated activatable antibodies provided herein
include a cleavable moiety. In some embodiments, the cleavable
moiety includes an amino acid sequence that is a substrate for a
protease, usually an extracellular protease. Suitable substrates
are identified using any of a variety of known techniques. For
example, peptide substrates are identified using the methods
described in U.S. Pat. No. 7,666,817 by Daugherty et al., the
contents of which are hereby incorporated by reference in their
entirety. (See also Boulware et al. "Evolutionary optimization of
peptide substrates for proteases that exhibit rapid hydrolysis
kinetics." Biotechnol Bioeng. 106.3 (2010): 339-46).
[0147] In some embodiments, the CM is selected for use with a
specific protease. In some embodiments, the CM is a substrate for
at least one protease selected from the group consisting of an ADAM
17, a BMP-1, a cysteine protease such as a cathepsin, a HtrA1, a
legumain, a matriptase (MT-SP1), a matrix metalloprotease (MMP), a
neutrophil elastase, a TMPRSS, such as TMPRSS3 or TMPRSS4, a
thrombin, and a u-type plasminogen activator (uPA, also referred to
as urokinase).
[0148] In some embodiments, the CM is a substrate for an ADAM 17.
In some embodiments, the CM is a substrate for a BMP-1. In some
embodiments, the CM is a substrate for a cathepsin. In some
embodiments, the CM is a substrate for a cysteine protease. In some
embodiments, the CM is a substrate for a HtrA1. In some
embodiments, the CM is a substrate for a legumain. In some
embodiments, the CM is a substrate for a MT-SP1. In some
embodiments, the CM is a substrate for a MMP. In some embodiments,
the CM is a substrate for a neutrophil elastase. In some
embodiments, the CM is a substrate for a thrombin. In some
embodiments, the CM is a substrate for a TMPRSS. In some
embodiments, the CM is a substrate for TMPRSS3. In some
embodiments, the CM is a substrate for TMPRSS4. In some
embodiments, the CM is a substrate for uPA.
[0149] In some embodiments, the cleavable moiety is selected for
use with a specific protease, for example a protease that is known
to be co-localized with the target of the activatable antibody. For
example, suitable cleavable moieties for use in the activatable
antibodies of the disclosure include the sequence TGRGPSWV (SEQ ID
NO: 183); SARGPSRW (SEQ ID NO: 184); TARGPSFK (SEQ ID NO: 185);
LSGRSDNH (SEQ ID NO: 186); GGWHTGRN (SEQ ID NO: 187); HTGRSGAL (SEQ
ID NO: 188); PLTGRSGG (SEQ ID NO: 189); AARGPAIH (SEQ ID NO: 190);
RGPAFNPM (SEQ ID NO: 191); SSRGPAYL (SEQ ID NO: 192); RGPATPIM (SEQ
ID NO: 193); RGPA (SEQ ID NO: 194); GGQPSGMWGW (SEQ ID NO: 195);
FPRPLGITGL (SEQ ID NO: 196); VHMPLGFLGP (SEQ ID NO: 197); SPLTGRSG
(SEQ ID NO: 198); SAGFSLPA (SEQ ID NO: 199); LAPLGLQRR (SEQ ID NO:
200); SGGPLGVR (SEQ ID NO: 201); and/or PLGL (SEQ ID NO: 202).
[0150] In some embodiments, the CM is a substrate for at least one
matrix metalloprotease (MMP). Examples of MMPs include MMP1; MMP2;
MMP3; MMP7; MMP8; MMP9; MMP10; MMP11; MMP12; MMP13; MMP14; MMP15;
MMP16; MMP17; MMP19; MMP20; MMP23; MMP24; MMP26; and MMP27. In some
embodiments, the CM is a substrate for MMP9, MMP14, MMP1, MMP3,
MMP13, MMP17, MMP1, and MMP19. In some embodiments, the CM is a
substrate for MMP7. In some embodiments the CM is a substrate for
MMP9. In some embodiments, the CM is a substrate for MMP14. In some
embodiments, the CM is a substrate for two or more MMPs. In some
embodiments, the CM is a substrate for at least MMP9 and MMP14. In
some embodiments, the CM comprises two or more substrates for the
same MMP. In some embodiments, the CM comprises at least two or
more MMP9 substrates. In some embodiments, the CM comprises at
least two or more MMP14 substrates.
[0151] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLLSS (SEQ ID NO: 298); QNQALRMA (SEQ ID
NO: 299); AQNLLGMV (SEQ ID NO: 300); STFPFGMF (SEQ ID NO: 301);
PVGYTSSL (SEQ ID NO: 302); DWLYWPGI (SEQ ID NO: 303); MIAPVAYR (SEQ
ID NO: 304); RPSPMWAY (SEQ ID NO: 305); WATPRPMR (SEQ ID NO: 306);
FRLLDWQW (SEQ ID NO: 307); LKAAPRWA (SEQ ID NO: 308); GPSHLVLT (SEQ
ID NO: 309); LPGGLSPW (SEQ ID NO: 310); MGLFSEAG (SEQ ID NO: 311);
SPLPLRVP (SEQ ID NO: 312); RMHLRSLG (SEQ ID NO: 313); LAAPLGLL (SEQ
ID NO: 314); AVGLLAPP (SEQ ID NO: 315); LLAPSHRA (SEQ ID NO: 316);
PAGLWLDP (SEQ ID NO: 317); and/or ISSGLSS (SEQ ID NO: 318).
[0152] In some embodiments, activatable antibodies for use in the
conjugated activatable antibodies of the disclosure may be made
biosynthetically using recombinant DNA technology and expression in
eukaryotic or prokaryotic species. The cDNAs encoding the masking
moiety, linker sequence (that may include a cleavable moiety (CM),
and antibody chain (heavy or light)) can be linked in an 5' to 3'
(N- to C-terminal in the translated product) sequence to create the
nucleic acid construct, which is expressed as the activatable
antibody protein following a conventional antibody expression
process. In some embodiments, the activatable antibody could be
semi-synthetically produced by expressing a CM-antibody and then
coupling the mask chemically at or near the N-terminus of the
protein. In some embodiments, the activatable antibody could be
produced by expressing an antibody and then coupling the mask and
the CM chemically at or near the N-terminus of the protein such
that the activatable antibody in the uncleaved state has the
structural arrangement from N-terminus to C-terminus as follows:
MM-CM-AB or AB-CM-MM.
[0153] The conjugated activatable antibodies described herein also
include an agent conjugated to the activatable antibody. In some
embodiments, the conjugated agent is a therapeutic agent, such as
an antineoplastic agent. In some embodiments, the agent is
conjugated to a sulfhydryl group of the antibody or antigen-binding
fragment in the activatable antibody. In some embodiments, the
agent is a thiol-containing agent. In some embodiments, the agent
is engineered to include one or more thiol groups.
[0154] In some embodiments, the agent is a cytotoxic agent such as
a 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). Suitable cytotoxic agents
include, for example, any of the cytotoxic agents listed in Table
4.
[0155] In some embodiments, the agent is a thiol-containing agent.
In some embodiments, the agent is engineered to include one or more
thiol groups. In some embodiments, the agent is a microtubule
inhibitor. In some embodiments, the agent is a dolastatin or a
derivative thereof (e.g. auristatin E, AFP, MMAF, MMAE, MMAD, DMAF,
DMAE). In some embodiments, the agent is monomethyl auristatin E
(MMAE). In some embodiments, the agent is monomethyl auristatin D
(MMAD). In some embodiments, the agent is a maytansinoid or
maytansinoid derivative. In some embodiments, the agent is DM1 or
DM4. In some embodiments, the agent is a nucleic acid damaging
agent. In some embodiments, the agent is a duocarmycin or
derivative thereof. In some embodiments, the agent is a
calicheamicin or derivative thereof.
[0156] In some embodiments, the agent is linked to the AB using a
maleimide caproyl-valine-citrulline linker or a maleimide
PEG-valine-citrulline linker. In some embodiments, the agent is
linked to the AB using a maleimide caproyl-valine-citrulline
linker. In some embodiments, the agent is linked to the AB using a
maleimide PEG-valine-citrulline linker In some embodiments, the
agent is monomethyl auristatin D (MMAD) linked to the AB using a
maleimide PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker,
and this linker payload construct is referred to herein as
"vc-MMAD." In some embodiments, the agent is monomethyl auristatin
E (MMAE) linked to the AB using a maleimide
PEG-valine-citrulline-para-aminobenzyloxycarbonyl linker, and this
linker payload construct is referred to herein as "vc-MMAE." The
structures of vc-MMAD and vc-MMAE are shown below:
##STR00001##
[0157] In some embodiments, in addition to the compositions and
methods provided herein, the conjugated activatable antibody can
also be modified for site-specific conjugation through modified
amino acid sequences inserted or otherwise included in the
activatable antibody sequence. These modified amino acid sequences
are designed to allow for controlled placement and/or dosage of the
conjugated agent within a conjugated activatable antibody. For
example, the activatable antibody can be engineered to include
cysteine substitutions at positions on light and heavy chains that
provide reactive thiol groups and do not negatively impact protein
folding and assembly, nor alter antigen binding. In some
embodiments, the activatable antibody can be engineered to include
or otherwise introduce one or more non-natural amino acid residues
within the activatable antibody to provide suitable sites for
conjugation. In some embodiments, the activatable antibody can be
engineered to include or otherwise introduce enzymatically
activatable peptide sequences within the activatable antibody
sequence.
[0158] In some embodiments, the agent is a detectable moiety such
as, for example, a label or other marker. For example, the agent is
or includes a radiolabeled amino acid, one or more biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods), one or more
radioisotopes or radionuclides, one or more fluorescent labels, one
or more enzymatic labels, and/or one or more chemiluminescent
agents. In some embodiments, detectable moieties are attached by
spacer molecules.
[0159] 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, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are available for the
production of radioconjugated antibodies. Examples include
.sup.212Bi, .sup.131I, .sup.131In, .sup.90Y, and .sup.186Re.
[0160] Conjugates of the antibody and 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 glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 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
radionuclide to the antibody. (See WO94/11026).
[0161] Those of ordinary skill in the art will recognize that a
large variety of possible moieties can be coupled to the resultant
antibodies of the invention. (See, for example, "Conjugate
Vaccines", Contributions to Microbiology and Immunology, J. M.
Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989),
the entire contents of which are incorporated herein by
reference).
[0162] Table 4 lists some of the exemplary pharmaceutical agents
that may be employed in the herein described invention but in no
way is meant to be an exhaustive list.
TABLE-US-00008 TABLE 4 Exemplary Pharmaceutical Agents for
Conjugation CYTOTOXIC AGENTS Auristatins Auristatin E Monomethyl
auristatin D (MMAD) Monomethyl auristatin E (MMAE) Desmethyl
auristatin E (DMAE) Auristatin F Monomethyl auristatin F (MMAF)
Desmethyl auristatin F (DMAF) Auristatin derivatives, e.g., amides
thereof Auristatin tyramine Auristatin quinoline Dolastatins
Dolastatin derivatives Dolastatin 16 DmJ Dolastatin 16 Dpv
Maytansinoids, e.g. DM-1; DM-4 Maytansinoid derivatives Duocarmycin
Duocarmycin derivatives Alpha-amanitin Anthracyclines Doxorubicin
Daunorubicin Bryostatins Camptothecin Camptothecin derivatives
7-substituted Camptothecin 10,11-Difluoromethylenedioxycamptothecin
Combretastatins Debromoaplysiatoxin Kahalalide-F Discodermolide
Ecteinascidins ANTIVIRALS Acyclovir Vira A Symmetrel ANTIFUNGALS
Nystatin ADDITIONAL ANTI-NEOPLASTICS Adriamycin Cerubidine
Bleomycin Alkeran Velban Oncovin Fluorouracil Methotrexate Thiotepa
Bisantrene Novantrone Thioguanine Procarabizine Cytarabine
ANTI-BACTERIALS Aminoglycosides Streptomycin Neomycin Kanamycin
Amikacin Gentamicin Tobramycin Streptomycin B Spectinomycin
Ampicillin Sulfanilamide Polymyxin Chloramphenicol Turbostatin
Phenstatins Hydroxyphenstatin Spongistatin 5 Spongistatin 7
Halistatin 1 Halistatin 2 Halistatin 3 Modified Bryostatins
Halocomstatins Pyrrolobenzimidazoles (PBI) Cibrostatin6 Doxaliform
Anthracyclins analogues Anthracyclins analogues Cemadotin analogue
(CemCH2-SH) Pseudomonas toxin A (PE38) variant Pseudomonas toxin A
(ZZ-PE38) variant ZJ-101 OSW-1 4-Nitrobenzyloxycarbonyl Derivatives
of O6-Benzylguanine Topoisomerase inhibitors Hemiasterlin
Cephalotaxine Homoharringtonine Pyrrolobenzodiazepine dimers (PBDs)
Pyrrolobenzodiazepenes Functionalized pyrrolobenzodiazepenes
Calicheamicins Podophyllotoxins Taxanes Vinca alkaloids
CONJUGATABLE DETECTION REAGENTS Fluorescein and derivatives thereof
Fluorescein isothiocyanate (FITC) RADIOISOTOPES .sup.125I .sup.131I
.sup.89Zr .sup.111In .sup.123I .sup.131I .sup.99mTc .sup.201Tl
.sup.133Xe .sup.11C .sup.62Cu .sup.18F .sup.68Ga .sup.13N .sup.15O
.sup.38K .sup.82Rb .sup.99mTc (Technetium) HEAVY METALS Barium Gold
Platinum ANTI-MYCOPLASMALS Tylosine Spectinomycin
[0163] In some embodiments, in addition to the compositions and
methods provided herein, the conjugated activatable antibody can
also be coupled using any chemical reaction that will bind the two
molecules so long as the antibody and the other moiety retain their
respective activities. This linkage can include many chemical
mechanisms, for instance covalent binding, affinity binding,
intercalation, coordinate binding and complexation. In some
embodiments, the binding is covalent binding. Covalent binding can
be achieved either by direct condensation of existing side chains
or by the incorporation of external bridging molecules. Many
bivalent or polyvalent linking agents are useful in coupling
protein molecules, such as the activatable antibodies of the
present invention, to other molecules. For example, representative
coupling agents can include organic compounds such as thioesters,
carbodiimides, succinimide esters, diisocyanates, glutaraldehyde,
diazobenzenes and hexamethylene diamines. This listing is not
intended to be exhaustive of the various classes of coupling agents
known in the art but, rather, is exemplary of the more common
coupling agents. (See Killen and Lindstrom, Jour. Immun.
133:1335-2549 (1984); Jansen et al., Immunological Reviews
62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).
[0164] Suitable linkers are described in the literature. (See, for
example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984)
describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide
ester). See also, U.S. Pat. No. 5,030,719, describing use of
halogenated acetyl hydrazide derivative coupled to an antibody by
way of an oligopeptide linker. Suitable linkers include: (i) SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (ii) SPDP (succinimidyl-6
[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); and (iii) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.
#2165-G. Additional linkers include, but are not limited to, SMCC,
sulfo-SMCC, SPDB, or sulfo-SPDB.
[0165] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, the linker SMPT contains a
sterically hindered disulfide bond, and can form conjugates with
increased stability. Disulfide linkages, are in general, less
stable than other linkages because the disulfide linkage is cleaved
in vitro, resulting in less conjugate available.
[0166] The reagent EDC
(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is
useful to create a carboxamide starting with a carboxylic acid and
a primary or secondary amine. Thus, EDC may be used to link lysine
residues in an antibody with a carboxylic acid in a linker or
toxin, or to link aspartate or glutamate residues in an antibody
with an amine in a linker or toxin. Such conjugation reactions
utilizing EDC may be enhanced by addition of NHS
(N-hydroxysuccinimide) or sulfo-NHS
(N-hydroxy-3-oxysulfonylsuccinimide). Addition of NHS or sulfo-NHS
to such conjugation reactions may enhance the rate, completeness,
selectivity, and/or reproducibility of the conjugation
reactions.
[0167] In some embodiments, the linkers are cleavable. In some
embodiments, the linkers are non-cleavable. In some embodiments,
two or more linkers are present. The two or more linkers are all
the same, e.g., cleavable or non-cleavable, or the two or more
linkers are different, e.g., at least one cleavable and at least
one non-cleavable.
[0168] In some embodiments, in addition to the compositions and
methods provided herein, the conjugated activatable antibody can
also be further conjugated using any of several methods for
attaching agents to ABs: (a) attachment to the carbohydrate
moieties of the AB, or (b) attachment to sulfhydryl groups of the
AB, or (c) attachment to amino groups of the AB, or (d) attachment
to carboxylate groups of the AB. According to the invention, ABs
may be covalently attached to an agent through an intermediate
linker having at least two reactive groups, one to react with AB
and one to react with the agent. The linker, which may include any
compatible organic compound, can be chosen such that the reaction
with AB (or agent) does not adversely affect AB reactivity and
selectivity. Furthermore, the attachment of linker to agent might
not destroy the activity of the agent. Suitable linkers for
reaction with oxidized antibodies or oxidized antibody fragments
include those containing an amine selected from the group
consisting of primary amine, secondary amine, hydrazine, hydrazide,
hydroxylamine, phenylhydrazine, semicarbazide and thiosemicarbazide
groups. Such reactive functional groups may exist as part of the
structure of the linker, or may be introduced by suitable chemical
modification of linkers not containing such groups.
[0169] According to the present invention, suitable linkers for
attachment to reduced ABs include those having certain reactive
groups capable of reaction with a sulfhydryl group of a reduced
antibody or fragment. Such reactive groups include, but are not
limited to: reactive haloalkyl groups (including, for example,
haloacetyl groups), p-mercuribenzoate groups and groups capable of
Michael-type addition reactions (including, for example, maleimides
and groups of the type described by Mitra and Lawton, 1979, J.
Amer. Chem. Soc. 101: 3097-3110).
[0170] According to the present invention, suitable linkers for
attachment to neither oxidized nor reduced ABs include those having
certain functional groups capable of reaction with the primary
amino groups present in unmodified lysine residues in the AB. Such
reactive groups include, but are not limited to, NHS carboxylic or
carbonic esters, sulfo-NHS carboxylic or carbonic esters,
4-nitrophenyl carboxylic or carbonic esters, pentafluorophenyl
carboxylic or carbonic esters, acyl imidazoles, isocyanates, and
isothiocyanates.
[0171] According to the present invention, suitable linkers for
attachment to neither oxidized nor reduced ABs include those having
certain functional groups capable of reaction with the carboxylic
acid groups present in aspartate or glutamate residues in the AB,
which have been activated with suitable reagents. Suitable
activating reagents include EDC, with or without added NHS or
sulfo-NHS, and other dehydrating agents utilized for carboxamide
formation. In these instances, the functional groups present in the
suitable linkers would include primary and secondary amines,
hydrazines, hydroxylamines, and hydrazides.
[0172] The agent may be attached to the linker before or after the
linker is attached to the AB. In certain applications it may be
desirable to first produce an AB-linker intermediate in which the
linker is free of an associated agent. Depending upon the
particular application, a specific agent may then be covalently
attached to the linker. In some embodiments the AB is first
attached to the MM, CM and associated linkers and then attached to
the linker for conjugation purposes.
[0173] Branched Linkers:
[0174] In specific embodiments, branched linkers that have multiple
sites for attachment of agents are utilized. For multiple site
linkers, a single covalent attachment to an AB would result in an
AB-linker intermediate capable of binding an agent at a number of
sites. The sites may be aldehyde or sulfhydryl groups or any
chemical site to which agents can be attached.
[0175] In some embodiments, higher specific activity (or higher
ratio of agents to AB) can be achieved by attachment of a single
site linker at a plurality of sites on the AB. This plurality of
sites may be introduced into the AB by either of two methods.
First, one may generate multiple aldehyde groups and/or sulfhydryl
groups in the same AB. Second, one may attach to an aldehyde or
sulfhydryl of the AB a "branched linker" having multiple functional
sites for subsequent attachment to linkers. The functional sites of
the branched linker or multiple site linker may be aldehyde or
sulfhydryl groups, or may be any chemical site to which linkers may
be attached. Still higher specific activities may be obtained by
combining these two approaches, that is, attaching multiple site
linkers at several sites on the AB.
[0176] Cleavable Linkers:
[0177] Peptide linkers that are susceptible to cleavage by enzymes
of the complement system, such as but not limited to urokinase,
tissue plasminogen activator, trypsin, plasmin, or another enzyme
having proteolytic activity may be used in one embodiment of the
present invention. According to one method of the present
invention, an agent is attached via a linker susceptible to
cleavage by complement. The antibody is selected from a class that
can activate complement. The antibody-agent conjugate, thus,
activates the complement cascade and releases the agent at the
target site. According to another method of the present invention,
an agent is attached via a linker susceptible to cleavage by
enzymes having a proteolytic activity such as a urokinase, a tissue
plasminogen activator, plasmin, or trypsin. These cleavable linkers
are useful in conjugated activatable antibodies that include an
extracellular toxin, e.g., by way of non-limiting example, any of
the extracellular toxins shown in Table 4.
[0178] Non-limiting examples of cleavable linker sequences are
provided in Table 5.
TABLE-US-00009 TABLE 5 Exemplary Linker Sequences for Conjugation
Amino Acid Types of Cleavable Sequences Sequence Plasmin cleavable
sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 203) PRFRIIGG (SEQ ID
NO: 204) TGF.beta. SSRHRRALD (SEQ ID NO: 205) Plasminogen
RKSSIIIRMRDVVL (SEQ ID NO: 206) Staphylokinase SSSFDKGKYKKGDDA (SEQ
ID NO: 207) SSSFDKGKYKRGDDA (SEQ ID NO: 208) Factor Xa cleavable
sequences IEGR (SEQ ID NO: 209) IDGR (SEQ ID NO: 210) GGSIDGR (SEQ
ID NO: 211) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO:
212) Collagenase cleavable sequences Calf skin collagen GPQGIAGQ
(.alpha.1(I) chain) (SEQ ID NO: 213) Calf skin collagen GPQGLLGA
(.alpha.2(I) chain) (SEQ ID NO: 214) Bovine cartilage collagen
GIAGQ (.alpha.1(II) chain) (SEQ ID NO: 215) Human liver collagen
GPLGIAGI (.alpha.1(III) chain) (SEQ ID NO: 216) Human
.alpha..sub.2M GPEGLRVG (SEQ ID NO: 217) Human PZP YGAGLGVV (SEQ ID
NO: 218) AGLGVVER (SEQ ID NO: 219) AGLGISST (SEQ ID NO: 220) Rat
.alpha..sub.1M EPQALAMS (SEQ ID NO: 221) QALAMSAI (SEQ ID NO: 222)
Rat .alpha..sub.2M AAYHLVSQ (SEQ ID NO: 223) MDAFLESS (SEQ ID NO:
224) Rat .alpha..sub.1I.sub.3(2J) ESLPVVAV (SEQ ID NO: 225) Rat
.alpha..sub.1I.sub.3(27J) SAPAVESE (SEQ ID NO: 226) Human
fibroblast collagenase DVAQFVLT (SEQ ID NO: 227) (autolytic
cleavages) VAQFVLTE (SEQ ID NO: 228) AQFVLTEG (SEQ ID NO: 229)
PVQPIGPQ (SEQ ID NO: 230)
[0179] In addition, agents may be attached via disulfide bonds (for
example, the disulfide bonds on a cysteine molecule) to the AB.
Since many tumors naturally release high levels of glutathione (a
reducing agent) this can reduce the disulfide bonds with subsequent
release of the agent at the site of delivery. In certain specific
embodiments the reducing agent that would modify a CM would also
modify the linker of the conjugated activatable antibody.
[0180] Spacer Elements and Cleavable Elements:
[0181] In some embodiments, it may be necessary to construct the
linker in such a way as to optimize the spacing between the agent
and the AB of the activatable antibody. This may be accomplished by
use of a linker of the general structure:
W--(CH.sub.2)n-Q
wherein W is either --NH--CH.sub.2-- or --CH.sub.2--; Q is an amino
acid, peptide; and n is an integer from 0 to 20.
[0182] In some embodiments, the linker may comprise a spacer
element and a cleavable element. The spacer element serves to
position the cleavable element away from the core of the AB such
that the cleavable element is more accessible to the enzyme
responsible for cleavage. Certain of the branched linkers described
above may serve as spacer elements.
[0183] Throughout this discussion, it should be understood that the
attachment of linker to agent (or of spacer element to cleavable
element, or cleavable element to agent) need not be effected by a
particular mode of attachment or reaction. Any reaction providing a
product of suitable stability and biological compatibility is
acceptable.
[0184] Serum Complement and Selection of Linkers:
[0185] According to one method of the present invention, when
release of an agent is desired, an AB that is an antibody of a
class that can activate complement is used. The resulting conjugate
retains both the ability to bind antigen and activate the
complement cascade. Thus, according to this embodiment of the
present invention, an agent is joined to one end of the cleavable
linker or cleavable element and the other end of the linker group
is attached to a specific site on the AB. For example, if the agent
has an hydroxy group or an amino group, it may be attached to the
carboxy terminus of a peptide, amino acid or other suitably chosen
linker via an ester or amide bond, respectively. For example, such
agents may be attached to the linker peptide via a carbodimide
reaction. If the agent contains functional groups that would
interfere with attachment to the linker, these interfering
functional groups can be blocked before attachment and deblocked
once the product conjugate or intermediate is made. The opposite or
amino terminus of the linker is then used either directly or after
further modification for binding to an AB that is capable of
activating complement.
[0186] Linkers (or spacer elements of linkers) may be of any
desired length, one end of which can be covalently attached to
specific sites on the AB of the activatable antibody. The other end
of the linker or spacer element may be attached to an amino acid or
peptide linker.
[0187] Thus when these conjugates bind to antigen in the presence
of complement the amide or ester bond that attaches the agent to
the linker will be cleaved, resulting in release of the agent in
its active form. These conjugates, when administered to a subject,
will accomplish delivery and release of the agent at the target
site, and are particularly effective for the in vivo delivery of
pharmaceutical agents, antibiotics, antimetabolites,
antiproliferative agents and the like as presented in but not
limited to those in Table 4.
[0188] Linkers for Release without Complement Activation:
[0189] In yet another application of targeted delivery, release of
the agent without complement activation is desired since activation
of the complement cascade will ultimately lyse the target cell.
Hence, this approach is useful when delivery and release of the
agent should be accomplished without killing the target cell. Such
is the goal when delivery of cell mediators such as hormones,
enzymes, corticosteroids, neurotransmitters, genes or enzymes to
target cells is desired. These conjugates may be prepared by
attaching the agent to an AB that is not capable of activating
complement via a linker that is mildly susceptible to cleavage by
serum proteases. When this conjugate is administered to an
individual, antigen-antibody complexes will form quickly whereas
cleavage of the agent will occur slowly, thus resulting in release
of the compound at the target site.
[0190] Biochemical Cross Linkers:
[0191] In some embodiments, the activatable antibody may be
conjugated to one or more therapeutic agents using certain
biochemical cross-linkers. Cross-linking reagents form molecular
bridges that tie together functional groups of two different
molecules. To link two different proteins in a step-wise manner,
hetero-bifunctional cross-linkers can be used that eliminate
unwanted homopolymer formation.
[0192] Peptidyl linkers cleavable by lysosomal proteases are also
useful, for example, Val-Cit, Val-Ala or other dipeptides. In
addition, acid-labile linkers cleavable in the low-pH environment
of the lysosome may be used, for example: bis-sialyl ether. Other
suitable linkers include cathepsin-labile substrates, particularly
those that show optimal function at an acidic pH.
[0193] Exemplary hetero-bifunctional cross-linkers are referenced
in Table 6.
TABLE-US-00010 TABLE 6 Exemplary Hetero-Bifunctional Cross Linkers
HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm Length after Reactive
Advantages and cross-linking Linker Toward Applications (Angstroms)
SMPT Primary amines Greater stability 11.2 .ANG. Sulfhydryls SPDP
Primary amines Thiolation 6.8 .ANG. Sulfhydryls Cleavable cross-
linking LC-SPDP Primary amines Extended spacer arm 15.6 .ANG.
Sulfhydryls Sulfo-LC- Primary amines Extender spacer arm 15.6 .ANG.
SPDP Sulfhydryls Water-soluble SMCC Primary amines Stable maleimide
11.6 .ANG. reactive group Sulfhydryls Enzyme-antibody conjugation
Hapten-carrier protein conjugation Sulfo-SMCC Primary amines Stable
maleimide 11.6 .ANG. reactive group Sulfhydryls Water-soluble
Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody 9.9
.ANG. conjugation Sulfhydryls Hapten-carrier protein conjugation
Sulfo-MBS Primary amines Water-soluble 9.9 .ANG. Sulfhydryls SIAB
Primary amines Enzyme-antibody 10.6 .ANG. conjugation Sulfhydryls
Sulfo-SIAB Primary amines Water-soluble 10.6 .ANG. Sulfhydryls SMPB
Primary amines Extended spacer arm 14.5 .ANG. Sulfhydryls
Enzyme-antibody conjugation Sulfo-SMPB Primary amines Extended
spacer arm 14.5 .ANG. Sulfhydryls Water-soluble EDE/Sulfo- Primary
amines Hapten-Carrier 0 NHS conjugation Carboxyl groups ABH
Carbohydrates Reacts with sugar 11.9 .ANG. groups Nonselective
[0194] Non-Cleavable Linkers or Direct Attachment:
[0195] In some embodiments of the invention, the conjugate may be
designed so that the agent is delivered to the target but not
released. This may be accomplished by attaching an agent to an AB
either directly or via a non-cleavable linker.
[0196] These non-cleavable linkers may include amino acids,
peptides, D-amino acids or other organic compounds that may be
modified to include functional groups that can subsequently be
utilized in attachment to ABs by the methods described herein.
A-general formula for such an organic linker could be
W--(CH.sub.2)n-Q
wherein W is either --NH--CH.sub.2-- or --CH.sub.2--; Q is an amino
acid, peptide; and n is an integer from 0 to 20.
[0197] Non-Cleavable Conjugates:
[0198] In some embodiments, a compound may be attached to ABs that
do not activate complement. When using ABs that are incapable of
complement activation, this attachment may be accomplished using
linkers that are susceptible to cleavage by activated complement or
using linkers that are not susceptible to cleavage by activated
complement.
Reducing Agents
[0199] Reducing Agent:
[0200] Examples of reducing agents suitable for use in the
compositions and methods of the disclosure include, by way of
non-limiting example, BMS (bis(2-mercaptoethyl)sulfone),
cysteamine, cysteine, DMH (dimethyl-bis-mercaptoacetyl hydrazine),
DTBA (dithiobutylamine), DTT (dithiothreitol), GILT (gamma
interferon inducible lysosomal thiol reductase; for enzymatic
reduction), glutathione, .beta.-mercaptoethanol, MEA
(2-mercaptoethylamine), pyridine-2-thione, sodium borohydride,
sodium phosphorothioate, TCEP ((tris(2-carboxyethyl)phosphine)),
and thiopropyl-agarose. In some embodiments, the reducing agent is
DTT, .beta.-mercaptoethanol or TCEP.
[0201] The studies provided herein use the reducing agent TCEP
(tris(2-carboxyethyl)phosphine), which has the following
structure:
##STR00002##
[0202] TCEP is often used as a reducing agent to cleave disulfide
bonds within and between proteins. TCEP is very selective and does
not react toward other functional groups found within proteins.
TCEP does not react with buried disulfides.
[0203] Compared to the other two most common agents used for this
purpose (DTT and .beta.-mercaptoethanol), TCEP has the advantages
of being odorless, a more powerful reducing agent, an irreversible
reducing agent, more hydrophilic, and more resistant to oxidation
in air. Unlike DTT, TCEP is active at both alkaline and acidic
conditions. TCEP is particularly useful when labeling cysteine
residues with maleimides. TCEP can keep the cysteines from forming
disulfide bonds, and unlike DTT and f-mercaptoethanol, it will not
react as readily with the maleimide.
[0204] The ratio of reduction agent to activatable antibody will
vary depending on the activatable antibody. In some embodiments,
the ratio of reducing agent to activatable antibody will be in a
range from about 20:1 to 1:1, from about 10:1 to 1:1, from about
9:1 to 1:1, from about 8:1 to 1:1, from about 7:1 to 1:1, from
about 6:1 to 1:1, from about 5:1 to 1:1, from about 4:1 to 1:1,
from about 3:1 to 1:1, from about 2:1 to 1:1, from about 20:1 to
1:1.5, from about 10:1 to 1:1.5, from about 9:1 to 1:1.5, from
about 8:1 to 1:1.5, from about 7:1 to 1:1.5, from about 6:1 to
1:1.5, from about 5:1 to 1:1.5, from about 4:1 to 1:1.5, from about
3:1 to 1:1.5, from about 2:1 to 1:1.5, from about 1.5:1 to 1:1.5,
or from about 1:1 to 1:1.5. In some embodiments, the ratio is in a
range of from about 5:1 to 1:1. In some embodiments, the ratio is
in a range of from about 5:1 to 1.5:1. In some embodiments, the
ratio is in a range of from about 4:1 to 1:1. In some embodiments,
the ratio is in a range from about 4:1 to 1.5:1. In some
embodiments, the ratio is in a range from about 8:1 to about 1:1.
In some embodiments, the ratio is in a range of from about 2.5:1 to
1:1.
DEFINITIONS
[0205] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well-known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
foregoing techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See e.g.,
Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)). The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well-known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0206] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0207] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically bind" or "immunoreacts with" or "immunospecifically
bind" is meant that the antibody reacts with one or more antigenic
determinants of the desired antigen and does not react with other
polypeptides or binds at much lower affinity
(K.sub.d>10.sup.-6). Antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, domain antibody, single chain,
Fab, and F(ab').sub.2 fragments, scFvs, and an Fab expression
library.
[0208] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. In general, antibody
molecules obtained from humans relate to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain.
[0209] The term "monoclonal antibody" (mAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs contain an antigen
binding site capable of immunoreacting with a particular epitope of
the antigen characterized by a unique binding affinity for it.
[0210] The term "antigen-binding site" or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences that are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature
342:878-883 (1989).
[0211] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin, an
scFv, or a T-cell receptor. The term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin or
T-cell receptor. Epitopic determinants usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. For example, antibodies may be raised against
N-terminal or C-terminal peptides of a polypeptide. An antibody is
said to specifically bind an antigen when the dissociation constant
is .ltoreq.1 .mu.M; in some embodiments, the dissociation constant
is .ltoreq.100 nM; in some embodiments, the dissociation constant
is .ltoreq.10 nM.
[0212] As used herein, the terms "specific binding," "immunological
binding," and "immunological binding properties" refer to the
non-covalent interactions of the type that occur between an
immunoglobulin molecule and an antigen for which the immunoglobulin
is specific. The strength, or affinity of immunological binding
interactions can be expressed in terms of the dissociation constant
(K.sub.d) of the interaction, wherein a smaller K.sub.d represents
a greater affinity. Immunological binding properties of selected
polypeptides can be quantified using methods well known in the art.
One such method entails measuring the rates of antigen-binding
site/antigen complex formation and dissociation, wherein those
rates depend on the concentrations of the complex partners, the
affinity of the interaction, and geometric parameters that equally
influence the rate in both directions. Thus, both the "on rate
constant" (K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to a target, when the
dissociation binding constant (K.sub.d) is .ltoreq.1 .mu.M as
measured by assays such as radioligand binding assays or similar
assays known to those skilled in the art. In some embodiments, the
K.sub.d is .ltoreq.100 nM. In some embodiments, the K.sub.d is
.ltoreq.10 nM. In some embodiments, the K.sub.d is .ltoreq.1 nM. In
some embodiments, the K.sub.d is .ltoreq.100 .mu.M to about 1
.mu.M.
[0213] The compositions and methods provided herein enable the
attachment of one or more agents to one or more cysteine residues
in the AB without compromising the activity (e.g., the masking,
activating or binding activity) of the activatable antibody.
[0214] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide that it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. Polynucleotides in accordance with the invention
include the nucleic acid molecules encoding the heavy chain
immunoglobulin molecules shown herein, and nucleic acid molecules
encoding the light chain immunoglobulin molecules shown herein.
[0215] The term "isolated protein" referred to herein means a
protein expressed from cDNA or recombinant RNA, or a protein of
synthetic origin or some combination thereof, which by virtue of
its origin, or source of derivation, the "isolated protein" (1) is
not associated with proteins found in nature, (2) is free of other
proteins from the same source, (3) is expressed by a cell from a
different species, or (4) does not occur in nature.
[0216] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein fragments, and analogs are species
of the polypeptide genus. Polypeptides in accordance with the
invention comprise the heavy chain immunoglobulin molecules shown
herein, and the light chain immunoglobulin molecules shown herein,
as well as antibody molecules formed by combinations comprising the
heavy chain immunoglobulin molecules with light chain
immunoglobulin molecules, such as kappa light chain immunoglobulin
molecules, and vice versa, as well as fragments and analogs
thereof.
[0217] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and that has not been intentionally
modified by man in the laboratory or otherwise is
naturally-occurring.
[0218] The term "operably linked" as used herein refers to
positions of components so described are in a relationship
permitting them to function in their intended manner. A control
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
conditions compatible with the control sequences.
[0219] The term "control sequence" as used herein refers to
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. The nature of such control sequences differs depending
upon the host organism: in prokaryotes and eukaryotes, such control
sequences generally include promoter, ribosomal binding site, and
transcription termination sequence. The term "control sequences" is
intended to include, at a minimum, all components whose presence is
essential for expression and processing, and can also include
additional components whose presence is advantageous, for example,
leader sequences and fusion partner sequences. The term
"polynucleotide" as referred to herein means nucleotides of at
least 10 bases in length, either ribonucleotides or
deoxynucleotides or a modified form of either type of nucleotide.
The term includes single and double stranded forms of DNA.
[0220] The term oligonucleotide referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally
comprising a length of 200 bases or fewer. In some embodiments,
oligonucleotides are 10 to 60 bases in length. In some embodiments,
the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to
40 bases in length. Oligonucleotides are usually single stranded,
e.g., for probes, although oligonucleotides may be double stranded,
e.g., for use in the construction of a gene mutant.
Oligonucleotides of the invention are either sense or antisense
oligonucleotides.
[0221] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotide linkages such as phosphorothioate,
phosphorodithioate, phosphoroselerloate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoronmidate, and the
like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984), Stein et al. Nucl.
Acids Res. 16:3209 (1988), Zon et al. Anti Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990). An
oligonucleotide can include a label for detection, if desired.
[0222] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland7 Mass. (1991)). Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for polypeptides of the present
invention. Examples of unconventional amino acids include: 4
hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, a .epsilon.N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0223] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction. Sequence regions on the DNA strand having the same
sequence as the RNA and that are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences". Sequence
regions on the DNA strand having the same sequence as the RNA and
that are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0224] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity. In some embodiments, the two
peptide sequences share at least 90 percent sequence identity. In
some embodiments, the two peptide sequences share at least 95
percent sequence identity. In some embodiments, the two peptide
sequences share at least 99 percent sequence identity.
[0225] In some embodiments, residue positions that are not
identical differ by conservative amino acid substitutions.
[0226] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75% amino acid sequence identity to a reference sequence
(e.g., the wild-type sequence). In some embodiments, the variations
in the amino acid sequence maintain at least 80%, 90%, 95%, or 99%
amino acid identity to the reference sequence. In particular,
conservative amino acid replacements are contemplated. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains. Genetically encoded
amino acids are generally divided into families: (1) acidic amino
acids are aspartate, glutamate; (2) basic amino acids are lysine,
arginine, histidine; (3) non-polar amino acids are alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan, and (4) uncharged polar amino acids are glycine,
asparagine, glutamine, cysteine, serine, threonine, tyrosine. The
hydrophilic amino acids include arginine, asparagine, aspartate,
glutamine, glutamate, histidine, lysine, serine, and threonine. The
hydrophobic amino acids include alanine, cysteine, isoleucine,
leucine, methionine, phenylalanine, proline, tryptophan, tyrosine
and valine. Other families of amino acids include (i) serine and
threonine, which are the aliphatic-hydroxy family; (ii) asparagine
and glutamine, which are the amide containing family; (iii)
alanine, valine, leucine and isoleucine, which are the aliphatic
family; and (iv) phenylalanine, tryptophan, and tyrosine, which are
the aromatic family. For example, it is reasonable to expect that
an isolated replacement of a leucine with an isoleucine or valine,
an aspartate with a glutamate, a threonine with a serine, or a
similar replacement of an amino acid with a structurally related
amino acid will not have a major effect on the binding or other
properties of the resulting molecule, for example, in situations
where the replacement does not involve an amino acid within a
complementarity determining region (CDR) or other variable region.
Whether an amino acid change results in a functional peptide can
readily be determined by assaying the specific activity of the
polypeptide derivative. Assays are described in detail herein.
Fragments or analogs of antibodies or immunoglobulin molecules can
be readily prepared by those of ordinary skill in the art. In some
embodiments, amino- and carboxy-termini of fragments or analogs
occur near boundaries of functional domains. Structural and
functional domains can be identified by comparison of the
nucleotide and/or amino acid sequence data to public or proprietary
sequence databases. In some embodiments, computerized comparison
methods are used to identify sequence motifs or predicted protein
conformation domains that occur in other proteins of known
structure and/or function. Methods to identify protein sequences
that fold into a known three-dimensional structure are known. Bowie
et al. Science 253:164 (1991). Thus, the foregoing examples
demonstrate that those of skill in the art can recognize sequence
motifs and structural conformations that may be used to define
structural and functional domains in accordance with the
invention.
[0227] In some embodiments, amino acid substitutions are those
that: (1) decrease susceptibility to proteolysis, (2) decrease
susceptibility to oxidation, (3) alter binding affinity for forming
protein complexes, (4) alter binding affinities, and (4) confer or
modify other physicochemical or functional properties of such
analogs. Analogs can include various muteins of a sequence other
than the naturally-occurring peptide sequence. For example, single
or multiple amino acid substitutions (in some embodiments,
conservative amino acid substitutions) may be made in the
naturally-occurring sequence (in some embodiments, in the portion
of the polypeptide outside the domain(s) forming intermolecular
contacts. A conservative amino acid substitution should not
substantially change the structural characteristics of the parent
sequence (e.g., a replacement amino acid should not tend to break a
helix that occurs in the parent sequence, or disrupt other types of
secondary structure that characterizes the parent sequence).
Examples of art-recognized polypeptide secondary and tertiary
structures are described in Proteins, Structures and Molecular
Principles (Creighton, Ed., W. H. Freeman and Company, New York
(1984)); Introduction to Protein Structure (C. Branden and J.
Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and
Thornton et at. Nature 354:105 (1991).
[0228] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino terminal and/or carboxy-terminal
deletion and/or one or more internal deletion(s), but where the
remaining amino acid sequence is identical to the corresponding
positions in the naturally-occurring sequence deduced, for example,
from a full length cDNA sequence. Fragments typically are at least
2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids long. In some embodiments,
the fragment is an antibody fragment that is at least 14 amino
acids long. In some embodiments, the fragment is a fragment of the
AB that is least 20 amino acids long. In some embodiments, the
fragment is a fragment of the AB that is at least 50 amino acids
long. In some embodiments, the fragment is a fragment of the AB
that is at least 70 amino acids long. The term "analog" as used
herein refers to polypeptides that are comprised of a segment of at
least 25 amino acids that has substantial identity to a portion of
a deduced amino acid sequence and that has specific binding to a
target, under suitable binding conditions. Typically, polypeptide
analogs comprise a conservative amino acid substitution (or
addition or deletion) with respect to the naturally-occurring
sequence. Analogs typically are at least 20 amino acids long, in
some embodiments, at least 50 amino acids long or longer, and can
often be as long as a full-length naturally-occurring
polypeptide.
[0229] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0230] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods). In certain
situations, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I), fluorescent
labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic
labels (e.g., horseradish peroxidase, p-galactosidase, luciferase,
alkaline phosphatase), chemiluminescent, biotinyl groups,
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In some
embodiments, labels are attached by spacer arms of various lengths
to decrease potential steric hindrance. The term "pharmaceutical
agent or drug" as used herein refers to a chemical compound or
composition capable of inducing a desired therapeutic effect when
properly administered to a patient.
[0231] The term "drug" as used herein means an element, compound,
agent, or molecular entity, including, e.g., a pharmaceutical,
therapeutic, or pharmacologic compound. Drugs can be natural or
synthetic or a combination thereof. A "therapeutic drug" is an
agent that exerts a therapeutic (e.g., beneficial) effect on cancer
cells or immune cells (e.g., activated immune cells), either alone
or in combination with another agent (e.g., a prodrug converting
enzyme in combination with a prodrug). Typically, therapeutic drugs
useful in accordance with the methods and compositions described
herein are those that exert a cytotoxic, cytostatic, or
immunosuppressive effect. In certain embodiments, a drug is not a
radioactive element. The drug can be a thiol-containing agent
and/or the drug can be engineered to include one or more thiol
groups.
[0232] "Cytotoxic agent," in reference to the effect of an agent on
a cell, means killing of the cell. "Cytostatic agent" means an
inhibition of cell proliferation.
[0233] The term "interchain disulfide bond," in the context of an
antibody, refers to a disulfide bond between two heavy chains, or a
heavy and a light chain.
[0234] The term "interchain thiol" refers to a thiol group of an
antibody heavy or light chain that can participate in the formation
of an interchain disulfide bond.
[0235] A protein is referred to as "fully-loaded" when all points
of conjugation of a particular type and/or of similar reactivity
are conjugated to drugs, resulting in a homogeneous population of
protein-drug conjugate. A protein is referred to as
"partially-loaded" when only some of the possible points of
conjugation of a particular type and/or of a similar reactivity are
conjugated to drugs, resulting in formation of a certain isomer or
isomers of the protein-drug conjugate.
[0236] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San
Francisco (1985)).
[0237] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and in some embodiments, a substantially purified
fraction is a composition wherein the object species comprises at
least about 50 percent (on a molar basis) of all macromolecular
species present.
[0238] Generally, a substantially pure composition will comprise
more than about 80 percent of all macromolecular species present in
the composition, in some embodiments, more than about 85%, 90%,
95%, and 99%. In some embodiments, the object species is purified
to essential homogeneity (contaminant species cannot be detected in
the composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0239] The term patient includes human and veterinary subjects.
[0240] Use of Conjugated Activatable Antibodies
[0241] It will be appreciated that administration of conjugated
activatable antibodies in accordance with the invention will be
administered with suitable carriers, excipients, and other agents
that are incorporated into formulations to provide improved
transfer, delivery, tolerance, and the like. A multitude of
appropriate formulations can be found in the formulary known to all
pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th
ed, Mack Publishing Company, Easton, Pa. (1975)), particularly
Chapter 87 by Blaug, Seymour, therein. These formulations include,
for example, powders, pastes, ointments, jellies, waxes, oils,
lipids, lipid (cationic or anionic) containing vesicles (such as
Lipofectin.TM.), DNA conjugates, anhydrous absorption pastes,
oil-in-water and water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of various molecular weights), semi-solid
gels, and semi-solid mixtures containing carbowax. Any of the
foregoing mixtures may be appropriate in treatments and therapies
in accordance with the present invention, provided that the active
ingredient in the formulation is not inactivated by the formulation
and the formulation is physiologically compatible and tolerable
with the route of administration. See also Baldrick P.
"Pharmaceutical excipient development: the need for preclinical
guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.
"Lyophilization and development of solid protein pharmaceuticals."
Int. J. Pharm. 203(1-2): 1-60 (2000), Charman W N "Lipids,
lipophilic drugs, and oral drug delivery-some emerging concepts." J
Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol.
52:238-311 (1998) and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
[0242] Therapeutic formulations of the invention, which include a
conjugated activatable antibody, are used to prevent, treat or
otherwise ameliorate a disease or disorder associated with
expression and/or activity of a target. For example, therapeutic
formulations of the invention are used to treat or otherwise
ameliorate a cancer or other neoplastic condition. In some
embodiments the cancer is a solid tumor or a hematologic malignancy
where the target is expressed. In some embodiments the cancer is a
solid tumor where the target is expressed. In some embodiments the
cancer is a hematologic malignancy where the target is expressed.
In some embodiments, the target is expressed on parenchyma (e.g.,
in cancer, the portion of an organ or tissue that often carries out
function(s) of the organ or tissue). In some embodiments, the
target is expressed on a cell, tissue, or organ. In some
embodiments, the target is expressed on stroma (i.e., the
connective supportive framework of a cell, tissue, or organ). In
some embodiments, the target is expressed on an osteoblast. In some
embodiments, the target is expressed on the endothelium
(vasculature). In some embodiments, the target is expressed on a
cancer stem cell. In some embodiments, the agent to which the
activatable antibody is conjugated is a microtubule inhibitor. In
some embodiments, the agent to which the activatable antibody is
conjugated is a nucleic acid damaging agent.
[0243] Pathologies treated and/or prevented and/or for which the
progression is delayed and/or for which a symptom is ameliorated
using the conjugated activatable anti-EGFR antibodies of the
invention include, for example, diseases or disorders associated
with expression and/or activity of EGFR. In some embodiments, the
disease or disorder associated with expression and/or activity of
EGFR is a cancer. In some embodiments, the cancer is a breast
cancer, e.g., by way of non-limiting example, the breast cancer is
a triple-negative breast cancer. In some embodiments, the cancer is
a triple-negative breast cancer. In some embodiments, the cancer is
colorectal cancer. In some embodiments, the cancer is gastric
cancer. In some embodiments, the cancer is glioblastoma. In some
embodiments, the cancer is a head and neck cancer, e.g., by way of
non-limiting example, esophageal cancer. In some embodiments, the
cancer is an esophageal cancer. In some embodiments, the cancer is
a lung cancer, e.g., by way of non-limiting example, non-small cell
lung cancer. In some embodiments, the cancer is a non-small cell
lung cancer. In some embodiments, the cancer is ovarian/endometrial
cancer. In some embodiments, the cancer is ovarian cancer. In some
embodiments, the cancer is endometrial cancer. In some embodiments,
the cancer is pancreatic cancer. In some embodiments, the cancer is
prostate cancer. In some embodiments, the cancer is a renal cancer.
In some embodiments, the cancer is a sarcoma, e.g., by way of
non-limiting example, osteosarcoma. In some embodiments, the cancer
is an osteosarcoma. In some embodiments, the cancer is a skin
cancer, e.g., by way of non-limiting example, squamous cell cancer,
basal cell carcinoma, and/or melanoma. In some embodiments, the
cancer is a squamous cell cancer. In some embodiments, the cancer
is a skin squamous cell carcinoma. In some embodiments, the cancer
is an esophageal squamous cell carcinoma. In some embodiments, the
cancer is a head and neck squamous cell carcinoma. In some
embodiments, the cancer is a lung squamous cell carcinoma. In some
embodiments, the cancer is a basal cell carcinoma. In some
embodiments, the cancer is a melanoma. In some embodiments, the
agent to which the activatable antibody is conjugated is a
microtubule inhibitor. In some embodiments, the agent to which the
activatable antibody is conjugated is a nucleic acid damaging
agent.
[0244] In some embodiments, the indication, e.g., disease or
disorder associated with expression and/or activity of EGFR is an
inflammatory disorder and/or an autoimmune disease. In some
embodiments, the inflammatory and/or autoimmune disease is
psoriasis. In some embodiments, the agent to which the activatable
antibody is conjugated is a microtubule inhibitor. In some
embodiments, the agent to which the activatable antibody is
conjugated is a nucleic acid damaging agent.
[0245] Pathologies treated and/or prevented and/or for which the
progression is delayed and/or for which a symptom is ameliorated
using the conjugated activatable anti-Jagged antibodies of the
invention include, for example, cancer. In some embodiments, the
conjugated activatable anti-Jagged antibodies of the invention are
used to treat, prevent, delay the progression of, and/or ameliorate
a symptom of a pathology such as, for example, leukemias, including
T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic
leukemia (CLL), lymphoblastic diseases including multiple myeloma,
and solid tumors, including lung, colorectal, prostate, pancreatic
and breast, including triple negative breast cancer. In addition,
since notch signaling is important for the survival and growth of
cancer stem cells, inhibition of Jagged dependent notch signaling
would impact stem cell growth and survival.
[0246] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, bone disease or metastasis in cancer, regardless of
primary tumor origin.
[0247] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, breast cancer, including by way of non-limiting
example, ER/PR+ breast cancer, Her2+ breast cancer, triple-negative
breast cancer.
[0248] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, colorectal cancer.
[0249] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, gastric cancer.
[0250] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, glioblastoma.
[0251] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, head and neck cancer.
[0252] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, lung cancer, such as by way of non-limiting example,
non-small cell lung cancer.
[0253] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, multiple myeloma.
[0254] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, ovarian cancer.
[0255] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, pancreatic cancer.
[0256] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, prostate cancer.
[0257] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, sarcoma.
[0258] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, renal cancer, such as by way of nonlimiting example,
renal cell carcinoma.
[0259] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, thyroid cancer.
[0260] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, a urogenital cancer, such as bladder cancer, kidney
cancer, or uterine cancer. In some embodiments, the pathology is
bladder cancer. In some embodiments, the pathology is kidney
cancer. In some embodiments, the pathology is uterine cancer.
[0261] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, skin cancer, such as by way of nonlimiting example,
skin squamous cell cancer, such as esophageal squamous cell
carcinoma (also known as squamous cell cancer of the esophagus),
head and neck squamous cell carcinoma (also known as squamous cell
cancer of the head and neck) or lung squamous cell carcinoma (also
known as squamous cell cancer of the lung), basal cell carcinoma,
or melanoma. In some embodiments, the cancer is a squamous cell
cancer. In some embodiments, the cancer is a skin squamous cell
carcinoma. In some embodiments, the cancer is an esophageal
squamous cell carcinoma. In some embodiments, the cancer is a head
and neck squamous cell carcinoma. In some embodiments, the cancer
is a lung squamous cell carcinoma. In some embodiments, the cancer
is a basal cell carcinoma. In some embodiments, the cancer is a
melanoma.
[0262] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, acute lymphoblastic leukemia (ALL), acute myelogenous
leukemia (AML), chronic lymphoblastic leukemia (CLL) or
myelodysplastic syndrome (MDS). In some embodiments, the pathology
is ALL. In some embodiments, the pathology is AML. In some
embodiments, the pathology is CLL. In some embodiments, the
pathology is MDS.
[0263] In addition to cancer, Jagged-dependent notch signaling is
critical to epithelial and fibroblast differentiation to
myofibroblasts, cells with a central role in the development of
fibrotic disease. Inhibition of Jagged dependent notch signaling,
and therefore inhibition of the emergence of myofibroblasts, would
be an effective treatment for fibrotic diseases of the kidney,
liver, lung, and skin. In some embodiments, the conjugated
activatable anti-Jagged antibodies are used to treat a fibrotic
disorder, such as idiopathic pulmonary fibrosis (IPF).
[0264] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, fibrotic disease.
[0265] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, idiopathic pulmonary fibrosis, kidney fibrotic
disease, liver fibrotic disease, peritoneal dialysis-induced
fibrosis, scleroderma.
[0266] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of a pathology such as,
for example, hearing loss.
[0267] In some embodiments, the conjugated activatable anti-Jagged
antibodies of the invention that are used to treat, prevent, delay
the progression of, and/or ameliorate a symptom of such pathologies
are conjugated to a microtubule inhibitor agent. In some
embodiments, the conjugated activatable anti-Jagged antibodies of
the invention that are used to treat, prevent, delay the
progression of, and/or ameliorate a symptom of such pathologies are
conjugated to a nucleic acid damaging agent.
[0268] Pathologies treated and/or prevented and/or for which the
progression is delayed and/or for which a symptom is ameliorated
using the conjugated activatable anti-interleukin 6 receptor (IL-6)
antibodies of the invention include, for example, diseases or
disorders associated with expression and/or activity of IL-6R. In
some embodiments, the disease or disorder associated with
expression and/or activity of IL-6R is cancer. In some embodiments,
the cancer is breast cancer, including but not limited to, triple
negative breast cancer (TNBC). In some embodiments, the cancer is
Castleman's disease. In some embodiments, the cancer is
hepatocellular carcinoma. In some embodiments, the cancer is lung
cancer. In some embodiments, the cancer is multiple myeloma. In
some embodiments, the cancer is ovarian cancer. In some
embodiments, the cancer is prostate cancer. In some embodiments,
the agent to which the activatable antibody is conjugated is a
microtubule inhibitor. In some embodiments, the agent to which the
activatable antibody is conjugated is a nucleic acid damaging
agent.
[0269] In some embodiments, the disease or disorder is inflammation
and/or an inflammatory disorder. In some embodiments, the disease
or disorder is an autoimmune disease. In some embodiments, the
agent to which the activatable antibody is conjugated is a
microtubule inhibitor. In some embodiments, the agent to which the
activatable antibody is conjugated is a nucleic acid damaging
agent.
[0270] Increased proteolysis is known to be a hallmark of cancer.
(See e.g., Affara N I, et al. "Delineating protease functions
during cancer development." Methods Mol Biol. 539 (2009): 1-32).
Progression, invasion and metastasis of tumors result from several
interdependent processes in which proteases are implicated.
[0271] Efficaciousness of prevention, amelioration or treatment is
determined in association with any known method for diagnosing or
treating the disease or disorder associated with target expression
and/or activity. Prolonging the survival of a subject or otherwise
delaying the progression of the disease or disorder associated with
target expression and/or activity in a subject indicates that the
activatable antibody confers a clinical benefit.
[0272] Conjugated activatable antibodies can be administered in the
form of pharmaceutical compositions. Principles and considerations
involved in preparing such compositions, as well as guidance in the
choice of components are provided, for example, in Remington: The
Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et
al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption
Enhancement: Concepts, Possibilities, Limitations, And Trends,
Harwood Academic Publishers, Langhome, Pa., 1994; and Peptide And
Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),
1991, M. Dekker, New York.
[0273] One embodiment of an activatable antibody fragment is the
smallest fragment that specifically binds to the binding domain of
the target protein. For example, based upon the variable-region
sequences of an antibody, peptide molecules can be designed that
retain the ability to bind the target protein sequence. Such
peptides can be synthesized chemically and/or produced by
recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl.
Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also
contain more than one active compound as necessary for the
particular indication being treated, and in some embodiments, those
with complementary activities that do not adversely affect each
other. Alternatively, or in addition, the composition can comprise
an agent that enhances its function, such as, for example, a
cytotoxic agent, cytokine, chemotherapeutic agent, or
growth-inhibitory agent. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0274] The active ingredients can also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions.
[0275] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0276] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
where matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0277] In some embodiments, the activatable antibody contains a
detectable label. An intact antibody, or a fragment thereof (e.g.,
Fab, scFv, or F(ab).sub.2) is used. The term "labeled", with regard
to the probe or antibody, is intended to encompass direct labeling
of the probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a
fluorescently-labeled secondary antibody and end-labeling of a DNA
probe with biotin such that it can be detected with
fluorescently-labeled streptavidin. The term "biological sample" is
intended to include tissues, cells and biological fluids isolated
from a subject, as well as tissues, cells and fluids present within
a subject. Included within the usage of the term "biological
sample", therefore, is blood and a fraction or component of blood
including blood serum, blood plasma, or lymph. That is, the
detection method of the invention can be used to detect a protein,
polypeptide or peptide in a biological sample in vitro as well as
in vivo. For example, in vitro techniques for detection of an
analyte protein include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations, immunochemical
staining, and immunofluorescence. Procedures for conducting
immunoassays are described, for example in "ELISA: Theory and
Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther
(Ed.) Human Press, Totowa, N.J., 1995; "Immunoassay", E. Diamandis
and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,
1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen,
Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo
techniques for detection of an analyte protein include introducing
into a subject a labeled anti-analyte protein antibody. For
example, the antibody can be labeled with a radioactive marker
whose presence and location in a subject can be detected by
standard imaging techniques.
[0278] Diagnostic and Prophylactic Formulations
[0279] The conjugated activatable antibodies of the invention are
used in diagnostic and prophylactic formulations. In one
embodiment, a conjugated activatable antibody is administered to
patients that are at risk of developing one or more of the
aforementioned cancer or other disorders. A patient's or organ's
predisposition to one or more of the aforementioned disorders can
be determined using genotypic, serological or biochemical
markers.
[0280] In some embodiments of the invention, a conjugated
activatable antibody is administered to human individuals diagnosed
with a clinical indication associated with one or more of the
aforementioned disorders. Upon diagnosis, a conjugated activatable
antibody is administered to mitigate or reverse the effects of the
clinical indication.
[0281] Conjugated activatable antibodies of the invention are also
useful in the detection of a target in patient samples and
accordingly are useful as diagnostics. For example, the conjugated
activatable antibodies of the invention are used in in vitro
assays, e.g., ELISA, to detect target levels in a patient
sample.
[0282] Conjugated activatable antibodies can also be used in
diagnostic and/or imaging methods. In some embodiments, such
methods are in vitro methods. In some embodiments, such methods are
in vivo methods. In some embodiments, such methods are in situ
methods. In some embodiments, such methods are ex vivo methods. For
example, a conjugated activatable antibody having an enzymatically
cleavable CM can be used to detect the presence or absence of an
enzyme that is capable of cleaving the CM. Such conjugated
activatable antibodies can be used in diagnostics, which can
include in vivo detection (e.g., qualitative or quantitative) of
enzyme activity (or, in some embodiments, an environment of
increased reduction potential such as that which can provide for
reduction of a disulfide bond) through measured accumulation of
activated conjugated activatable antibodies (i.e., antibodies
resulting from cleavage of a conjugated activatable antibody) in a
given cell or tissue of a given host organism. Such accumulation of
activated conjugated antibodies indicates not only that the tissue
expresses enzymatic activity (or an increased reduction potential
depending on the nature of the CM) but also that the tissue
expresses target to which the activated antibody binds.
[0283] For example, the CM can be selected to be a protease
substrate for a protease found at the site of a tumor, at the site
of a viral or bacterial infection at a biologically confined site
(e.g., such as in an abscess, in an organ, and the like), and the
like. The AB can be one that binds a target antigen. Using methods
as disclosed herein or, when appropriate, methods familiar to one
skilled in the art, a detectable label (e.g., a fluorescent label
or radioactive label or radiotracer) can be conjugated to an AB or
other region of an activatable antibody. Suitable detectable labels
are discussed in the context of the above screening methods and
additional specific examples are provided below. Using an AB
specific to a protein or peptide of the disease state, along with a
protease whose activity is elevated in the disease tissue of
interest, protease-activated activatable antibodies will exhibit an
increased rate of binding to disease tissue relative to tissues
where the CM specific enzyme is not present at a detectable level
or is present at a lower level than in disease tissue or is
inactive (e.g., in zymogen form or in complex with an inhibitor).
Since small proteins and peptides are rapidly cleared from the
blood by the renal filtration system, and because the enzyme
specific for the CM is not present at a detectable level (or is
present at lower levels in non-disease tissues or is present in
inactive conformation), accumulation of activated antibodies in the
disease tissue is enhanced relative to non-disease tissues.
[0284] In another example, conjugated activatable antibodies can be
used to detect the presence or absence of a cleaving agent in a
sample. For example, where the conjugated activatable antibodies
contain a CM susceptible to cleavage by an enzyme, the conjugated
activatable antibodies can be used to detect (either qualitatively
or quantitatively) the presence of an enzyme in the sample. In
another example, where the conjugated activatable antibodies
contain a CM susceptible to cleavage by reducing agent, the
conjugated activatable antibodies can be used to detect (either
qualitatively or quantitatively) the presence of reducing
conditions in a sample. To facilitate analysis in these methods,
the conjugated activatable antibodies can be detectably labeled and
can be bound to a support (e.g., a solid support, such as a slide
or bead). The detectable label can be positioned on a portion of
the activatable antibody that is not released following cleavage,
for example, the detectable label can be a quenched fluorescent
label or other label that is not detectable until cleavage has
occurred. The assay can be conducted by, for example, contacting
the immobilized, detectably labeled activatable antibodies with a
sample suspected of containing an enzyme and/or reducing agent for
a time sufficient for cleavage to occur, then washing to remove
excess sample and contaminants. The presence or absence of the
cleaving agent (e.g., enzyme or reducing agent) in the sample is
then assessed by a change in detectable signal of the activatable
antibodies prior to contacting with the sample e.g., the presence
of and/or an increase in detectable signal due to cleavage of the
activatable antibody by the cleaving agent in the sample.
[0285] Such detection methods can be adapted to also provide for
detection of the presence or absence of a target that is capable of
binding the AB of the conjugated activatable antibodies when
cleaved. Thus, the assays can be adapted to assess the presence or
absence of a cleaving agent and the presence or absence of a target
of interest. The presence or absence of the cleaving agent can be
detected by the presence of and/or an increase in detectable label
of the activatable antibodies as described above, and the presence
or absence of the target can be detected by detection of a
target-AB complex e.g., by use of a detectably labeled anti-target
antibody.
[0286] Conjugated activatable antibodies are also useful in in situ
imaging for the validation of activatable antibody activation,
e.g., by protease cleavage, and binding to a particular target. In
situ imaging is a technique that enables localization of
proteolytic activity and target in biological samples such as cell
cultures or tissue sections. Using this technique, it is possible
to confirm both binding to a given target and proteolytic activity
based on the presence of a detectable label (e.g., a fluorescent
label).
[0287] These techniques are useful with any frozen cells or tissue
derived from a disease site (e.g. tumor tissue) or healthy tissues.
These techniques are also useful with fresh cell or tissue
samples.
[0288] In these techniques, an activatable antibody is labeled with
a detectable label. The detectable label may be a fluorescent dye,
(e.g. Fluor.RTM. Isothiocyanate (FITC), Rhodamine Isothiocyanate
(TRITC), an Alexa Fluor.RTM. label, such as Alexa Fluor.RTM. 680 or
Alexa Fluor.RTM. 750), a near infrared (NIR) dye (e.g., Qdot.RTM.
nanocrystals), a colloidal metal, a hapten, a radioactive marker,
biotin and an amplification reagent such as streptavidin, or an
enzyme (e.g., horseradish peroxidase or alkaline phosphatase).
[0289] Detection of the label in a sample that has been incubated
with the labeled, activatable antibody indicates that the sample
contains the target and contains a protease that is specific for
the CM of the activatable antibody. In some embodiments, the
presence of the protease can be confirmed using broad spectrum
protease inhibitors and/or using an agent that is specific for the
protease, for example, an antibody such as A11, which is specific
for the protease matriptase (MT-SP1) and inhibits the proteolytic
activity of MT-SP1; see e.g., International Publication Number WO
2010/129609, published 11 Nov. 2010. The same approach of using
broad spectrum protease inhibitors and/or by using a more selective
inhibitory agent can be used to identify a protease or class of
proteases specific for the CM of the activatable antibody. In some
embodiments, the presence of the target can be confirmed using an
agent that is specific for the target, e.g., another antibody, or
the detectable label can be competed with unlabeled target. In some
embodiments, unlabeled activatable antibody could be used, with
detection by a labeled secondary antibody or more complex detection
system.
[0290] Similar techniques are also useful for in vivo imaging where
detection of the fluorescent signal in a subject, e.g., a mammal,
including a human, indicates that the disease site contains the
target and contains a protease that is specific for the CM of the
activatable antibody.
[0291] These techniques are also useful in kits and/or as reagents
for the detection, identification or characterization of protease
activity in a variety of cells, tissues, and organisms based on the
protease-specific CM in the activatable antibody.
[0292] In some embodiments, in situ imaging and/or in vivo imaging
are useful in methods to identify which patients to treat. For
example, in in situ imaging, the activatable antibodies are used to
screen patient samples to identify those patients having the
appropriate protease(s) and target(s) at the appropriate location,
e.g., at a tumor site.
[0293] In some embodiments in situ imaging is used to identify or
otherwise refine a patient population suitable for treatment with a
conjugated activatable antibody of the disclosure. For example,
patients that test positive for both the target and a protease that
cleaves the substrate in the cleavable moiety (CM) of the
activatable antibody being tested (e.g., accumulate activated
antibodies at the disease site) are identified as suitable
candidates for treatment with such an activatable antibody
comprising such a CM. Likewise, patients that test negative for
either or both of the target and the protease that cleaves the
substrate in the CM in the activatable antibody being tested using
these methods might be identified as suitable candidates for
another form of therapy. In some embodiments, such patients that
test negative with respect to a first activatable antibody can be
tested with other activatable antibodies comprising different CMs
until a suitable activatable antibody for treatment is identified
(e.g., an activatable antibody comprising a CM that is cleaved by
the patient at the site of disease). In some embodiments, the
patient is then administered a therapeutically effective amount of
the conjugated activatable antibody for which the patient tested
positive.
[0294] In some embodiments in vivo imaging is used to identify or
otherwise refine a patient population suitable for treatment with
an activatable antibody of the disclosure. For example, patients
that test positive for both the target and a protease that cleaves
the substrate in the cleavable moiety (CM) of the activatable
antibody being tested (e.g., accumulate activated antibodies at the
disease site) are identified as suitable candidates for treatment
with such an activatable antibody comprising such a CM. Likewise,
patients that test negative might be identified as suitable
candidates for another form of therapy. In some embodiments, such
patients that test negative with respect to a first activatable
antibody can be tested with other activatable antibodies comprising
different CMs until a suitable activatable antibody for treatment
is identified (e.g., an activatable antibody comprising a CM that
is cleaved by the patient at the site of disease). In some
embodiments, the patient is then administered a therapeutically
effective amount of the conjugated activatable antibody for which
the patient tested positive.
[0295] Pharmaceutical Compositions
[0296] The conjugated activatable antibodies of the invention (also
referred to herein as "active compounds"), and derivatives,
fragments, analogs and homologs thereof, can be incorporated into
pharmaceutical compositions suitable for administration. Such
compositions typically comprise the conjugated activatable antibody
and a pharmaceutically acceptable carrier. As used herein, the term
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. Suitable
carriers are described in the most recent edition of Remington's
Pharmaceutical Sciences, a standard reference text in the field,
which is incorporated herein by reference. Examples of such
carriers or diluents include, but are not limited to, water,
saline, ringer's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0297] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (i.e., topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid (EDTA); buffers such as acetates,
citrates or phosphates, and agents for the adjustment of tonicity
such as sodium chloride or dextrose. The pH can be adjusted with
acids or bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0298] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringeability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity 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. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, aluminum monostearate and
gelatin.
[0299] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, 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, 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.
[0300] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0301] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser that contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0302] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0303] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0304] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0305] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0306] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0307] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Materials and Methods
[0308] The examples provided herein use an anti-EGFR activatable
antibody referred to herein as activatable antibody
3954-1204-C225v5 (also referred to herein as 3954-1204-C225v5
activatable antibody or 3954-1204-C225v5) that includes an
EGFR-binding sequence, a masking moiety (MM), and a cleavable
moiety (CM) that is a substrate for a protease. These examples also
use an activatable anti-EGFR antibody construct referred to herein
as masked antibody 3954-NSUB-C225v5 (also referred to herein as
3954-NSUB-C225v5 masked antibody or 3954-NSUB-C225v5) that includes
a non-cleavable moiety located between the MM and the EGFR-binding
sequence. It is to be understood that while the examples provided
herein use these anti-EGFR activatable antibody constructs, these
methods are applicable to any activatable antibody having two or
more cysteine residues, where it is desired that only a portion of
the total number of cysteine residues in the activatable antibody
be reduced prior to conjugation. This is referred to herein as
"partial reduction."
[0309] It should be further understood that the examples provided
herein use a fluorescent agent, Alexa-680 Fluor.RTM. (also referred
to herein as Alexa 680.RTM.), as the agent that is to be conjugated
to an activatable antibody. This particular dye was chosen because
it has a molecular weight that is similar to a known cytotoxic
agent, MMAE. However, this fluorescent agent is merely used as an
example, and the compositions and methods used herein are useful
with any number of conjugated agents, including by way of
non-limiting example, toxins and other payload agents. The
compositions and methods are not limited to agents of any
particular molecular weight, size or other such characteristic.
[0310] Anti-EGFR Activatable Antibody Constructs:
[0311] The 3954-1204-C225v5 activatable anti-EGFR antibody
construct includes the following heavy and light chain
sequences:
TABLE-US-00011 3954-1204-C225v5 Activatable Antibody Heavy Chain
Nucleotide Sequence: [C225v5 (SEQ ID NO: 1)] (SEQ ID NO: 1)
[caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccaga
gcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggc
gtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgt
gatttggagcggcggcaacaccgattataacaccccgtttaccagccgcc
tgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaac
agcctgcaaagccaggataccgcgatttattattgcgcgcgcgcgctgac
ctattatgattatgaatttgcgtattggggccagggcaccctggtgaccg
tgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcc
tccaagagcacctctgggggcacagcggccctgggctgcctggtcaagga
ctacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgacca
gcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactcc
ctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagaccta
catctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaag
ttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagca
cctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaa
ggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtgg
acgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggc
gtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacag
cacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctga
atggcaaggagtacaagtgcaaggtctccaacaaagccctcccagccccc
atcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggt
gtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcc
tgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgg
gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgct
ggactccgacggctccttcttcctctacagcaagctcaccgtggacaaga
gcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggct
ctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatg a]
3954-1204-C225v5 Activatable Antibody Heavy Chain Amino Acid
Sequence: [C225v5 (SEQ ID NO: 2)] (SEQ ID NO: 2)
[QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLG
VIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARAL
TYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVK
DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT
YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFHWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K*]
3954-1204-C225v5 Activatable Antibody Light Chain Nucleotide
Sequence: [Spacer (SEQ ID NO: 5)][Mask (SEQ ID NO: 6)] [Linker 1
(SEQ ID NO: 7)][1204 Substrate (SEQ ID NO: 8)][ (SEQ ID NO: 9)]
[C225 (SEQ ID NO: 10)] (SEQ ID NO: 3)
[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacgg
cccatacgtcatgtac][ggctcgagcggtggcagcggtggctctggtgg
atccggt][ctgagcggccgttccgataatcat] [
][cagatcttgctgacccagagcccggtgattct
gagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccaga
gcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccg
cgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccg
ctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcg
tggaaagcgaagatattgcggattattattgccagcagaacaacaactgg
ccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggc
tgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctg
gaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcc
aaagtacagtggaaggtggataacgccctccaatcgggtaactcccagga
gagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagca
ccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgc
gaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacag gggagagtgttag]
Bold: Spacer Underline: Mask Italics and Underline: Linker 1 Bold
and Underline: 1204 Substrate : Linker Normal text: anti-EGFR
antibody de- rived sequence 3954-1204-C225v5 Activatable Antibody
Light Chain Amino Acid Sequence: [Spacer (SEQ ID NO: 11)][Mask (SEQ
ID NO: 12)] [Linker 1 (SEQ ID NO: 13)][1204 Substrate (SEQ ID NO:
14)][ (SEQ ID NO: 15)] [C225 (SEQ ID NO: 16)] (SEQ ID NO: 4)
[QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGSGGSG][LSGRSDNH] [
][QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNG
SPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNN
NWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR
EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and
Underline: Linker 1 Bold and Underline: 1204 Substrate : Linker 2
Normal text: anti-EGFR antibody de- rived sequence
[0312] In some embodiments, the spacer sequence for the light chain
3954-1204-C225v5 activatable antibody can include an N-terminal
variant, such as for example, a spacer selected from the group
consisting of GQSGQ (SEQ ID NO: 235), QSGQ (SEQ ID NO: 236), SGQ
(SEQ ID NO: 237), GQ and Q. In these embodiments, all other
elements of the 3954-1204-C225v5 activatable antibodies, e.g., the
heavy chain sequence, the light chain sequence, the 3954 mask,
linker 1, the 1204 substrate, and linker 2, all remain the same as
shown above in SEQ ID NO: 4.
[0313] The 3954-NSUB-C225v5 masked anti-EGFR antibody construct
includes the same heavy chain as the 3954-1204-C225v5 activatable
anti-EGFR antibody shown above. The 3954-NSUB-C225v5 masked
anti-EGFR antibody construct includes the following light chain
sequence:
TABLE-US-00012 3954-NSUB-C225v5 Masked Antibody Light Chain
Nucleotide Sequence: [Spacer (SEQ ID NO: 5)][Mask (SEQ ID NO: 6)]
[Linker 1-Noncleavable Substrate-Linker 2 (SEQ ID NO: 19)][C225
(SEQ ID NO: 10)] (SEQ ID NO: 17)
[caaggccagtctggccag][tgcatctcacctcgtggttgtccggacgg
cccatacgtcatgtac][ggctcgagcggtggcagcggtggctctggtgg
ctcaggtggaggctcgggcggtgggagcggcggttct][cagatcttgct
gacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagct
ttagctgccgcgcgagccagagcattggcaccaacattcattggtatcag
cagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaag
cattagcggcattccgagccgctttagcggcagcggcagcggcaccgatt
ttaccctgagcattaacagcgtggaaagcgaagatattgcggattattat
tgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaact
ggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccat
ctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaat
aacttctatcccagagaggccaaagtacagtggaaggtggataacgccct
ccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggaca
gcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag
aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcc
cgtcacaaagagcttcaacaggggagagtgttag] Bold: Spacer Underline: Mask
Italics and Underline: Linker 1-Noncleavable substrate-Linker 2
Normal text: anti-EGFR antibody derived sequence 3954-NSUB-C225v5
Masked Antibody Light Chain Amino Acid Sequence: [Spacer (SEQ ID
NO: 11)][Mask (SEQ ID NO: 12)] [Linker 1-Noncleavable
Substrate-Linker 2 (SEQ ID NO: 20)][C225 (SEQ ID NO: 16)] (SEQ ID
NO: 18) [QGQSGQ][CISPRGCPDGPYVMY][GSSGGSGGSGGSGGGSGGGSGGS]
[QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK
YASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFG
AGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ
GLSSPVTKSFNRGEC*] Bold: Spacer Underline: Mask Italics and
Underline: Linker 1-Noncleavable substrate-Linker 2 Normal text:
anti-EGFR antibody derived sequence
[0314] Reducing Agent:
[0315] The studies provided herein use the reducing agent TCEP
(tris(2-carboxyethyl)phosphine).
[0316] Protocol for TCEP Partial Reduction of Anti-EGFR Activatable
Antibody and Subsequent Conjugation to Maleimide Alexa-680:
[0317] Bond-Breaker@ TCEP Solution (neutral pH solution, Thermo
Scientific) is used at various molar ratios of TCEP to an
activatable antibody that, in the cleaved state (i.e., activated
state), binds Epidermal Growth Factor Receptor, and the anti-EGFR
activatable antibody is formulated in PBS. For example, the ratio
of reducing agent, e.g., TCEP, to activatable antibody to be tested
can include a ratio in a range from about 20:1 to 1:1, from about
10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from
about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1,
from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to
1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about
9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5,
from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to
1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about
1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments,
the ratio is in a range of from about 5:1 to 1:1. In some
embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In
some embodiments, the ratio is in a range of from about 4:1 to 1:1.
In some embodiments, the ratio is in a range from about 4:1 to
1.5:1. In some embodiments, the ratio is in a range from about 8:1
to about 1:1. In some embodiments, the ratio is in a range of from
about 2.5:1 to 1:1. It is to be understood that while the examples
provided herein use an anti-EGFR activatable antibody referred to
herein as 3954-1204-C225v5, these methods are applicable to any
activatable antibody having two or more cysteine residues, where it
is desired that only a portion of the total number of cysteine
residues in the activatable antibody be reduced prior to
conjugation. This is referred to herein as "partial reduction."
[0318] Briefly, a TCEP solution at twice the final concentration
was mixed 1:1 (volume:volume) with 3954-1204-C225v5 to result in
the final TCEP:(3954-1204-C225v5) ratio desired. The final solution
was then incubated at 37.degree. C. for specified periods of time
for the reduction reaction to progress. At the end of the reduction
reaction, the solution was cooled to room temperature and Maleimide
Alexa-680 (Invitrogen) was added into the solution (Maleimide
Alexa-680 was used at half of the reduction volume and at a
concentration equal to 10.times. molar concentration of TCEP during
the reduction reaction; for example, if the original reduction
reaction comprised 50 microliters (ul) of 13.2 uM 3954-1204-C225v5
and 50 ul of 52.8 uM TCEP, then 50 ul 264 uM Maleimide Alexa-680
would be used) to begin the Alexa-680 conjugation. The conjugation
reaction proceeded for 2 hours at room temperature in a light tight
container. After the 2-hour reaction, the solution was spun down
and buffer exchanged into PBS using a PD-10 column (GE Healthcare)
or equivalent using manufacturer's instructions. The final
conjugated product was analyzed using a UV Spectrophotometer to
determine final protein concentration and the degree of labeling of
the Alexa-680 dye.
[0319] Protocol for the Analysis of Maleimide Alexa-680 Conjugated
Anti-EGFR Activatable Antibody Using LabChip GXII:
[0320] A HT Protein Express LabChip (Perkin Elmer) was prepared
according to manufacturer's instructions using either the Pico
Protein Express protocol or the HT Protein Express protocol: the
Pico Protein Express protocol was used to analyze the Alexa-680
conjugated portion of the TCEP reduced 3954-1204-C225v5; the HT
Protein Express protocol was used to analyze the total protein in
the TCEP reduced, Alexa-680 conjugated 3954-1204-C225v5. TCEP
reduced, Alexa-680 conjugated 3954-1204-C225v5 was prepared for the
GXII analysis using Perkin Elmer's instructions. The sample was
analyzed using the 200 series of the LabChip GXII analysis protocol
(High sensitivity for the HT protocol and Pico for the Pico
protocol). Resulting data was analyzed using the LabChip GXII
software.
[0321] Protocol for EGFR Binding ELISA:
[0322] NUNC Maxisorp flatbottom 96 well plates were coated with 50
ul/well, 2 ug/ml human EGFR-Fc fusion protein (R&D Systems) in
Hank's Balanced Salt Solution (HBSS, Teknova) for 2 hours at room
temperature. At the end of the 2 hour coating, the liquid contents
of the plate were evacuated and 250 ul/well of HBSS containing 1%
BSA was introduced and allowed to block the plate for 30 minutes at
room temperature. At the end of the blocking period, liquid
contents of the 96-well plate were removed and serially diluted
samples (i.e., 3954-1204-C225v5, Alexa-680 conjugated
3954-1204-C225v5, uPA-activated 3954-1204-C225v5, uPA-activated
Alexa-680 conjugated 3954-1204-C225v5, and C225 (a cetuximab
antibody), starting at a concentration of 100 ug/ml and diluted by
a factor of 3 per dilution step) were introduced at 50 ul/well. The
plate was incubated at room temperature for 1 hour. At the end of
the hour, the plate was washed with HBSS containing 0.05% Tween-20
using a BioTek ELx450 Select CW plate washer (300 ul/well wash
volume, 6 cycles of aspiration and wash). Washed plates were tapped
dry and 50 ul/well of 400 ng/ml Horse Radish Peroxidase conjugated
Goat anti-Human IgG Fab'.sub.2 specific antibody (Jackson
ImmunoResearch) were introduced and incubated for 30 minutes at
room temperature. The plates were washed as previously stated and
100 ul/well of 1-Step TMB Substrate (Thermo Scientific) was
introduced. Color change was observed and the reaction was stopped
by the addition of 100 ul/well of 1M HCl (Fisher Scientific). The
reacted plate was analyzed using a BioTek EL800 plate reader at
O.D. 450. Data were computed using Excel (Microsoft) and the result
was plotted using Prism 6 (GraphPad).
Example 2
TCEP-Mediated Reduction of Activatable Antibodies
[0323] The compositions and methods provided herein determine the
combination of reagents and reaction conditions that produce the
desired partial reduction followed by conjugation. When reduction
and subsequent conjugation is not controlled properly, activatable
antibodies will be completely reduced, and the masking efficiency
of the activatable antibody is compromised. For example, when the
reducing agent is used at a ratio of 20:1 (reducing agent to
activatable antibody), the activatable antibody was completely
reduced into free heavy chain and free light chain. Attempts to
produce a milder reduction (i.e., less than complete reduction),
for example, by immobilizing a reducing agent, were too mild and
did not sufficiently reduce the activatable antibody to allow for
subsequent conjugation. In these studies, bands corresponding to
predominantly intact IgG (high molecular weight band >150 kDa)
were observed in all reduction conditions.
[0324] Studies were conducted to determine the range of reducing
agent to activatable antibody. At lower ratios, for example, in the
range of 0.5:1 to 2:1 (reducing agent to activatable antibody),
some reduction was achieved, and the activatable antibody integrity
and masking efficiency were retained. At ratios of 1.5:1 to 5:1
(reducing agent to activatable antibody), reduction time from 30
minutes to 2 hours, there was an increasing amount of reduced
activatable antibody species corresponding to the molecular weight
of one heavy chain and one light chain activatable antibody. The
partially reduced activatable antibody maintained the EGFR binding
characteristics of the original non-reduced and masked activatable
antibody demonstrating that the activatable antibody partially
reduced under these conditions was capable of maintaining the
original masking efficiency. At the identified ratio of reducing
agent to activatable antibody and reduction time, an inter-chain
disulfide-reduced activatable antibody can be produced to allow for
subsequent maximum conjugation through free cysteines while
maintaining the masking efficiency of the original, non-reduced
activatable antibody.
[0325] At a reduction time of 2 hours, a ratio of reducing agent to
activatable antibody ratio above 5:1 was too reductive to maintain
the original masking efficiency of the tested activatable
antibodies. The varied shift in masking efficiency loss and the
varied amounts of partially reduced activatable antibody subspecies
showed that the tested activatable antibodies have different
tolerance to reducing agent-mediated reduction, for example,
TCEP-mediated reduction. The varied combination of antibody,
linkers, cleavable moiety (CM) and masking moiety (MM) results in a
spectrum in the tolerance of the activatable antibody for reducing
agent-mediated reduction, for example, TCEP-mediated reduction.
[0326] In one set of studies described herein, an activatable
anti-EGFR antibody referred to as 3954-1204-C225v5 was reduced at
various ratios of TCEP to activatable antibody (e.g., from about
1.5:1 to about 4:1) using a 90-minute reduction time. In some
instances, reduction was followed by conjugation to a fluorescent
dye, Alexa 680. The results of these studies (at TCEP to
activatable antibody ratios of 1.5:1, 2:1, and 4:1) are shown in
FIG. 1.
[0327] In another set of studies described herein, an anti-EGFR
antibody construct that includes an antigen-binding portion that
specifically binds EGFR, a masking moiety, and a non-cleavable
linker (referred to as 3954-NSUB-C225v5) was reduced at various
ratios of TCEP to activatable antibody (e.g., from about 1.5:1 to
about 4:1) using a 90-minute reduction time. In some instances,
reduction was followed by conjugation to Alexa 680. The results of
these studies (at TCEP to activatable antibody ratios of 1.5:1,
2:1, and 4:1) are shown in FIG. 2.
[0328] Using thiol conjugatable Alexa 680 as a surrogate for thiol
conjugatable toxin, these studies demonstrate varying degrees of
Alexa 680 conjugation dependent on both TCEP to activatable
antibody ratio and time of reduction. The conjugation of thiol
conjugatable Alexa 680 to TCEP-partially reduced 3954-1204-C225v5
or 3954-NSUB-C225v5 does not significantly change the titration
profile of 3954-1204-C225v5 or 3954-NSUB-C225v5 to EGFR. Thus,
partial reduction and subsequent thiol conjugation of Alexa 680 can
be done in such a way as to maintain the masking efficiency of
activatable antibodies. FIGS. 3 through 6 demonstrate that partial
reduction and subsequent thiol conjugation of Alexa 680 Fluor.RTM.
can be done in such a way as to also maintain activation of
3954-1204-C225v5 by uPA and not to lead to activation of
3954-NSUB-C225v5.
[0329] Further studies indicated that a degree of labeling (molar
ratio of Alexa 680 Fluor.RTM. vs. activatable antibody) of 3.8 was
achieved for 3954-1204-C225v5 and a degree of labeling of 3.5 was
achieved for 3954-NSUB-C225v5.
Example 3
Mass Spectrometry Analysis of Conjugated Activatable Antibodies
[0330] Molecular Weight Determination by MALDI Mass Spectrometry
(MALDI MS):
[0331] In MALDI MS, the dissolved sample is deposited on a metal
target and the peptides and proteins are co-crystallized with a
light-absorbing matrix. A laser beam is directed at the dry matrix
sample, the sample molecules are desorbed and ionized and the
masses are measured in a time-of-flight (TOF) mass analyzer.
Proteins are observed in the mass spectrum (mass-over-charge
spectrum m/z) as singly (m/z MH+) as well as multiple charged
ions.
[0332] In the present analysis, the partially reduced and
conjugated activatable antibodies (intact and DTT-reduced) were
purified using C4 ziptips from Millipore. Each purified sample was
mixed with 2,5-dihydroxyacetophonone/diammonium hydrogen citrate
(DHAP/DAHC) matrix and spotted onto a Big Anchor target from
Bruker. Mass spectra were obtained on an Autoflex Speed MALDI
TOF/TOF mass spectrometer in linear mode using Compas 1.4 control
and processing software. The mass spectra were calibrated by
external quadratic calibration using Bruker Protein Calibration
Standard 1 or 2. The sample mass was calculated from the least
charged ion within the calibrated range of the mass spectrum.
[0333] Each Alexa-680 molecule added approximately 1000 dalton
(.about.1 kDa) of mass to the activatable antibody. A comparison of
the molecular weight of the unconjugated 3954-1204-C225v5 to the
conjugated 3954-1204-C225v5 enables estimation of the number of
Alexa-680.RTM. molecules that has been conjugated upon
3954-1204-C225v5.
[0334] The MALDI-MW data indicated that up to four 1-kDa molecules
were attached to the activatable anti-EGFR antibody
3954-1204-C225v5. From the reduced samples, it was determined that
this modification was most likely heterogeneous, as the light chain
was observed with 0 and 1 modification(s) and the heavy chain was
observed with 0, 1 or 2 modifications.
[0335] It was determined from the MALDI-MS data on digested samples
that all peptides containing cysteine residues were still observed
in the conjugated sample (Cyt04-680 high). The MALDI-MW data also
confirmed that the antibody was not fully labeled by
Alexa-680.RTM..
Example 4
Materials and Methods
[0336] The examples provided herein use an anti-Jagged activatable
antibody referred to herein as activatable antibody 5342-1204-4D11
(also referred to herein as 5342-1204-4D11 activatable antibody or
5342-1204-4D11) that includes a Jagged-binding sequence, a masking
moiety (MM), and a cleavable moiety (CM) that is a substrate for a
protease. It is to be understood that while the examples provided
herein use these anti-Jagged activatable antibody constructs, these
methods are applicable to any activatable antibody having two or
more cysteine residues, where it is desired that only a portion of
the total number of cysteine residues in the activatable antibody
be reduced prior to conjugation. This is referred to herein as
"partial reduction."
[0337] It should be further understood that the examples provided
herein use a fluorescent agent, Alexa-680 Fluor.RTM. (also referred
to herein as Alexa 680.RTM.), as the agent that is to be conjugated
to an activatable antibody. This particular dye was chosen because
it has a molecular weight that is similar to a known cytotoxic
agent, MMAE. However, this fluorescent agent is merely used as an
example, and the compositions and methods used herein are useful
with any number of conjugated agents, including by way of
non-limiting example, toxins and other payload agents. The
compositions and methods are not limited to agents of any
particular molecular weight, size or other such characteristic.
[0338] Anti-Jagged Activatable Antibody Constructs:
[0339] The 5342-1204-4D11 activatable anti-Jagged antibody
construct includes the following heavy and light chain
sequences:
TABLE-US-00013 5342-1204-4D11 Activatable Antibody Heavy Chain
Nucleotide Sequence: (SEQ ID NO: 231)
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCA
TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGT
ATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATC
GGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT
CTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT
CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTAC
ATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCAC
CTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA
CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 5342-1204-4D11
Activatable Antibody Heavy Chain Amino Acid Sequence: (SEQ ID NO:
245) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVIGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
VTRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 5342-1204-4D11
Activatable Antibody Light Chain Nucleotide Sequence: (SEQ ID NO:
233) CAAGGCCAGTCTGGCCAGTGCAATATTTGGCTCGTAGGTGGTGATTGCAG
GGGCTGGCAGGGGGGCTCGAGCGGTGGCAGCGGTGGCTCTGGTGGTCTGA
GCGGCCGTTCCGATAATCATGGCGGCGGTTCTGACATCCAGATGACCCAG
TCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTG
CCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAAC
CAGGGAAAGCCCCTAAGCTCCTGATCTATGCGGCATCCAGTTTGCAAAGT
GGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCT
CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAAC
AGACGGTTGTGGCGCCTCCGTTATTCGGCCAAGGGACCAAGGTGGAAATC
AAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGA
GCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCT
ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG
GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA
CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACA
AAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACA
AAGAGCTTCAACAGGGGAGAGTGT 5342-1204-4D11 Activatable Antibody Light
Chain Amino Acid Sequence: (SEQ ID NO: 234)
QGQSGQCNIWLVGGDCRGWQGGSSGGSGGSGGLSGRSDNHGGGSDIQMTQ
SPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEI
KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS
GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC
[0340] In some embodiments, the spacer sequence for the light chain
5342-1204-4D11 activatable antibody can include an N-terminal
variant, such as for example, a spacer selected from the group
consisting of GQSGQ (SEQ ID NO: 235), QSGQ (SEQ ID NO: 236), SGQ
(SEQ ID NO: 237), GQ and Q. In these embodiments, all other
elements of the 5342-1204-4D11 activatable antibodies, e.g., the
heavy chain sequence, the light chain sequence, the 5342 mask,
linker 1, the 1204 substrate, and linker 2, all remain the same as
shown above in SEQ ID NO: 234.
[0341] Reducing Agent:
[0342] The studies provided herein use the reducing agent TCEP
(tris(2-carboxyethyl)phosphine).
[0343] Protocol for TCEP Partial Reduction of Anti-Jagged
Activatable Antibody and Subsequent Conjugation to Maleimide
Alexa-680:
[0344] Bond-Breaker@ TCEP Solution (neutral pH solution, Thermo
Scientific) is used at various molar ratios of TCEP to an
activatable antibody that, in the cleaved state (i.e., activated
state), binds Epidermal Growth Factor Receptor, and the anti-Jagged
activatable antibody is formulated in PBS. For example, the ratio
of reducing agent, e.g., TCEP, to activatable antibody to be tested
can include a ratio in a range from about 20:1 to 1:1, from about
10:1 to 1:1, from about 9:1 to 1:1, from about 8:1 to 1:1, from
about 7:1 to 1:1, from about 6:1 to 1:1, from about 5:1 to 1:1,
from about 4:1 to 1:1, from about 3:1 to 1:1, from about 2:1 to
1:1, from about 20:1 to 1:1.5, from about 10:1 to 1:1.5, from about
9:1 to 1:1.5, from about 8:1 to 1:1.5, from about 7:1 to 1:1.5,
from about 6:1 to 1:1.5, from about 5:1 to 1:1.5, from about 4:1 to
1:1.5, from about 3:1 to 1:1.5, from about 2:1 to 1:1.5, from about
1.5:1 to 1:1.5, or from about 1:1 to 1:1.5. In some embodiments,
the ratio is in a range of from about 5:1 to 1:1. In some
embodiments, the ratio is in a range of from about 5:1 to 1.5:1. In
some embodiments, the ratio is in a range of from about 4:1 to 1:1.
In some embodiments, the ratio is in a range from about 4:1 to
1.5:1. In some embodiments, the ratio is in a range from about 8:1
to about 1:1. In some embodiments, the ratio is in a range of from
about 2.5:1 to 1:1. It is to be understood that while the examples
provided herein use an anti-Jagged activatable antibody referred to
herein as 5342-1204-4D11, these methods are applicable to any
activatable antibody having two or more cysteine residues, where it
is desired that only a portion of the total number of cysteine
residues in the activatable antibody be reduced prior to
conjugation. This is referred to herein as "partial reduction."
[0345] Briefly, a TCEP solution at twice the final concentration
was mixed 1:1 (volume:volume) with 5342-1204-4D11 to result in the
final TCEP:(5342-1204-4D11) ratio desired. The final solution was
then incubated at 37.degree. C. for specified periods of time for
the reduction reaction to progress. At the end of the reduction
reaction, the solution was cooled to room temperature and Maleimide
Alexa-680 (Invitrogen) was added into the solution (Maleimide
Alexa-680 was used at half of the reduction volume and at a
concentration equal to 10.times. molar concentration of TCEP during
the reduction reaction; for example, if the original reduction
reaction comprised 50 microliters (ul) of 13.2 uM 5342-1204-4D11
and 50 ul of 52.8 uM TCEP, then 50 ul 264 uM Maleimide Alexa-680
would be used) to begin the Alexa-680 conjugation. The conjugation
reaction proceeded for 2 hours at room temperature in a light tight
container. After the 2-hour reaction, the solution was spun down
and buffer exchanged into PBS using a PD-10 column (GE Healthcare)
or equivalent using manufacturer's instructions. The final
conjugated product was analyzed using a UV Spectrophotometer to
determine final protein concentration and the degree of labeling of
the Alexa-680 dye.
[0346] Protocol for the Analysis of Maleimide Alexa-680 Conjugated
Anti-Jagged Activatable Antibody Using LabChip GXII:
[0347] A HT Protein Express LabChip (Perkin Elmer) was prepared
according to manufacturer's instructions using either the Pico
Protein Express protocol or the HT Protein Express protocol: the
Pico Protein Express protocol was used to analyze the Alexa-680
conjugated portion of the TCEP reduced 5342-1204-4D11; the HT
Protein Express protocol was used to analyze the total protein in
the TCEP reduced, Alexa-680 conjugated 5342-1204-4D11. TCEP
reduced, Alexa-680 conjugated 5342-1204-4D11 was prepared for the
GXII analysis using Perkin Elmer's instructions. The sample was
analyzed using the 200 series of the LabChip GXII analysis protocol
(High sensitivity for the HT protocol and Pico for the Pico
protocol). Resulting data was analyzed using the LabChip GXII
software.
[0348] Protocol for Jagged Binding ELISA:
[0349] NUNC Maxisorp flatbottom 96 well plates were coated with 50
ul/well, 2 ug/ml human Jagged-Fc fusion protein (R&D Systems)
in Hank's Balanced Salt Solution (HBSS, Teknova) for 2 hours at
room temperature. At the end of the 2 hour coating, the liquid
contents of the plate were evacuated and 250 ul/well of HBSS
containing 1% BSA was introduced and allowed to block the plate for
30 minutes at room temperature. At the end of the blocking period,
liquid contents of the 96-well plate were removed and serially
diluted samples (i.e., 5342-1204-4D11, Alexa-680 conjugated
5342-1204-4D11, uPA-activated 5342-1204-4D11, uPA-activated
Alexa-680 conjugated 5342-1204-4D11, and 4D111 (an anti-Jagged
antibody), starting at a concentration of 100 ug/ml and diluted by
a factor of 3 per dilution step) were introduced at 50 ul/well. The
plate was incubated at room temperature for 1 hour. At the end of
the hour, the plate was washed with HBSS containing 0.05% Tween-20
using a BioTek ELx450 Select CW plate washer (300 ul/well wash
volume, 6 cycles of aspiration and wash). Washed plates were tapped
dry and 50 ul/well of 400 ng/ml Horse Radish Peroxidase conjugated
Goat anti-Human IgG Fab'.sub.2 specific antibody (Jackson
ImmunoResearch) were introduced and incubated for 30 minutes at
room temperature. The plates were washed as previously stated and
100 ul/well of 1-Step TMB Substrate (Thermo Scientific) was
introduced. Color change was observed and the reaction was stopped
by the addition of 100 ul/well of 1 M HCl (Fisher Scientific). The
reacted plate was analyzed using a BioTek EL800 plate reader at
O.D. 450. Data were computed using Excel (Microsoft) and the result
was plotted using Prism 6 (GraphPad).
Example 5
TCEP-Mediated Reduction of Activatable Antibodies
[0350] The compositions and methods provided herein determine the
combination of reagents and reaction conditions that produce the
desired partial reduction followed by conjugation. When reduction
and subsequent conjugation is not controlled properly, activatable
antibodies will be completely reduced, and the masking efficiency
of the activatable antibody is compromised.
[0351] Studies were conducted to determine the range of reducing
agent to activatable antibody. At lower ratios, for example, in the
range of 0.5:1 to 2:1 (reducing agent to activatable antibody),
some reduction was achieved, and the activatable antibody integrity
and masking efficiency were retained. At ratios of 1.5:1 to 5:1
(reducing agent to activatable antibody), reduction time from 30
minutes to 2 hours, there was an increasing amount of reduced
activatable antibody species corresponding to the molecular weight
of one heavy chain and one light chain activatable antibody. The
partially reduced activatable antibody maintained the Jagged
binding characteristics of the original non-reduced and masked
activatable antibody demonstrating that the activatable antibody
partially reduced under these conditions was capable of maintaining
the original masking efficiency. At the identified ratio of
reducing agent to activatable antibody and reduction time, an
inter-chain disulfide-reduced activatable antibody can be produced
to allow for subsequent maximum conjugation through free cysteines
while maintaining the masking efficiency of the original,
non-reduced activatable antibody.
[0352] In one set of studies described herein, an activatable
anti-Jagged antibody referred to as 5342-1204-4D11 was reduced at a
ratio of TCEP to activatable antibody equaling 4; 1 using a
120-minute reduction time. In some instances, reduction was
followed by conjugation to a fluorescent dye, Alexa 680. The
results of these studies (at TCEP to activatable antibody ratios of
4:1) are shown in FIG. 7.
[0353] Using thiol conjugatable Alexa 680 as a surrogate for thiol
conjugatable toxin, these studies demonstrate varying degrees of
Alexa 680 conjugation dependent on both TCEP to activatable
antibody ratio and time of reduction. The conjugation of thiol
conjugatable Alexa 680 to TCEP-partially reduced 5342-1204-4D11
does not significantly change the titration profile of
5342-1204-4D11 to Jagged. Thus, partial reduction and subsequent
thiol conjugation of Alexa 680 can be done in such a way as to
maintain the masking efficiency of activatable antibodies. FIG. 8
demonstrates that partial reduction and subsequent thiol
conjugation of Alexa 680 Fluor.RTM. can be done in such a way as to
also maintain activation of 5342-1204-4D11 by uPA.
Example 6
Materials and Methods
[0354] Antibodies and Activatable Antibodies:
[0355] The examples provided herein use an anti-Jagged activatable
antibody, referred to herein as activatable antibody
5342-1204-4D11, which is described herein. It is to be understood
that while the examples provided herein use these anti-Jagged
activatable antibody constructs, these methods are applicable to
any activatable antibody having two or more cysteine residues,
where it is desired that only a portion of the total number of
cysteine residues in the activatable antibody be reduced prior to
conjugation. This is referred to herein as "partial reduction."
[0356] The examples provided herein also use an anti-Jagged
antibody, referred to herein as anti-Jagged antibody 4D11 (also
referred to as antibody 411 and 411 antibody). The antibody 4D11
includes the following heavy and light chain sequences:
TABLE-US-00014 4D11 Antibody Heavy Chain Nucleotide Sequence: (SEQ
ID NO: 231) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC
CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCA
TGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAAGT
ATTGACCCGGAAGGTCGGCAGACATATTACGCAGACTCCGTGAAGGGCCG
GTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGA
ACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATC
GGCGGCAGGTCGGCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGT
CTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCT
CCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG
CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCC
TCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTAC
ATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGT
TGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCAC
CTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA
CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCG
TGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTG
TACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGG
AGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG
GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAG
CAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTC
TGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 4D11 Antibody Heavy
Chain Amino Acid Sequence: (SEQ ID NO: 245)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11 Antibody
Light Chain Nucleotide Sequence: (nucleotides 133-774 of SEQ ID NO:
233) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAA
ATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCG
GCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACTTACTACTGTCAACAGACGGTTGTGGCGCCTCCGTTATTCGGCCAA
GGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCAT
CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT
GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG
GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA
CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC
CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 4D11 Antibody Light
Chain Amino Acid Sequence: (SEQ ID NO: 244)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0357] Additional antibodies used herein include Synagis.RTM.
(palivizumab) and rituximab, both of which were purchased from Drug
Products Service Labs (UCSF) and used as human IgG isotype
controls.
[0358] SDS Gel:
[0359] Five to ten micrograms (ug) of sample were diluted in 7.5
microliters (ul) of water, final volume. 2.5 ul of 4.times. sample
loading buffer (Invitrogen) with or without 1 ul of 10.times.
reducing agent (Invitrogen) were added and the samples heated at
.about.90.degree. C. for .about.10 min in a heating block. Samples
were loaded onto a 10% Bis-Tris (Novex) gel and run in MOPS buffer
at 200 mV for .about.40 minutes. Gels were stained in Instant Blue
(Expedion) for .about.1 hr followed by de-staining in multiple
washes of water. Gel images were captured on the Imagequant.
[0360] Cell Line:
[0361] BxPC3 cells (ATCC CRL 1687) were maintained in CM (RPMI 1640
supplemented with 10% heat-inactivated fetal bovine serum (FBS).
Prior to FACS binding cell dissociation buffer (Sigma #C5914) was
used to dissociate the adherent cells.
[0362] Protocol for FACS Cell Binding:
[0363] The binding of antibodies, activatable antibodies, or their
immunoconjugate derivatives on BxPC3 cells was evaluated by an
indirect immunofluorescence assay. Dissociated cells
(50,000-100,000 per well) were pelleted in a 96-well v-bottom plate
and incubated at 4.degree. C. for 45 minutes with serial dilutions
of test article in 50 to 100 ul of FACS buffer (FB; HBSS
supplemented with 2% FBS). Control wells included human IgG1
isotype control (Synagis or rituximab) and no IgG containing wells.
Cells were washed twice in cold FB and stained with Alexa 647
conjugated goat anti-human IgG (AF-647 conjugated affinity pure
F(ab')2 fragment goat anti human IgG Fc gamma (.gamma.) fragment
specific, Jackson labs, #1909-606-170) for 30 minutes at 4.degree.
C. Cells were washed as before, fixed in 1% paraformaldehyde/FB and
analyzed using a FACSAria flow cytometer (BD Biosciences). FCS
files were analyzed in FCS Express (DeNovo) and mean fluorescence
intensity (MFI) against dose titration plotted in GraphPad
PRISM.
[0364] Protocol for In Vitro Cytotoxicity Assay:
[0365] Cells (4,000 per well) were plated in white walled 96-well
plates in 50 ul CM. Cells were treated with an equal volume of
serial dilutions of test article for 3 to 5 days. An equal volume
(100 ul) of Cell Titer Glo reagent (Promega) was added to each
well, according to manufacturer's instructions, and relative
luminescence units (RLU) was measured on the Tecan Infinite M200
Pro. RLU against dose titration was plotted and curve fitting
generated in GraphPad PRISM.
Example 7
Production of Activatable Antibody Conjugates by TCEP-Mediated
Partial Reduction and Conjugation
[0366] This example describes the use of partial reduction and
conjugation methods of the invention to produce antibody conjugates
and activatable antibody conjugates of the invention. It is to be
understood that while the examples provided herein use anti-Jagged
activatable antibody constructs, these methods are applicable to
any activatable antibody having two or more cysteine residues,
where it is desired that only a portion of the total number of
cysteine residues in the activatable antibody be reduced prior to
conjugation. This is referred to herein as "partial reduction." In
addition, these methods are applicable to any cleavable or
non-cleavable linker and agent combination.
[0367] This example presents conjugates that display a maleimide
PEG-valine-citrulline-para-aminobenzyloxycarbonyl-monomethyl
auristatin D linker payload, referred to herein as "vc-MMAD," or a
maleimide
PEG-valine-citrulline-para-aminobenzyloxycarbonyl-monomethyl
auristatin E linker payload, referred to herein as "vc-MMAE."
[0368] Conjugates of anti-Jagged activatable antibody
5342-1204-4D11 comprising the maleimide caproyl-valine-citruline
monomethyl auristatin D linker payload (vc-MMAD) or the maleimide
caproyl-valine-citruline monomethyl auristatin E linker payload
(vc-MMAE), referred to herein as activatable antibody conjugate
5342-1204-4D11-vc-MMAD and activatable antibody conjugate
5342-1204-4D11-vc-MMAE, respectively, were prepared as follows.
[0369] The vc-MMAD and vc-MMAE reagents were prepared at Bayside
Chemicals (Burlingame, Calif.). N,N, Dimethylacetamide (DMA),
tris(2-carboxyethyl)phosphine (TCEP; cat #646547), and IN NaOH were
purchased from Sigma. Prior to performing a conjugation, the
following stock solutions were prepared: 5.0 mg/mL antibody or
activatable antibody in PBS (total phosphate=4.25 mM), 5.0 mg/mL
vc-MMAD or vc-MMAE in DMA, 1.0 mM TCEP in water, and 10 mM NaOH in
water. TCEP was used at a range from 1:1 to 8:1 ratio, typically at
a 2.5:1 equimolar ratio, of TCEP to antibody or activatable
antibody.
[0370] A typical partial reduction and conjugation was performed as
follows, wherein equivalents (eq) are reported relative to the
antibody or activatable antibody. To 200 uL of antibody or
activatable antibody in PBS in a tube was added 2.5 eq TCEP. The
tube was closed and swirled, to generate a homogeneous solution,
which was allowed to stand for 90 minutes at room temperature. The
tube was opened, and 13 eq NaOH and 6.0 eq vc-MMAD or vc-MMAE were
added. The tube was closed and swirled, to generate a homogeneous
solution, which was allowed to stand at room temperature for 120
minutes. The tube was opened, and the reaction mixture was passed
through a Zeba de-salting column (Thermo Scientific). The protein
concentration in the filtrate was analyzed by UV spectrophotometry,
and the product was analyzed by SDS gel and HIC-HPLC, and then
tested for cell-binding and cell-killing activities. For the
cell-binding and cell-killing assays, activatable antibody
conjugates were activated by incubating a 0.5-mg sample of the
activatable antibody conjugate in a 10% solution of recombinant
human uPA protease (R and D systems MN-207-16) at 37.degree. C. for
16 hr. Protease was removed from the thus activated activatable
antibody conjugate by running the sample through a pre-washed
MabSelect (GE Healthcare) column and eluting with 0.1M glycine,
followed by neutralization using Tris-HCl. In other conjugations,
the TCEP eq varied from 1.0 to 8.0, and/or the NaOH eq varied from
0 to 13, and/or the reaction times after the addition of TCEP, or
the additions of NaOH and vc-MMAD or vc-MMAD, varied from 60 min to
16 hours.
Example 8
Use of Activatable Antibody Conjugates that Include Microtubule
Inhibitor Monomethyl Auristatin D (MMAD)
[0371] This example demonstrates that activatable antibody
conjugates comprising microtubule inhibitor MMAD display potent in
vitro killing activity. The example also demonstrates that addition
of the linker payload to the activatable antibody does not
interfere with the ability of the masking moiety to block binding
of the activatable antibody comprising such masking moiety to its
target.
[0372] FIG. 9A demonstrates that partial reduction and subsequent
thiol conjugation of anti-Jagged activatable antibody
5342-1204-4D11 or anti-Jagged antibody 4D11 with the vc-MMAD linker
payload did not affect the binding behavior of the resultant
conjugates to BxPC3 cells: There was a 38-fold reduction in IC50 of
binding of activatable antibody conjugate 5342-1204-4D11-vc-MMAD
(27 nM) compared to antibody conjugate 4D11-vc-MMAD (0.7 nM). Upon
activation with uPA, binding of activated activatable antibody
conjugate 5342-1204-4D11-vc-MMAD was comparable to that of antibody
conjugate 4D11-vc-MMAD. The figure also demonstrates that Synagis
(a human IgG isotype control) did not bind to BxPC3 cells.
[0373] Conjugation of activatable antibody 5342-1204-4D11 and
antibody 4D11 was observed by the increased shift in MW of the HC
and LC under reduced conditions by SDS analysis. HIC-HPLC analysis
demonstrated between 40 to 75% yield of toxin-conjugated
material.
[0374] FIG. 9B demonstrates the potent killing activity of
activatable antibody conjugate 5342-1204-4D11-vc-MMAD upon
activation with uPA and antibody conjugate 4D11-vc-MMAD.
Non-activated activatable antibody conjugate 5342-1204-4D11-vc-MMAD
killed BxPC3 cells at a 17-fold reduced potency (IC50=7 nM)
compared to antibody conjugate 4D11-vc-MMAD (IC50=0.4 nM). The lack
of cell killing activity of rituximab antibody (an unrelated IgG1
isotype antibody) conjugated with the vc-MMAD linker is also shown.
Antibody 4D11 alone (i.e., not conjugated) did not exhibit any
cytotoxic activity on BxPC3.
Example 9
Use of Activatable Antibody Conjugates that Include Microtubule
Inhibitor Monomethyl Auristatin E (MMAE)
[0375] This example demonstrates that activatable antibody
conjugates comprising microtubule inhibitor MMAE display potent in
vitro killing activity. The example also demonstrates that addition
of the linker payload to the activatable antibody does not
interfere with the ability of the masking moiety to block binding
of the activatable antibody comprising such masking moiety to its
target.
[0376] FIG. 10A demonstrates that partial reduction and subsequent
thiol conjugation of anti-Jagged activatable antibody
5342-1204-4D11 or anti-Jagged antibody 4D11 with the vc-MMAE linker
payload did not affect the binding behavior of the resultant
conjugates to BxPC3 cells: There was a 33-fold reduction in IC50 of
binding of activatable antibody conjugate 5342-1204-4D11-vc-MMAE
(.about.30 nM) (which was similar to the binding activity of
non-conjugated activatable antibody 5342-1404-4D11) compared to
antibody conjugate 4D11-vc-MMAE (0.9 nM). Upon activation with uPA,
binding of activated activatable antibody conjugate
5342-1204-4D11-vc-MMAE was comparable to that of antibody conjugate
4D11-vc-MMAE. The figure also demonstrates that Synagis conjugated
to vc-MMAE did not bind to BxPC3 cells.
[0377] FIG. 10B demonstrates the potent killing activity of
activatable antibody conjugate 5342-1204-4D11-vc-MMAE upon
activation with uPA and antibody conjugate 4D11-vc-MMAE.
Non-activated activatable antibody conjugate 5342-1204-4D11-vc-MMAE
killed BxPC3 cells at a 12-fold reduced potency (IC50=5 nM)
compared to antibody conjugate 4D11-vc-MMAE (IC50=0.4 nM). The lack
of cell killing activity of Synagis conjugated with the vc-MMAE
linker is also shown.
Other Embodiments
[0378] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 318 <210> SEQ ID NO 1 <211> LENGTH: 1350
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C225v5 Antibody
Heavy Chain <400> SEQUENCE: 1 caggtgcagc tgaaacagag
cggcccgggc ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg
tgagcggctt tagcctgacc aactatggcg tgcattgggt gcgccagagc 120
ccgggcaaag gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac
180 accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca
ggtgtttttt 240 aaaatgaaca gcctgcaaag ccaggatacc gcgatttatt
attgcgcgcg cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc
cagggcaccc tggtgaccgt gagcgcggct 360 agcaccaagg gcccatcggt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc
tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480
aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg
acaagaaagt tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg
tgcccagcac ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 960 tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga accacaggtg
tacaccctgc ccccatcccg ggatgaactg 1080 accaagaacc aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1260 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1320 aagagcctct ccctgtctcc gggtaaatga 1350 <210>
SEQ ID NO 2 <211> LENGTH: 449 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v5 Antibody Heavy Chain
<400> SEQUENCE: 2 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu
Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser
Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu
Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys
Met Asn Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90
95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
<210> SEQ ID NO 3 <211> LENGTH: 786 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 3954-1204-C225v5 Activatable
Antibody Light Chain <400> SEQUENCE: 3 caaggccagt ctggccagtg
catctcacct cgtggttgtc cggacggccc atacgtcatg 60 tacggctcga
gcggtggcag cggtggctct ggtggatccg gtctgagcgg ccgttccgat 120
aatcatggca gtagcggtac ccagatcttg ctgacccaga gcccggtgat tctgagcgtg
180 agcccgggcg aacgtgtgag ctttagctgc cgcgcgagcc agagcattgg
caccaacatt 240 cattggtatc agcagcgcac caacggcagc ccgcgcctgc
tgattaaata tgcgagcgaa 300 agcattagcg gcattccgag ccgctttagc
ggcagcggca gcggcaccga ttttaccctg 360 agcattaaca gcgtggaaag
cgaagatatt gcggattatt attgccagca gaacaacaac 420 tggccgacca
cctttggcgc gggcaccaaa ctggaactga aacgtacggt ggctgcacca 480
tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg
540 tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt
ggataacgcc 600 ctccaatcgg gtaactccca ggagagtgtc acagagcagg
acagcaagga cagcacctac 660 agcctcagca gcaccctgac gctgagcaaa
gcagactacg agaaacacaa agtctacgcc 720 tgcgaagtca cccatcaggg
cctgagctcg cccgtcacaa agagcttcaa caggggagag 780 tgttag 786
<210> SEQ ID NO 4 <211> LENGTH: 261 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 3954-1204-C225v5 Activatable
Antibody Light Chain <400> SEQUENCE: 4 Gln Gly Gln Ser Gly
Gln Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly 1 5 10 15 Pro Tyr Val
Met Tyr Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Gly Leu Ser Gly Arg Ser Asp Asn His Gly Ser Ser Gly Thr Gln 35 40
45 Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu
50 55 60 Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr
Asn Ile 65 70 75 80 His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg
Leu Leu Ile Lys 85 90 95 Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
Ser Arg Phe Ser Gly Ser 100 105 110 Gly Ser Gly Thr Asp Phe Thr Leu
Ser Ile Asn Ser Val Glu Ser Glu 115 120 125 Asp Ile Ala Asp Tyr Tyr
Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr 130 135 140 Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro 145 150 155 160 Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170
175 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
180 185 190 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln Glu 195 200 205 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser 210 215 220 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr Ala 225 230 235 240 Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser Phe 245 250 255 Asn Arg Gly Glu Cys
260 <210> SEQ ID NO 5 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: spacer peptide encoding
sequence <400> SEQUENCE: 5 caaggccagt ctggccag 18 <210>
SEQ ID NO 6 <211> LENGTH: 45 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety encoding sequence
<400> SEQUENCE: 6 tgcatctcac ctcgtggttg tccggacggc ccatacgtca
tgtac 45 <210> SEQ ID NO 7 <211> LENGTH: 39 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: linker peptide encoding
sequence <400> SEQUENCE: 7 ggctcgagcg gtggcagcgg tggctctggt
ggatccggt 39 <210> SEQ ID NO 8 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 1204 substrate
encoding sequence <400> SEQUENCE: 8 ctgagcggcc gttccgataa
tcat 24 <210> SEQ ID NO 9 <211> LENGTH: 15 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: linker peptide encoding
sequence <400> SEQUENCE: 9 ggcagtagcg gtacc 15 <210>
SEQ ID NO 10 <211> LENGTH: 645 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v5 Antibody Light Chain
<400> SEQUENCE: 10 cagatcttgc tgacccagag cccggtgatt
ctgagcgtga gcccgggcga acgtgtgagc 60 tttagctgcc gcgcgagcca
gagcattggc accaacattc attggtatca gcagcgcacc 120 aacggcagcc
cgcgcctgct gattaaatat gcgagcgaaa gcattagcgg cattccgagc 180
cgctttagcg gcagcggcag cggcaccgat tttaccctga gcattaacag cgtggaaagc
240 gaagatattg cggattatta ttgccagcag aacaacaact ggccgaccac
ctttggcgcg 300 ggcaccaaac tggaactgaa acgtacggtg gctgcaccat
ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540
ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag 645
<210> SEQ ID NO 11 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: spacer peptide <400> SEQUENCE:
11 Gln Gly Gln Ser Gly Gln 1 5 <210> SEQ ID NO 12 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 12 Cys Ile Ser Pro Arg Gly Cys
Pro Asp Gly Pro Tyr Val Met Tyr 1 5 10 15 <210> SEQ ID NO 13
<211> LENGTH: 13 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linker peptide <400> SEQUENCE: 13 Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 <210> SEQ ID
NO 14 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: 1204 substrate <400> SEQUENCE: 14 Leu Ser
Gly Arg Ser Asp Asn His 1 5 <210> SEQ ID NO 15 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: linker
peptide <400> SEQUENCE: 15 Gly Ser Ser Gly Thr 1 5
<210> SEQ ID NO 16 <211> LENGTH: 214 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v5 Antibody Light Chain
<400> SEQUENCE: 16 Gln Ile Leu Leu Thr Gln Ser Pro Val Ile
Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg
Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln
Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser
Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65 70 75 80
Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr 85
90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205
Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID NO 17 <211>
LENGTH: 777 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
3954-NSUB-C225v5 Masked Antibody Light Chain <400> SEQUENCE:
17 caaggccagt ctggccagtg catctcacct cgtggttgtc cggacggccc
atacgtcatg 60 tacggctcga gcggtggcag cggtggctct ggtggctcag
gtggaggctc gggcggtggg 120 agcggcggtt ctcagatctt gctgacccag
agcccggtga ttctgagcgt gagcccgggc 180 gaacgtgtga gctttagctg
ccgcgcgagc cagagcattg gcaccaacat tcattggtat 240 cagcagcgca
ccaacggcag cccgcgcctg ctgattaaat atgcgagcga aagcattagc 300
ggcattccga gccgctttag cggcagcggc agcggcaccg attttaccct gagcattaac
360 agcgtggaaa gcgaagatat tgcggattat tattgccagc agaacaacaa
ctggccgacc 420 acctttggcg cgggcaccaa actggaactg aaacgtacgg
tggctgcacc atctgtcttc 480 atcttcccgc catctgatga gcagttgaaa
tctggaactg cctctgttgt gtgcctgctg 540 aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 600 ggtaactccc
aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 660
agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc
720 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag
777 <210> SEQ ID NO 18 <211> LENGTH: 258 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: 3954-NSUB-C225v5 Masked
Antibody Light Chain <400> SEQUENCE: 18 Gln Gly Gln Ser Gly
Gln Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly 1 5 10 15 Pro Tyr Val
Met Tyr Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Gln Ile Leu Leu 35 40
45 Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser
50 55 60 Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His
Trp Tyr 65 70 75 80 Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile
Lys Tyr Ala Ser 85 90 95 Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Leu Ser Ile Asn
Ser Val Glu Ser Glu Asp Ile Ala 115 120 125 Asp Tyr Tyr Cys Gln Gln
Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala 130 135 140 Gly Thr Lys Leu
Glu Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe 145 150 155 160 Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170
175 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
180 185 190 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr 195 200 205 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr 210 215 220 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val 225 230 235 240 Thr His Gln Gly Leu Ser Ser
Pro Val Thr Lys Ser Phe Asn Arg Gly 245 250 255 Glu Cys <210>
SEQ ID NO 19 <211> LENGTH: 69 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: linker - non-cleavable substrate -
linker peptide encoding sequence <400> SEQUENCE: 19
ggctcgagcg gtggcagcgg tggctctggt ggctcaggtg gaggctcggg cggtgggagc
60 ggcggttct 69 <210> SEQ ID NO 20 <211> LENGTH: 23
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linker -
non-cleavable substrate - linker peptide <400> SEQUENCE: 20
Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser 1 5
10 15 Gly Gly Gly Ser Gly Gly Ser 20 <210> SEQ ID NO 21
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linking peptide <220> FEATURE: <221>
NAME/KEY: REPEAT <222> LOCATION: (1)..(5) <223> OTHER
INFORMATION: May be repeated <400> SEQUENCE: 21 Gly Ser Gly
Gly Ser 1 5 <210> SEQ ID NO 22 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linking peptide
<220> FEATURE: <221> NAME/KEY: REPEAT <222>
LOCATION: (1)..(4) <223> OTHER INFORMATION: May be repeated
<400> SEQUENCE: 22 Gly Gly Gly Ser 1 <210> SEQ ID NO 23
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linking peptide <400> SEQUENCE: 23 Gly Gly Ser
Gly 1 <210> SEQ ID NO 24 <211> LENGTH: 5 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: linking peptide <400>
SEQUENCE: 24 Gly Gly Ser Gly Gly 1 5 <210> SEQ ID NO 25
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linking peptide <400> SEQUENCE: 25 Gly Ser Gly
Ser Gly 1 5 <210> SEQ ID NO 26 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linking peptide
<400> SEQUENCE: 26 Gly Ser Gly Gly Gly 1 5 <210> SEQ ID
NO 27 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: linking peptide <400> SEQUENCE: 27 Gly Gly
Gly Ser Gly 1 5 <210> SEQ ID NO 28 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linking peptide
<400> SEQUENCE: 28 Gly Ser Ser Ser Gly 1 5 <210> SEQ ID
NO 29 <211> LENGTH: 6 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 29 Cys Ile
Ser Pro Arg Gly 1 5 <210> SEQ ID NO 30 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 30 Cys Ile Ser Pro Arg Gly Cys Gly 1 5
<210> SEQ ID NO 31 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
31 Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly Pro Tyr Val Met Tyr 1 5
10 15 <210> SEQ ID NO 32 <211> LENGTH: 14 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 32 Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly Pro Tyr Val
Met 1 5 10 <210> SEQ ID NO 33 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 33 Cys Ile Ser Pro Arg Gly Cys Glu Pro Gly
Thr Tyr Val Pro Thr 1 5 10 15 <210> SEQ ID NO 34 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 34 Cys Ile Ser Pro Arg Gly Cys
Pro Gly Gln Ile Trp His Pro Pro 1 5 10 15 <210> SEQ ID NO 35
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 35 Gly Ser His
Cys Leu Ile Pro Ile Asn Met Gly Ala Pro Ser Cys 1 5 10 15
<210> SEQ ID NO 36 <211> LENGTH: 32 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
36 Cys Ile Ser Pro Arg Gly Cys Gly Gly Ser Ser Ala Ser Gln Ser Gly
1 5 10 15 Gln Gly Ser His Cys Leu Ile Pro Ile Asn Met Gly Ala Pro
Ser Cys 20 25 30 <210> SEQ ID NO 37 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 37 Cys Asn His His Tyr Phe Tyr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
38 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 38 Ala Asp
His Val Phe Trp Gly Ser Tyr Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 39 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 39 Cys His His Val Tyr Trp Gly His Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
40 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 40 Cys Pro
His Phe Thr Thr Thr Ser Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 41 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 41 Cys Asn His His Tyr His Tyr Tyr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
42 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 42 Cys Pro
His Val Ser Phe Gly Ser Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 43 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 43 Cys Pro Tyr Tyr Thr Leu Ser Tyr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
44 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 44 Cys Asn
His Val Tyr Phe Gly Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 45 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 45 Cys Asn His Phe Thr Leu Thr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
46 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 46 Cys His
His Phe Thr Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 47 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 47 Tyr Asn Pro Cys Ala Thr Pro Met Cys Cys
Ile Ser Pro Arg Gly Cys 1 5 10 15 Pro Gly <210> SEQ ID NO 48
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 48 Cys Asn His
His Tyr Phe Tyr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys
Gly <210> SEQ ID NO 49 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 49 Cys Asn His His Tyr His Tyr Tyr Cys Gly Cys Ile Ser
Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 50 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 50 Cys Asn His Val Tyr Phe Gly
Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210>
SEQ ID NO 51 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
51 Cys His His Val Tyr Trp Gly His Cys Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Gly <210> SEQ ID NO 52 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 52 Cys Pro His Phe Thr Thr Thr Ser Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 53
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 53 Cys Asn His
Phe Thr Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys
Gly <210> SEQ ID NO 54 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 54 Cys His His Phe Thr Leu Thr Thr Cys Gly Cys Ile Ser
Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 55 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 55 Cys Pro Tyr Tyr Thr Leu Ser
Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210>
SEQ ID NO 56 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
56 Cys Pro His Val Ser Phe Gly Ser Cys Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Gly <210> SEQ ID NO 57 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 57 Ala Asp His Val Phe Trp Gly Ser Tyr Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 58
<211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 58 Tyr Asn Pro
Cys Ala Thr Pro Met Cys Cys Ile Ser Pro Arg Gly Cys 1 5 10 15 Gly
<210> SEQ ID NO 59 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
59 Cys His His Val Tyr Trp Gly His Cys Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Gly <210> SEQ ID NO 60 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa may be Asn or
Phe <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa
may be His, Val or Phe <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa may be Tyr or Thr <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6) <223>
OTHER INFORMATION: Xaa may be Phe, Trp, Thr or Leu <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(7)..(7) <223> OTHER INFORMATION: Xaa may be Tyr, Gly, Thr or
Ser <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (8)..(8) <223> OTHER INFORMATION: Xaa
may be Thr, Ser, Tyr or His <400> SEQUENCE: 60 Cys Xaa His
Xaa Xaa Xaa Xaa Xaa Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys
Gly <210> SEQ ID NO 61 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 61 Cys Ile Ser Pro Arg Gly Cys Gly Gln Pro Ile Pro Ser
Val Lys 1 5 10 15 <210> SEQ ID NO 62 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 62 Cys Ile Ser Pro Arg Gly Cys Thr Gln Pro
Tyr His Val Ser Arg 1 5 10 15 <210> SEQ ID NO 63 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 63 Cys Ile Ser Pro Arg Gly Cys
Asn Ala Val Ser Gly Leu Gly Ser 1 5 10 15 <210> SEQ ID NO 64
<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 64 Gln Gly Gln
Ser Gly Gln Gly Gln Gln Gln Trp Cys Asn Ile Trp Ile 1 5 10 15 Asn
Gly Gly Asp Cys Arg Gly Trp Asn Gly 20 25 <210> SEQ ID NO 65
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 65 Pro Trp Cys
Met Gln Arg Gln Asp Phe Leu Arg Cys Pro Gln Pro 1 5 10 15
<210> SEQ ID NO 66 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
66 Gln Leu Gly Leu Pro Ala Tyr Met Cys Thr Phe Glu Cys Leu Arg 1 5
10 15 <210> SEQ ID NO 67 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 67 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys Gly Gly Leu
Gln Gly 1 5 10 15 <210> SEQ ID NO 68 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 68 Ser Cys Ser Leu Trp Thr Ser Gly Ser Cys
Leu Pro His Ser Pro 1 5 10 15 <210> SEQ ID NO 69 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 69 Tyr Cys Leu Gln Leu Pro His
Tyr Met Gln Ala Met Cys Gly Arg 1 5 10 15 <210> SEQ ID NO 70
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 70 Cys Phe Leu
Tyr Ser Cys Thr Asp Val Ser Tyr Trp Asn Asn Thr 1 5 10 15
<210> SEQ ID NO 71 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
71 Pro Trp Cys Met Gln Arg Gln Asp Tyr Leu Arg Cys Pro Gln Pro 1 5
10 15 <210> SEQ ID NO 72 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 72 Cys Asn Leu Trp Ile Ser Gly Gly Asp Cys Arg Gly Leu
Ala Gly 1 5 10 15 <210> SEQ ID NO 73 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 73 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys
Arg Gly Val Gln Gly 1 5 10 15 <210> SEQ ID NO 74 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 74 Cys Asn Leu Trp Val Ser Gly
Gly Asp Cys Arg Gly Leu Arg Gly 1 5 10 15 <210> SEQ ID NO 75
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 75 Cys Asn Leu
Trp Ile Ser Gly Gly Asp Cys Arg Gly Leu Pro Gly 1 5 10 15
<210> SEQ ID NO 76 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
76 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys Arg Asp Ala Pro Trp 1 5
10 15 <210> SEQ ID NO 77 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 77 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys Arg Asp Leu
Leu Gly 1 5 10 15 <210> SEQ ID NO 78 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 78 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys
Arg Gly Leu Gln Gly 1 5 10 15 <210> SEQ ID NO 79 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 79 Cys Asn Leu Trp Leu His Gly
Gly Asp Cys Arg Gly Trp Gln Gly 1 5 10 15 <210> SEQ ID NO 80
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 80 Cys Asn Ile
Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Gln Gly 1 5 10 15
<210> SEQ ID NO 81 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
81 Cys Thr Thr Trp Phe Cys Gly Gly Asp Cys Gly Val Met Arg Gly 1 5
10 15 <210> SEQ ID NO 82 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 82 Cys Asn Ile Trp Gly Pro Ser Val Asp Cys Gly Ala Leu
Leu Gly 1 5 10 15 <210> SEQ ID NO 83 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 83 Cys Asn Ile Trp Val Asn Gly Gly Asp Cys
Arg Ser Phe Glu Gly 1 5 10 15 <210> SEQ ID NO 84 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 84 Tyr Cys Leu Asn Leu Pro Arg
Tyr Met Gln Asp Met Cys Trp Ala 1 5 10 15 <210> SEQ ID NO 85
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 85 Tyr Cys Leu
Ala Leu Pro His Tyr Met Gln Ala Asp Cys Ala Arg 1 5 10 15
<210> SEQ ID NO 86 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
86 Cys Phe Leu Tyr Ser Cys Gly Asp Val Ser Tyr Trp Gly Ser Ala 1 5
10 15 <210> SEQ ID NO 87 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 87 Cys Tyr Leu Tyr Ser Cys Thr Asp Ser Ala Phe Trp Asn
Asn Arg 1 5 10 15 <210> SEQ ID NO 88 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 88 Cys Tyr Leu Tyr Ser Cys Asn Asp Val Ser
Tyr Trp Ser Asn Thr 1 5 10 15 <210> SEQ ID NO 89 <211>
LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 89 Cys Phe Leu Tyr Ser Cys Thr
Asp Val Ser Tyr Trp 1 5 10 <210> SEQ ID NO 90 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 90 Cys Phe Leu Tyr Ser Cys Thr
Asp Val Ala Tyr Trp Asn Ser Ala 1 5 10 15 <210> SEQ ID NO 91
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 91 Cys Phe Leu
Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Asp Thr 1 5 10 15
<210> SEQ ID NO 92 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
92 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Asn Ser 1 5
10 15 <210> SEQ ID NO 93 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 93 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala Tyr Trp Asn
Asn Thr 1 5 10 15 <210> SEQ ID NO 94 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 94 Cys Phe Leu Tyr Ser Cys Gly Asp Val Ser
Tyr Trp Gly Asn Pro Gly 1 5 10 15 Leu Ser <210> SEQ ID NO 95
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 95 Cys Phe Leu
Tyr Ser Cys Thr Asp Val Ala Tyr Trp Ser Gly Leu 1 5 10 15
<210> SEQ ID NO 96 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
96 Cys Tyr Leu Tyr Ser Cys Thr Asp Gly Ser Tyr Trp Asn Ser Thr 1 5
10 15 <210> SEQ ID NO 97 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 97 Cys Phe Leu Tyr Ser Cys Ser Asp Val Ser Tyr Trp Gly
Asn Ile 1 5 10 15 <210> SEQ ID NO 98 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 98 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala
Tyr Trp 1 5 10 <210> SEQ ID NO 99 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 99 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser
Tyr Trp Gly Ser Thr 1 5 10 15 <210> SEQ ID NO 100 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 100 Cys Phe Leu Tyr Ser Cys
Thr Asp Val Ala Tyr Trp Gly Asp Thr 1 5 10 15 <210> SEQ ID NO
101 <211> LENGTH: 20 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 101 Gly Cys
Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg Gly Trp Val Asp 1 5 10 15
Pro Leu Gln Gly 20 <210> SEQ ID NO 102 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 102 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp
Cys Arg Gly Trp Ile Gly 1 5 10 15 Asp Thr Asn Gly 20 <210>
SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
103 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Ile Glu
1 5 10 15 Asp Ser Asn Gly 20 <210> SEQ ID NO 104 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 104 Gly Cys Asn Ile Trp Ala
Asn Gly Gly Asp Cys Arg Gly Trp Ile Asp 1 5 10 15 Asn Ile Asp Gly
20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 105 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly
Trp Leu Gly 1 5 10 15 Glu Ala Val Gly 20 <210> SEQ ID NO 106
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 106 Gly Cys Asn
Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Leu Glu 1 5 10 15 Glu
Ala Val Gly 20 <210> SEQ ID NO 107 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 107 Gly Gly Pro Ala Leu Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Ser Gly 20 <210>
SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
108 Gly Ala Pro Val Phe Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg
1 5 10 15 Gly Trp Met Gly 20 <210> SEQ ID NO 109 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 109 Gly Gln Gln Gln Trp Cys
Asn Ile Trp Ile Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Asn Gly
20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 110 Gly Lys Ser Glu Phe Cys Asn Ile Trp Leu Asn Gly Gly
Asp Cys Arg 1 5 10 15 Gly Trp Ile Gly 20 <210> SEQ ID NO 111
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 111 Gly Thr Pro
Gly Gly Cys Asn Ile Trp Ala Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly
Trp Glu Gly 20 <210> SEQ ID NO 112 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 112 Gly Ala Ser Gln Tyr Cys Asn Leu Trp Ile
Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Arg Gly 20 <210>
SEQ ID NO 113 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
113 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Pro Trp Val Glu
1 5 10 15 Gly Gly <210> SEQ ID NO 114 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 114 Gly Cys Asn Ile Trp Ala Val Gly Gly Asp
Cys Arg Pro Phe Val Asp 1 5 10 15 Gly Gly <210> SEQ ID NO 115
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 115 Gly Cys Asn
Ile Trp Leu Asn Gly Gly Asp Cys Arg Ala Trp Val Asp 1 5 10 15 Thr
Gly <210> SEQ ID NO 116 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 116 Gly Cys Asn Ile Trp Ile Val Gly Gly Asp Cys Arg Pro
Phe Ile Asn 1 5 10 15 Asp Gly <210> SEQ ID NO 117 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 117 Gly Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg Pro Val Val Phe 1 5 10 15 Gly Gly
<210> SEQ ID NO 118 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
118 Gly Cys Asn Ile Trp Leu Ser Gly Gly Asp Cys Arg Met Phe Met Asn
1 5 10 15 Glu Gly <210> SEQ ID NO 119 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 119 Gly Cys Asn Ile Trp Val Asn Gly Gly Asp
Cys Arg Ser Phe Val Tyr 1 5 10 15 Ser Gly <210> SEQ ID NO 120
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 120 Gly Cys Asn
Ile Trp Leu Asn Gly Gly Asp Cys Arg Gly Trp Glu Ala 1 5 10 15 Ser
Gly <210> SEQ ID NO 121 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 121 Gly Cys Asn Ile Trp Ala His Gly Gly Asp Cys Arg Gly
Phe Ile Glu 1 5 10 15 Pro Gly <210> SEQ ID NO 122 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 122 Gly Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg Thr Phe Val Ala 1 5 10 15 Ser Gly
<210> SEQ ID NO 123 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
123 Gly Cys Asn Ile Trp Ala His Gly Gly Asp Cys Arg Gly Phe Ile Glu
1 5 10 15 Pro Gly <210> SEQ ID NO 124 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 124 Gly Phe Leu Glu Asn Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Thr Gly <210> SEQ ID NO 125
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 125 Gly Ile Tyr
Glu Asn Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg 1 5 10 15 Met
Gly <210> SEQ ID NO 126 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 126 Gly Ile Pro Asp Asn Cys Asn Ile Trp Ile Asn Gly Gly
Asp Cys Arg 1 5 10 15 Tyr Gly <210> SEQ ID NO 127 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 127 Gln Gly Gln Ser Gly Gln
Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met Asp
Cys 20 <210> SEQ ID NO 128 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 128 Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His 1 5 10 15 Trp Val Pro Ile Thr 20 <210> SEQ ID NO
129 <211> LENGTH: 24 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 129 Gln Gly
Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn 1 5 10 15
Tyr Ala His Ile Phe Met Asp Cys 20 <210> SEQ ID NO 130
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 130 Tyr Arg Ser
Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 131 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
131 Pro Gly Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr
1 5 10 15 <210> SEQ ID NO 132 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 132 Glu Ser Ser Cys Val Trp Asn Tyr Val His
Ile Tyr Met Asp Cys 1 5 10 15 <210> SEQ ID NO 133 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 133 Tyr Pro Gly Cys Lys Trp
Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
134 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 134 Tyr Arg
Thr Cys Ser Trp Asn Tyr Val Gly Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 135 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
135 Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Met Asp Cys 1 5
10 15 <210> SEQ ID NO 136 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 136 Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 137 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 137 Tyr Gly Ser Cys Asn Trp Asn Tyr Val His
Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO 138 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 138 Tyr Thr Ser Cys Asn Trp
Asn Tyr Val His Ile Phe Met Asp Cys 1 5 10 15 <210> SEQ ID NO
139 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 139 Tyr Pro
Gly Cys Lys Trp Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 140 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
140 Trp Arg Ser Cys Asn Trp Asn Tyr Ala His Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 141 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 141 Trp Ser Asn Cys His Trp Asn Tyr Val His Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 142 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 142 Asp Arg Ser Cys Thr Trp Asn Tyr Val Arg
Ile Ser Tyr Asp Cys 1 5 10 15 <210> SEQ ID NO 143 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 143 Ser Gly Ser Cys Lys Trp
Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
144 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 144 Ser Arg
Ser Cys Ile Trp Asn Tyr Ala His Ile His Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 145 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
145 Ser Met Ser Cys Tyr Trp Gln Tyr Glu Arg Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 146 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 146 Tyr Arg Ser Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 147 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 147 Tyr Gly Ser Cys Ser Trp Asn Tyr Val His
Ile Phe Met Asp Cys 1 5 10 15 <210> SEQ ID NO 148 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 148 Ser Gly Ser Cys Lys Trp
Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
149 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 149 Tyr Lys
Ser Cys His Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 150 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
150 Tyr Gly Ser Cys Thr Trp Asn Tyr Val His Ile Phe Met Glu Cys 1 5
10 15 <210> SEQ ID NO 151 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 151 Phe Ser Ser Cys Asn Trp Asn Tyr Val His Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 152 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 152 Trp Arg Ser Cys Asn Trp Asn Tyr Ala His
Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO 153 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 153 Tyr Gly Ser Cys Gln Trp
Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
154 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 154 Tyr Arg
Ser Cys Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 155 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
155 Asn Met Ser Cys His Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 156 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 156 Phe Gly Pro Cys Thr Trp Asn Tyr Ala Arg Ile Ser Trp
Asp Cys 1 5 10 15 <210> SEQ ID NO 157 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(2) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 157 Xaa Xaa Ser Cys Xaa Trp Xaa Tyr Val His
Ile Phe Xaa Asp Cys 1 5 10 15 <210> SEQ ID NO 158 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 158 Met Gly Val Pro Ala Gly
Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Asp Cys
<210> SEQ ID NO 159 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
159 Arg Asp Thr Gly Gly Gln Cys Arg Trp Asp Tyr Val His Ile Phe Met
1 5 10 15 Asp Cys <210> SEQ ID NO 160 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 160 Ala Gly Val Pro Ala Gly Cys Thr Trp Asn
Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 161
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 161 Val Gly Val
Pro Asn Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu
Cys <210> SEQ ID NO 162 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 162 Asp Gly Gly Pro Ala Gly Cys Ser Trp Asn Tyr Val His
Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 163 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 163 Ala Val Gly Pro Ala Gly
Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
<210> SEQ ID NO 164 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
164 Cys Thr Trp Asn Tyr Val His Ile Phe Met Asp Cys Gly Glu Gly Glu
1 5 10 15 Gly Pro <210> SEQ ID NO 165 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 165 Gly Gly Val Pro Glu Gly Cys Thr Trp Asn
Tyr Ala His Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 166
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 166 Ala Glu Val
Pro Ala Gly Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu
Cys <210> SEQ ID NO 167 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 167 Ala Gly Val Pro Ala Gly Cys Thr Trp Asn Tyr Val His
Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 168 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 168 Ser Gly Ala Ser Gly Gly
Cys Lys Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Asp Cys
<210> SEQ ID NO 169 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
169 Met Gly Val Pro Ala Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met
1 5 10 15 Asp Cys <210> SEQ ID NO 170 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 170 Thr Pro Gly Cys Arg Trp Asn Tyr Val His
Ile Phe Met Glu Cys Glu 1 5 10 15 Ala Leu <210> SEQ ID NO 171
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 171 Val Gly Val
Pro Asn Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu
Cys <210> SEQ ID NO 172 <211> LENGTH: 16 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 172 Pro Gly Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val
Pro Asn Thr 1 5 10 15 <210> SEQ ID NO 173 <211> LENGTH:
16 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 173 Arg Gly Ala Cys Asp Ile Pro Phe Pro Ala
His Trp Ile Pro Asn Thr 1 5 10 15 <210> SEQ ID NO 174
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 174 Gln Gly Asp
Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Ile Thr 1 5 10 15
<210> SEQ ID NO 175 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa can be any naturally occurring
amino acid <400> SEQUENCE: 175 Xaa Gly Ala Phe Asp Ile Pro
Phe Pro Ala His Trp Val Pro Asn Thr 1 5 10 15 <210> SEQ ID NO
176 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 176 Arg Gly
Asp Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15
Pro Arg Thr <210> SEQ ID NO 177 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 177 Ser Gly Val Gly Arg Asp Arg Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr <210> SEQ ID NO
178 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 178 Trp Ala
Gly Gly Asn Asp Cys Asp Ile Pro Phe Pro Ala His Trp Ile 1 5 10 15
Pro Asn Thr <210> SEQ ID NO 179 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 179 Trp Gly Asp Gly Met Asp Val Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Val Thr <210> SEQ ID NO
180 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 180 Ala Gly
Ser Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15
Pro Arg Thr <210> SEQ ID NO 181 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 181 Glu Ser Arg Ser Gly Tyr Ala Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr <210> SEQ ID NO
182 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 182 Arg Glu
Cys Gly Arg Cys Gly Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15
Pro Arg Thr <210> SEQ ID NO 183 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 183 Thr Gly Arg Gly Pro Ser Trp Val 1
5 <210> SEQ ID NO 184 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 184 Ser Ala Arg Gly Pro Ser Arg Trp 1 5 <210> SEQ
ID NO 185 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 185 Thr
Ala Arg Gly Pro Ser Phe Lys 1 5 <210> SEQ ID NO 186
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 186 Leu Ser Gly
Arg Ser Asp Asn His 1 5 <210> SEQ ID NO 187 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 187 Gly Gly Trp His Thr Gly
Arg Asn 1 5 <210> SEQ ID NO 188 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 188 His Thr Gly Arg Ser Gly Ala Leu 1
5 <210> SEQ ID NO 189 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 189 Pro Leu Thr Gly Arg Ser Gly Gly 1 5 <210> SEQ
ID NO 190 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 190 Ala
Ala Arg Gly Pro Ala Ile His 1 5 <210> SEQ ID NO 191
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 191 Arg Gly Pro
Ala Phe Asn Pro Met 1 5 <210> SEQ ID NO 192 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 192 Ser Ser Arg Gly Pro Ala
Tyr Leu 1 5 <210> SEQ ID NO 193 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 193 Arg Gly Pro Ala Thr Pro Ile Met 1
5 <210> SEQ ID NO 194 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 194 Arg Gly Pro Ala 1 <210> SEQ ID NO 195
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 195 Gly Gly Gln
Pro Ser Gly Met Trp Gly Trp 1 5 10 <210> SEQ ID NO 196
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 196 Phe Pro Arg
Pro Leu Gly Ile Thr Gly Leu 1 5 10 <210> SEQ ID NO 197
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 197 Val His Met
Pro Leu Gly Phe Leu Gly Pro 1 5 10 <210> SEQ ID NO 198
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 198 Ser Pro Leu
Thr Gly Arg Ser Gly 1 5 <210> SEQ ID NO 199 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 199 Ser Ala Gly Phe Ser Leu
Pro Ala 1 5 <210> SEQ ID NO 200 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 200 Leu Ala Pro Leu Gly Leu Gln Arg
Arg 1 5 <210> SEQ ID NO 201 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: cleavable moiety
<400> SEQUENCE: 201 Ser Gly Gly Pro Leu Gly Val Arg 1 5
<210> SEQ ID NO 202 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 202 Pro Leu Gly Leu 1 <210> SEQ ID NO 203
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 203 Pro Arg Phe
Lys Ile Ile Gly Gly 1 5 <210> SEQ ID NO 204 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 204 Pro Arg Phe Arg Ile Ile
Gly Gly 1 5 <210> SEQ ID NO 205 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 205 Ser Ser Arg His Arg Arg Ala Leu
Asp 1 5 <210> SEQ ID NO 206 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 206 Arg Lys Ser Ser Ile Ile Ile Arg
Met Arg Asp Val Val Leu 1 5 10 <210> SEQ ID NO 207
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 207 Ser Ser Ser
Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala 1 5 10 15
<210> SEQ ID NO 208 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 208 Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Arg Gly Asp
Asp Ala 1 5 10 15 <210> SEQ ID NO 209 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 209 Ile Glu Gly Arg 1 <210> SEQ
ID NO 210 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 210 Ile
Asp Gly Arg 1 <210> SEQ ID NO 211 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 211 Gly Gly Ser Ile Asp Gly Arg 1 5
<210> SEQ ID NO 212 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 212 Pro Leu Gly Leu Trp Ala 1 5 <210> SEQ ID NO 213
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 213 Gly Pro Gln
Gly Ile Ala Gly Gln 1 5 <210> SEQ ID NO 214 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 214 Gly Pro Gln Gly Leu Leu
Gly Ala 1 5 <210> SEQ ID NO 215 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 215 Gly Ile Ala Gly Gln 1 5
<210> SEQ ID NO 216 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 216 Gly Pro Leu Gly Ile Ala Gly Ile 1 5 <210> SEQ
ID NO 217 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 217 Gly
Pro Glu Gly Leu Arg Val Gly 1 5 <210> SEQ ID NO 218
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 218 Tyr Gly Ala
Gly Leu Gly Val Val 1 5 <210> SEQ ID NO 219 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 219 Ala Gly Leu Gly Val Val
Glu Arg 1 5 <210> SEQ ID NO 220 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 220 Ala Gly Leu Gly Ile Ser Ser Thr 1
5 <210> SEQ ID NO 221 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 221 Glu Pro Gln Ala Leu Ala Met Ser 1 5 <210> SEQ
ID NO 222 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 222 Gln
Ala Leu Ala Met Ser Ala Ile 1 5 <210> SEQ ID NO 223
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 223 Ala Ala Tyr
His Leu Val Ser Gln 1 5 <210> SEQ ID NO 224 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 224 Met Asp Ala Phe Leu Glu
Ser Ser 1 5 <210> SEQ ID NO 225 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 225 Glu Ser Leu Pro Val Val Ala Val 1
5 <210> SEQ ID NO 226 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 226 Ser Ala Pro Ala Val Glu Ser Glu 1 5 <210> SEQ
ID NO 227 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 227 Asp
Val Ala Gln Phe Val Leu Thr 1 5 <210> SEQ ID NO 228
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 228 Val Ala Gln
Phe Val Leu Thr Glu 1 5 <210> SEQ ID NO 229 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 229 Ala Gln Phe Val Leu Thr
Glu Gly 1 5 <210> SEQ ID NO 230 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 230 Pro Val Gln Pro Ile Gly Pro Gln 1
5 <210> SEQ ID NO 231 <211> LENGTH: 1347 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: 5342-1204-4D11 Activatable
Antibody Heavy Chain <400> SEQUENCE: 231 gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct
120 ccagggaagg ggctggagtg ggtgtcaagt attgacccgg aaggtcggca
gacatattac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagacatc 300 ggcggcaggt cggcctttga
ctactggggc cagggaaccc tggtcaccgt ctcctcagct 360 agcaccaagg
gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420
acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
480 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg acaagaaagt tgagcccaaa 660 tcttgtgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720 tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
840 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 960 tacaagtgca aggtctccaa caaagccctc
ccagccccca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggaggagatg 1080 accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1200 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1260 caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 1320 aagagcctct ccctgtctcc gggtaaa 1347
<210> SEQ ID NO 232 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
232 Gln Gly Gln Ser Gly Gln Cys Asn Ile Trp Leu Val Gly Gly Asp Cys
1 5 10 15 Arg Gly Trp Gln Gly 20 <210> SEQ ID NO 233
<211> LENGTH: 774 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 5342-1204-4D11 Activatable Antibody Light Chain
<400> SEQUENCE: 233 caaggccagt ctggccagtg caatatttgg
ctcgtaggtg gtgattgcag gggctggcag 60 gggggctcga gcggtggcag
cggtggctct ggtggtctga gcggccgttc cgataatcat 120 ggcggcggtt
ctgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga 180
gacagagtca ccatcacttg ccgggcaagt cagagcatta gcagctattt aaattggtat
240 cagcagaaac cagggaaagc ccctaagctc ctgatctatg cggcatccag
tttgcaaagt 300 ggggtcccat caaggttcag tggcagtgga tctgggacag
atttcactct caccatcagc 360 agtctgcaac ctgaagattt tgcaacttac
tactgtcaac agacggttgt ggcgcctccg 420 ttattcggcc aagggaccaa
ggtggaaatc aaacgtacgg tggctgcacc atctgtcttc 480 atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 540
aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
600 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 660 agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 720 acccatcagg gcctgagctc gcccgtcaca
aagagcttca acaggggaga gtgt 774 <210> SEQ ID NO 234
<211> LENGTH: 258 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 5342-1204-4D11 Activatable Antibody Light Chain
<400> SEQUENCE: 234 Gln Gly Gln Ser Gly Gln Cys Asn Ile Trp
Leu Val Gly Gly Asp Cys 1 5 10 15 Arg Gly Trp Gln Gly Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Leu Ser Gly Arg Ser Asp
Asn His Gly Gly Gly Ser Asp Ile Gln Met 35 40 45 Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60 Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 65 70 75 80
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 85
90 95 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly 100 105 110 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln Thr Val Val Ala Pro
Pro Leu Phe Gly Gln 130 135 140 Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe 145 150 155 160 Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175 Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190 Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210
215 220 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val 225 230 235 240 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly 245 250 255 Glu Cys <210> SEQ ID NO 235
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: spacer peptide <400> SEQUENCE: 235 Gly Gln Ser
Gly Gln 1 5 <210> SEQ ID NO 236 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: spacer peptide
<400> SEQUENCE: 236 Gln Ser Gly Gln 1 <210> SEQ ID NO
237 <211> LENGTH: 3 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: spacer peptide <400> SEQUENCE: 237 Ser Gly
Gln 1 <210> SEQ ID NO 238 <211> LENGTH: 1350
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C225v4 Antibody
Heavy Chain <400> SEQUENCE: 238 caggtgcagc tgaaacagag
cggcccgggc ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg
tgagcggctt tagcctgacc aactatggcg tgcattgggt gcgccagagc 120
ccgggcaaag gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac
180 accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca
ggtgtttttt 240 aaaatgaaca gcctgcaaag caacgatacc gcgatttatt
attgcgcgcg cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc
cagggcaccc tggtgaccgt gagcgcggct 360 agcaccaagg gcccatcggt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc
tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480
aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg
acaagaaagt tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg
tgcccagcac ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 960 tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga accacaggtg
tacaccctgc ccccatcccg ggatgaactg 1080 accaagaacc aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1260 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacgcag 1320 aagagcctct ccctgtctcc gggtaaatga 1350 <210>
SEQ ID NO 239 <211> LENGTH: 449 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v4 Antibody Heavy Chain
<400> SEQUENCE: 239 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly
Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg
Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp
Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg
Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80
Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85
90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210
215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
<210> SEQ ID NO 240 <211> LENGTH: 1350 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: C225v6 Antibody Heavy Chain
<400> SEQUENCE: 240 caggtgcagc tgaaacagag cggcccgggc
ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg tgagcggctt
tagcctgacc aactatggcg tgcattgggt gcgccagagc 120 ccgggcaaag
gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac 180
accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca ggtgtttttt
240 aaaatgaaca gcctgcaaag ccaggatacc gcgatttatt attgcgcgcg
cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc cagggcaccc
tggtgaccgt gagcgcggct 360 agcaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540
ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac
600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt
tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacgccagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
960 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 1020 gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggatgaactg 1080 accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320 aagagcctct ccctgtctcc gggtaaatga 1350 <210> SEQ ID NO
241 <211> LENGTH: 449 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: C225v6 Antibody Heavy Chain <400>
SEQUENCE: 241 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly
Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile
Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn
Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210>
SEQ ID NO 242 <211> LENGTH: 449 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Av1 Antibody Heavy Chain <400>
SEQUENCE: 242 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr
Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser
Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105
110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210>
SEQ ID NO 243 <211> LENGTH: 214 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Av1 Antibody Light Chain <400>
SEQUENCE: 243 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 <210> SEQ ID NO 244 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11 Light
Chain <400> SEQUENCE: 244 Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Val Val Ala Pro
Pro 85 90 95 Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID NO 245
<211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 4D11 Heavy Chain <400> SEQUENCE: 245 Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25
30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ser Ile Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser
Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405
410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 246 <211>
LENGTH: 449 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: 4D11v2
Heavy Chain <400> SEQUENCE: 246 Glu Val His Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310
315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435
440 445 Lys <210> SEQ ID NO 247 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11v2 Light
Chain <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (182)..(182) <223> OTHER INFORMATION:
Xaa can be any naturally occurring amino acid <400> SEQUENCE:
247 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Val Val Ala Pro Pro 85 90 95 Leu Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Xaa Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 <210> SEQ ID NO 248 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Variable Light Chain Lc4
<400> SEQUENCE: 248 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 249 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc4 <400>
SEQUENCE: 249 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly
Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 250
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Light Chain Lc5 <400> SEQUENCE: 250 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 251
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc5 <400> SEQUENCE: 251 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Tyr
His Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 <210> SEQ ID NO 252 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc7 <400> SEQUENCE: 252 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 253 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc7 <400> SEQUENCE: 253 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 254 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc8 <400>
SEQUENCE: 254 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 255 <211> LENGTH: 119 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Heavy Chain Hc8 <400> SEQUENCE:
255 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ile
Gly Arg Thr Asn Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 <210> SEQ ID NO 256 <211>
LENGTH: 108 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Variable Light Chain Lc13 <400> SEQUENCE: 256 Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser
Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg 100 105 <210> SEQ ID NO 257 <211> LENGTH:
116 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc13 <400> SEQUENCE: 257 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Glu Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 258 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc16 <400> SEQUENCE:
258 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 259
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc16 <400> SEQUENCE: 259
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro
Tyr Tyr Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 260 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc19 <400> SEQUENCE: 260 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 261 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc19 <400> SEQUENCE: 261 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 262 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc21
<400> SEQUENCE: 262 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 263 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc21
<400> SEQUENCE: 263 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 264 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc24 <400> SEQUENCE:
264 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 265
<211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc24 <400> SEQUENCE: 265
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Glu Met Gly Trp Gln Thr Leu
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Ala
Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 <210> SEQ ID NO 266 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc26 <400> SEQUENCE: 266 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 267 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc26 <400> SEQUENCE: 267 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 268 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc27
<400> SEQUENCE: 268 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 269 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc27
<400> SEQUENCE: 269 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ser Pro Pro Phe Tyr Gly Gln Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 270 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc28 <400> SEQUENCE:
270 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 271
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc28 <400> SEQUENCE: 271
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro
Phe Phe Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 272 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc30 <400> SEQUENCE: 272 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 273 <211> LENGTH: 115
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc30 <400> SEQUENCE: 273 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Glu Met Gly Trp Gln Thr Leu Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Ala 85 90 95 Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO
274 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc31 <400> SEQUENCE:
274 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 275
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc31 <400> SEQUENCE: 275
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly
Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 276 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc32 <400> SEQUENCE: 276 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 277 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc32 <400> SEQUENCE: 277 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Pro Glu Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 278 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc37 <400> SEQUENCE:
278 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 279
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc37 <400> SEQUENCE: 279
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro
His Asn Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 280 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc39 <400> SEQUENCE: 280 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 281 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc39 <400> SEQUENCE: 281 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Glu Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 282 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc40 <400> SEQUENCE:
282 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 283
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc40 <400> SEQUENCE: 283
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro
Phe Phe Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 284 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc47 <400> SEQUENCE: 284 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 285 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc47 <400> SEQUENCE: 285 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Glu Met Gly Trp Gln Thr Glu Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 286 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable 4B2 Light Chain <400> SEQUENCE:
286 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Leu Asp Ala Pro Pro 85 90 95 Gln Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 287
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 4B2 Heavy Chain <400> SEQUENCE: 287 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly
Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 <210> SEQ ID NO 288 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4D11
Light Chain <400> SEQUENCE: 288 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Val Val Ala
Pro Pro 85 90 95 Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 289 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4D11
Heavy Chain <400> SEQUENCE: 289 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 290 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable 4E7 Light Chain <400>
SEQUENCE: 290 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Val Ala Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 291 <211> LENGTH: 116 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable 4E7 Heavy Chain <400> SEQUENCE:
291 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Glu Met Gly Trp Gln Thr
Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala
Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ser 115 <210> SEQ ID NO 292 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4E11
Light Chain <400> SEQUENCE: 292 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Leu Asp Ala
Pro Leu 85 90 95 Met Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 293 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4E11
Heavy Chain <400> SEQUENCE: 293 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Pro Met Gly Gln Leu Thr Glu Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 294 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable 6B7 Light Chain <400>
SEQUENCE: 294 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ala Leu Val Ala Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 295 <211> LENGTH: 116 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable 6B7 Heavy Chain <400> SEQUENCE:
295 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp Glu Met Gly Trp Gln Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala
Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ser 115 <210> SEQ ID NO 296 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 6F8
Light Chain <400> SEQUENCE: 296 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Leu Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 297 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 6F8
Heavy Chain <400> SEQUENCE: 297 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Glu Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 298 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 298 Ile
Ser Ser Gly Leu Leu Ser Ser 1 5 <210> SEQ ID NO 299
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 299 Gln Asn Gln
Ala Leu Arg Met Ala 1 5 <210> SEQ ID NO 300 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 300 Ala Gln Asn Leu Leu Gly
Met Val 1 5 <210> SEQ ID NO 301 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 301 Ser Thr Phe Pro Phe Gly Met Phe 1
5 <210> SEQ ID NO 302 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 302 Pro Val Gly Tyr Thr Ser Ser Leu 1 5 <210> SEQ
ID NO 303 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 303 Asp
Trp Leu Tyr Trp Pro Gly Ile 1 5 <210> SEQ ID NO 304
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 304 Met Ile Ala
Pro Val Ala Tyr Arg 1 5 <210> SEQ ID NO 305 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 305 Arg Pro Ser Pro Met Trp
Ala Tyr 1 5 <210> SEQ ID NO 306 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 306 Trp Ala Thr Pro Arg Pro Met Arg 1
5 <210> SEQ ID NO 307 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 307 Phe Arg Leu Leu Asp Trp Gln Trp 1 5 <210> SEQ
ID NO 308 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 308 Leu
Lys Ala Ala Pro Arg Trp Ala 1 5 <210> SEQ ID NO 309
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 309 Gly Pro Ser
His Leu Val Leu Thr 1 5 <210> SEQ ID NO 310 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 310 Leu Pro Gly Gly Leu Ser
Pro Trp 1 5 <210> SEQ ID NO 311 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 311 Met Gly Leu Phe Ser Glu Ala Gly 1
5 <210> SEQ ID NO 312 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 312 Ser Pro Leu Pro Leu Arg Val Pro 1 5 <210> SEQ
ID NO 313 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 313 Arg
Met His Leu Arg Ser Leu Gly 1 5 <210> SEQ ID NO 314
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 314 Leu Ala Ala
Pro Leu Gly Leu Leu 1 5 <210> SEQ ID NO 315 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 315 Ala Val Gly Leu Leu Ala
Pro Pro 1 5 <210> SEQ ID NO 316 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 316 Leu Leu Ala Pro Ser His Arg Ala 1
5 <210> SEQ ID NO 317 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 317 Pro Ala Gly Leu Trp Leu Asp Pro 1 5 <210> SEQ
ID NO 318 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 318 Ile
Ser Ser Gly Leu Ser Ser 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 318
<210> SEQ ID NO 1 <211> LENGTH: 1350 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v5 Antibody Heavy Chain
<400> SEQUENCE: 1 caggtgcagc tgaaacagag cggcccgggc ctggtgcagc
cgagccagag cctgagcatt 60 acctgcaccg tgagcggctt tagcctgacc
aactatggcg tgcattgggt gcgccagagc 120 ccgggcaaag gcctggaatg
gctgggcgtg atttggagcg gcggcaacac cgattataac 180 accccgttta
ccagccgcct gagcattaac aaagataaca gcaaaagcca ggtgtttttt 240
aaaatgaaca gcctgcaaag ccaggatacc gcgatttatt attgcgcgcg cgcgctgacc
300 tattatgatt atgaatttgc gtattggggc cagggcaccc tggtgaccgt
gagcgcggct 360 agcaccaagg gcccatcggt cttccccctg gcaccctcct
ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct ggtcaaggac
tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg ccctgaccag
cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540 ctctactccc
tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600
atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa
660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct
ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag gacaccctca
tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga cgtgagccac
gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg tggaggtgca
taatgccaag acaaagccgc gggaggagca gtacaacagc 900 acgtaccgtg
tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa
1020 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg
ggatgaactg 1080 accaagaacc aggtcagcct gacctgcctg gtcaaaggct
tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg gctccttctt
cctctacagc aagctcaccg tggacaagag caggtggcag 1260 caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320
aagagcctct ccctgtctcc gggtaaatga 1350 <210> SEQ ID NO 2
<211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: C225v5 Antibody Heavy Chain <400> SEQUENCE: 2
Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5
10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn
Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu
Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr
Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn
Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ser
Gln Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Ala Leu Thr Tyr
Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> SEQ ID NO 3
<211> LENGTH: 786 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 3954-1204-C225v5 Activatable Antibody Light Chain
<400> SEQUENCE: 3 caaggccagt ctggccagtg catctcacct cgtggttgtc
cggacggccc atacgtcatg 60 tacggctcga gcggtggcag cggtggctct
ggtggatccg gtctgagcgg ccgttccgat 120 aatcatggca gtagcggtac
ccagatcttg ctgacccaga gcccggtgat tctgagcgtg 180 agcccgggcg
aacgtgtgag ctttagctgc cgcgcgagcc agagcattgg caccaacatt 240
cattggtatc agcagcgcac caacggcagc ccgcgcctgc tgattaaata tgcgagcgaa
300 agcattagcg gcattccgag ccgctttagc ggcagcggca gcggcaccga
ttttaccctg 360 agcattaaca gcgtggaaag cgaagatatt gcggattatt
attgccagca gaacaacaac 420 tggccgacca cctttggcgc gggcaccaaa
ctggaactga aacgtacggt ggctgcacca 480 tctgtcttca tcttcccgcc
atctgatgag cagttgaaat ctggaactgc ctctgttgtg 540 tgcctgctga
ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 600
ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac
660 agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa
agtctacgcc 720 tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa
agagcttcaa caggggagag 780 tgttag 786 <210> SEQ ID NO 4
<211> LENGTH: 261 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 3954-1204-C225v5 Activatable Antibody Light Chain
<400> SEQUENCE: 4 Gln Gly Gln Ser Gly Gln Cys Ile Ser Pro Arg
Gly Cys Pro Asp Gly 1 5 10 15 Pro Tyr Val Met Tyr Gly Ser Ser Gly
Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser Gly Leu Ser Gly Arg Ser
Asp Asn His Gly Ser Ser Gly Thr Gln 35 40 45 Ile Leu Leu Thr Gln
Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu 50 55 60 Arg Val Ser
Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile 65 70 75 80 His
Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys 85 90
95 Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser
100 105 110 Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu
Ser Glu 115 120 125 Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn
Trp Pro Thr Thr 130 135 140 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
Arg Thr Val Ala Ala Pro 145 150 155 160 Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly Thr 165 170 175 Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 180 185 190 Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 195 200 205 Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 210 215
220
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 225
230 235 240 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe 245 250 255 Asn Arg Gly Glu Cys 260 <210> SEQ ID NO 5
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: spacer peptide encoding sequence <400> SEQUENCE:
5 caaggccagt ctggccag 18 <210> SEQ ID NO 6 <211>
LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety encoding sequence <400> SEQUENCE: 6 tgcatctcac
ctcgtggttg tccggacggc ccatacgtca tgtac 45 <210> SEQ ID NO 7
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linker peptide encoding sequence <400> SEQUENCE:
7 ggctcgagcg gtggcagcgg tggctctggt ggatccggt 39 <210> SEQ ID
NO 8 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: 1204 substrate encoding sequence <400>
SEQUENCE: 8 ctgagcggcc gttccgataa tcat 24 <210> SEQ ID NO 9
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linker peptide encoding sequence <400> SEQUENCE:
9 ggcagtagcg gtacc 15 <210> SEQ ID NO 10 <211> LENGTH:
645 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C225v5 Antibody
Light Chain <400> SEQUENCE: 10 cagatcttgc tgacccagag
cccggtgatt ctgagcgtga gcccgggcga acgtgtgagc 60 tttagctgcc
gcgcgagcca gagcattggc accaacattc attggtatca gcagcgcacc 120
aacggcagcc cgcgcctgct gattaaatat gcgagcgaaa gcattagcgg cattccgagc
180 cgctttagcg gcagcggcag cggcaccgat tttaccctga gcattaacag
cgtggaaagc 240 gaagatattg cggattatta ttgccagcag aacaacaact
ggccgaccac ctttggcgcg 300 ggcaccaaac tggaactgaa acgtacggtg
gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc
tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg
ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480
gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
645 <210> SEQ ID NO 11 <211> LENGTH: 6 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: spacer peptide <400>
SEQUENCE: 11 Gln Gly Gln Ser Gly Gln 1 5 <210> SEQ ID NO 12
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 12 Cys Ile Ser
Pro Arg Gly Cys Pro Asp Gly Pro Tyr Val Met Tyr 1 5 10 15
<210> SEQ ID NO 13 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: linker peptide <400> SEQUENCE:
13 Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10
<210> SEQ ID NO 14 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 1204 substrate <400> SEQUENCE:
14 Leu Ser Gly Arg Ser Asp Asn His 1 5 <210> SEQ ID NO 15
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linker peptide <400> SEQUENCE: 15 Gly Ser Ser
Gly Thr 1 5 <210> SEQ ID NO 16 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: C225v5 Antibody
Light Chain <400> SEQUENCE: 16 Gln Ile Leu Leu Thr Gln Ser
Pro Val Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe
Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp
Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys
Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser
65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp
Pro Thr 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID NO 17
<211> LENGTH: 777 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 3954-NSUB-C225v5 Masked Antibody Light Chain
<400> SEQUENCE: 17 caaggccagt ctggccagtg catctcacct
cgtggttgtc cggacggccc atacgtcatg 60 tacggctcga gcggtggcag
cggtggctct ggtggctcag gtggaggctc gggcggtggg 120 agcggcggtt
ctcagatctt gctgacccag agcccggtga ttctgagcgt gagcccgggc 180
gaacgtgtga gctttagctg ccgcgcgagc cagagcattg gcaccaacat tcattggtat
240 cagcagcgca ccaacggcag cccgcgcctg ctgattaaat atgcgagcga
aagcattagc 300 ggcattccga gccgctttag cggcagcggc agcggcaccg
attttaccct gagcattaac 360 agcgtggaaa gcgaagatat tgcggattat
tattgccagc agaacaacaa ctggccgacc 420 acctttggcg cgggcaccaa
actggaactg aaacgtacgg tggctgcacc atctgtcttc 480 atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 540
aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
600 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 660 agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 720 acccatcagg gcctgagctc gcccgtcaca
aagagcttca acaggggaga gtgttag 777
<210> SEQ ID NO 18 <211> LENGTH: 258 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: 3954-NSUB-C225v5 Masked Antibody
Light Chain <400> SEQUENCE: 18 Gln Gly Gln Ser Gly Gln Cys
Ile Ser Pro Arg Gly Cys Pro Asp Gly 1 5 10 15 Pro Tyr Val Met Tyr
Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser Gly Gly
Gly Ser Gly Gly Gly Ser Gly Gly Ser Gln Ile Leu Leu 35 40 45 Thr
Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser 50 55
60 Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr
65 70 75 80 Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile Lys Tyr
Ala Ser 85 90 95 Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Leu Ser Ile Asn Ser Val
Glu Ser Glu Asp Ile Ala 115 120 125 Asp Tyr Tyr Cys Gln Gln Asn Asn
Asn Trp Pro Thr Thr Phe Gly Ala 130 135 140 Gly Thr Lys Leu Glu Leu
Lys Arg Thr Val Ala Ala Pro Ser Val Phe 145 150 155 160 Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175 Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185
190 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
195 200 205 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
Leu Thr 210 215 220 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
Ala Cys Glu Val 225 230 235 240 Thr His Gln Gly Leu Ser Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly 245 250 255 Glu Cys <210> SEQ ID
NO 19 <211> LENGTH: 69 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: linker - non-cleavable substrate - linker
peptide encoding sequence <400> SEQUENCE: 19 ggctcgagcg
gtggcagcgg tggctctggt ggctcaggtg gaggctcggg cggtgggagc 60 ggcggttct
69 <210> SEQ ID NO 20 <211> LENGTH: 23 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: linker - non-cleavable
substrate - linker peptide <400> SEQUENCE: 20 Gly Ser Ser Gly
Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Gly
Gly Ser Gly Gly Ser 20 <210> SEQ ID NO 21 <211> LENGTH:
5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linking peptide
<220> FEATURE: <221> NAME/KEY: REPEAT <222>
LOCATION: (1)..(5) <223> OTHER INFORMATION: May be repeated
<400> SEQUENCE: 21 Gly Ser Gly Gly Ser 1 5 <210> SEQ ID
NO 22 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: linking peptide <220> FEATURE: <221>
NAME/KEY: REPEAT <222> LOCATION: (1)..(4) <223> OTHER
INFORMATION: May be repeated <400> SEQUENCE: 22 Gly Gly Gly
Ser 1 <210> SEQ ID NO 23 <211> LENGTH: 4 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: linking peptide <400>
SEQUENCE: 23 Gly Gly Ser Gly 1 <210> SEQ ID NO 24 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
linking peptide <400> SEQUENCE: 24 Gly Gly Ser Gly Gly 1 5
<210> SEQ ID NO 25 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: linking peptide <400>
SEQUENCE: 25 Gly Ser Gly Ser Gly 1 5 <210> SEQ ID NO 26
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: linking peptide <400> SEQUENCE: 26 Gly Ser Gly
Gly Gly 1 5 <210> SEQ ID NO 27 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: linking peptide
<400> SEQUENCE: 27 Gly Gly Gly Ser Gly 1 5 <210> SEQ ID
NO 28 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: linking peptide <400> SEQUENCE: 28 Gly Ser
Ser Ser Gly 1 5 <210> SEQ ID NO 29 <211> LENGTH: 6
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 29 Cys Ile Ser Pro Arg Gly 1 5 <210>
SEQ ID NO 30 <211> LENGTH: 8 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
30 Cys Ile Ser Pro Arg Gly Cys Gly 1 5 <210> SEQ ID NO 31
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 31 Cys Ile Ser
Pro Arg Gly Cys Pro Asp Gly Pro Tyr Val Met Tyr 1 5 10 15
<210> SEQ ID NO 32 <211> LENGTH: 14 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
32
Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly Pro Tyr Val Met 1 5 10
<210> SEQ ID NO 33 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
33 Cys Ile Ser Pro Arg Gly Cys Glu Pro Gly Thr Tyr Val Pro Thr 1 5
10 15 <210> SEQ ID NO 34 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 34 Cys Ile Ser Pro Arg Gly Cys Pro Gly Gln Ile Trp His
Pro Pro 1 5 10 15 <210> SEQ ID NO 35 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 35 Gly Ser His Cys Leu Ile Pro Ile Asn Met
Gly Ala Pro Ser Cys 1 5 10 15 <210> SEQ ID NO 36 <211>
LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 36 Cys Ile Ser Pro Arg Gly Cys
Gly Gly Ser Ser Ala Ser Gln Ser Gly 1 5 10 15 Gln Gly Ser His Cys
Leu Ile Pro Ile Asn Met Gly Ala Pro Ser Cys 20 25 30 <210>
SEQ ID NO 37 <211> LENGTH: 19 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
37 Cys Asn His His Tyr Phe Tyr Thr Cys Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Pro Gly <210> SEQ ID NO 38 <211> LENGTH:
19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 38 Ala Asp His Val Phe Trp Gly Ser Tyr Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
39 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 39 Cys His
His Val Tyr Trp Gly His Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 40 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 40 Cys Pro His Phe Thr Thr Thr Ser Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
41 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 41 Cys Asn
His His Tyr His Tyr Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 42 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 42 Cys Pro His Val Ser Phe Gly Ser Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
43 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 43 Cys Pro
Tyr Tyr Thr Leu Ser Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 44 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 44 Cys Asn His Val Tyr Phe Gly Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
45 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 45 Cys Asn
His Phe Thr Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Pro Gly <210> SEQ ID NO 46 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 46 Cys His His Phe Thr Leu Thr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly <210> SEQ ID NO
47 <211> LENGTH: 18 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 47 Tyr Asn
Pro Cys Ala Thr Pro Met Cys Cys Ile Ser Pro Arg Gly Cys 1 5 10 15
Pro Gly <210> SEQ ID NO 48 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 48 Cys Asn His His Tyr Phe Tyr Thr Cys Gly Cys Ile Ser
Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 49 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 49 Cys Asn His His Tyr His Tyr
Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15
Cys Gly <210> SEQ ID NO 50 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 50 Cys Asn His Val Tyr Phe Gly Thr Cys Gly Cys Ile Ser
Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 51 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 51 Cys His His Val Tyr Trp Gly
His Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210>
SEQ ID NO 52 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
52 Cys Pro His Phe Thr Thr Thr Ser Cys Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Gly <210> SEQ ID NO 53 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 53 Cys Asn His Phe Thr Leu Thr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 54
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 54 Cys His His
Phe Thr Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys
Gly <210> SEQ ID NO 55 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 55 Cys Pro Tyr Tyr Thr Leu Ser Tyr Cys Gly Cys Ile Ser
Pro Arg Gly 1 5 10 15 Cys Gly <210> SEQ ID NO 56 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 56 Cys Pro His Val Ser Phe Gly
Ser Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly <210>
SEQ ID NO 57 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
57 Ala Asp His Val Phe Trp Gly Ser Tyr Gly Cys Ile Ser Pro Arg Gly
1 5 10 15 Cys Gly <210> SEQ ID NO 58 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 58 Tyr Asn Pro Cys Ala Thr Pro Met Cys Cys
Ile Ser Pro Arg Gly Cys 1 5 10 15 Gly <210> SEQ ID NO 59
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 59 Cys His His
Val Tyr Trp Gly His Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys
Gly <210> SEQ ID NO 60 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa may be Asn or Phe
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (4)..(4) <223> OTHER INFORMATION: Xaa may be His,
Val or Phe <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (5)..(5) <223> OTHER INFORMATION: Xaa
may be Tyr or Thr <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (6)..(6) <223> OTHER
INFORMATION: Xaa may be Phe, Trp, Thr or Leu <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (7)..(7)
<223> OTHER INFORMATION: Xaa may be Tyr, Gly, Thr or Ser
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (8)..(8) <223> OTHER INFORMATION: Xaa may be Thr,
Ser, Tyr or His <400> SEQUENCE: 60 Cys Xaa His Xaa Xaa Xaa
Xaa Xaa Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly
<210> SEQ ID NO 61 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
61 Cys Ile Ser Pro Arg Gly Cys Gly Gln Pro Ile Pro Ser Val Lys 1 5
10 15 <210> SEQ ID NO 62 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 62 Cys Ile Ser Pro Arg Gly Cys Thr Gln Pro Tyr His Val
Ser Arg 1 5 10 15 <210> SEQ ID NO 63 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 63 Cys Ile Ser Pro Arg Gly Cys Asn Ala Val
Ser Gly Leu Gly Ser 1 5 10 15 <210> SEQ ID NO 64 <211>
LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 64 Gln Gly Gln Ser Gly Gln Gly
Gln Gln Gln Trp Cys Asn Ile Trp Ile 1 5 10 15 Asn Gly Gly Asp Cys
Arg Gly Trp Asn Gly 20 25 <210> SEQ ID NO 65 <211>
LENGTH: 15 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
65 Pro Trp Cys Met Gln Arg Gln Asp Phe Leu Arg Cys Pro Gln Pro 1 5
10 15 <210> SEQ ID NO 66 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 66 Gln Leu Gly Leu Pro Ala Tyr Met Cys Thr Phe Glu Cys
Leu Arg 1 5 10 15 <210> SEQ ID NO 67 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 67 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys
Gly Gly Leu Gln Gly 1 5 10 15 <210> SEQ ID NO 68 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 68 Ser Cys Ser Leu Trp Thr Ser
Gly Ser Cys Leu Pro His Ser Pro 1 5 10 15 <210> SEQ ID NO 69
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 69 Tyr Cys Leu
Gln Leu Pro His Tyr Met Gln Ala Met Cys Gly Arg 1 5 10 15
<210> SEQ ID NO 70 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
70 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser Tyr Trp Asn Asn Thr 1 5
10 15 <210> SEQ ID NO 71 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 71 Pro Trp Cys Met Gln Arg Gln Asp Tyr Leu Arg Cys Pro
Gln Pro 1 5 10 15 <210> SEQ ID NO 72 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 72 Cys Asn Leu Trp Ile Ser Gly Gly Asp Cys
Arg Gly Leu Ala Gly 1 5 10 15 <210> SEQ ID NO 73 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 73 Cys Asn Leu Trp Val Ser Gly
Gly Asp Cys Arg Gly Val Gln Gly 1 5 10 15 <210> SEQ ID NO 74
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 74 Cys Asn Leu
Trp Val Ser Gly Gly Asp Cys Arg Gly Leu Arg Gly 1 5 10 15
<210> SEQ ID NO 75 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
75 Cys Asn Leu Trp Ile Ser Gly Gly Asp Cys Arg Gly Leu Pro Gly 1 5
10 15 <210> SEQ ID NO 76 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 76 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys Arg Asp Ala
Pro Trp 1 5 10 15 <210> SEQ ID NO 77 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 77 Cys Asn Leu Trp Val Ser Gly Gly Asp Cys
Arg Asp Leu Leu Gly 1 5 10 15 <210> SEQ ID NO 78 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 78 Cys Asn Leu Trp Val Ser Gly
Gly Asp Cys Arg Gly Leu Gln Gly 1 5 10 15 <210> SEQ ID NO 79
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 79 Cys Asn Leu
Trp Leu His Gly Gly Asp Cys Arg Gly Trp Gln Gly 1 5 10 15
<210> SEQ ID NO 80 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
80 Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Gln Gly 1 5
10 15 <210> SEQ ID NO 81 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 81 Cys Thr Thr Trp Phe Cys Gly Gly Asp Cys Gly Val Met
Arg Gly 1 5 10 15 <210> SEQ ID NO 82 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 82 Cys Asn Ile Trp Gly Pro Ser Val Asp Cys
Gly Ala Leu Leu Gly 1 5 10 15 <210> SEQ ID NO 83 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 83 Cys Asn Ile Trp Val Asn Gly
Gly Asp Cys Arg Ser Phe Glu Gly 1 5 10 15 <210> SEQ ID NO 84
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety
<400> SEQUENCE: 84 Tyr Cys Leu Asn Leu Pro Arg Tyr Met Gln
Asp Met Cys Trp Ala 1 5 10 15 <210> SEQ ID NO 85 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 85 Tyr Cys Leu Ala Leu Pro His
Tyr Met Gln Ala Asp Cys Ala Arg 1 5 10 15 <210> SEQ ID NO 86
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 86 Cys Phe Leu
Tyr Ser Cys Gly Asp Val Ser Tyr Trp Gly Ser Ala 1 5 10 15
<210> SEQ ID NO 87 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
87 Cys Tyr Leu Tyr Ser Cys Thr Asp Ser Ala Phe Trp Asn Asn Arg 1 5
10 15 <210> SEQ ID NO 88 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 88 Cys Tyr Leu Tyr Ser Cys Asn Asp Val Ser Tyr Trp Ser
Asn Thr 1 5 10 15 <210> SEQ ID NO 89 <211> LENGTH: 12
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 89 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser
Tyr Trp 1 5 10 <210> SEQ ID NO 90 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 90 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala
Tyr Trp Asn Ser Ala 1 5 10 15 <210> SEQ ID NO 91 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 91 Cys Phe Leu Tyr Ser Cys Thr
Asp Val Ser Tyr Trp Gly Asp Thr 1 5 10 15 <210> SEQ ID NO 92
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 92 Cys Phe Leu
Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Asn Ser 1 5 10 15
<210> SEQ ID NO 93 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
93 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala Tyr Trp Asn Asn Thr 1 5
10 15 <210> SEQ ID NO 94 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 94 Cys Phe Leu Tyr Ser Cys Gly Asp Val Ser Tyr Trp Gly
Asn Pro Gly 1 5 10 15 Leu Ser <210> SEQ ID NO 95 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 95 Cys Phe Leu Tyr Ser Cys Thr
Asp Val Ala Tyr Trp Ser Gly Leu 1 5 10 15 <210> SEQ ID NO 96
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 96 Cys Tyr Leu
Tyr Ser Cys Thr Asp Gly Ser Tyr Trp Asn Ser Thr 1 5 10 15
<210> SEQ ID NO 97 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
97 Cys Phe Leu Tyr Ser Cys Ser Asp Val Ser Tyr Trp Gly Asn Ile 1 5
10 15 <210> SEQ ID NO 98 <211> LENGTH: 12 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 98 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala Tyr Trp 1 5 10
<210> SEQ ID NO 99 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
99 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Ser Thr 1 5
10 15 <210> SEQ ID NO 100 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 100 Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala Tyr Trp Gly
Asp Thr 1 5 10 15 <210> SEQ ID NO 101 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 101 Gly Cys Asn Ile Trp Leu Asn Gly Gly Asp
Cys Arg Gly Trp Val Asp 1 5 10 15 Pro Leu Gln Gly 20 <210>
SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
102 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Ile Gly
1 5 10 15 Asp Thr Asn Gly 20 <210> SEQ ID NO 103 <211>
LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
103 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Ile Glu
1 5 10 15 Asp Ser Asn Gly 20 <210> SEQ ID NO 104 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 104 Gly Cys Asn Ile Trp Ala
Asn Gly Gly Asp Cys Arg Gly Trp Ile Asp 1 5 10 15 Asn Ile Asp Gly
20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 105 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly
Trp Leu Gly 1 5 10 15 Glu Ala Val Gly 20 <210> SEQ ID NO 106
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 106 Gly Cys Asn
Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Leu Glu 1 5 10 15 Glu
Ala Val Gly 20 <210> SEQ ID NO 107 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 107 Gly Gly Pro Ala Leu Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Ser Gly 20 <210>
SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
108 Gly Ala Pro Val Phe Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg
1 5 10 15 Gly Trp Met Gly 20 <210> SEQ ID NO 109 <211>
LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 109 Gly Gln Gln Gln Trp Cys
Asn Ile Trp Ile Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Asn Gly
20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 110 Gly Lys Ser Glu Phe Cys Asn Ile Trp Leu Asn Gly Gly
Asp Cys Arg 1 5 10 15 Gly Trp Ile Gly 20 <210> SEQ ID NO 111
<211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 111 Gly Thr Pro
Gly Gly Cys Asn Ile Trp Ala Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly
Trp Glu Gly 20 <210> SEQ ID NO 112 <211> LENGTH: 20
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 112 Gly Ala Ser Gln Tyr Cys Asn Leu Trp Ile
Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp Arg Gly 20 <210>
SEQ ID NO 113 <211> LENGTH: 18 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
113 Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Pro Trp Val Glu
1 5 10 15 Gly Gly <210> SEQ ID NO 114 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 114 Gly Cys Asn Ile Trp Ala Val Gly Gly Asp
Cys Arg Pro Phe Val Asp 1 5 10 15 Gly Gly <210> SEQ ID NO 115
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 115 Gly Cys Asn
Ile Trp Leu Asn Gly Gly Asp Cys Arg Ala Trp Val Asp 1 5 10 15 Thr
Gly <210> SEQ ID NO 116 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 116 Gly Cys Asn Ile Trp Ile Val Gly Gly Asp Cys Arg Pro
Phe Ile Asn 1 5 10 15 Asp Gly <210> SEQ ID NO 117 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 117 Gly Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg Pro Val Val Phe 1 5 10 15 Gly Gly
<210> SEQ ID NO 118 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
118 Gly Cys Asn Ile Trp Leu Ser Gly Gly Asp Cys Arg Met Phe Met Asn
1 5 10 15 Glu Gly <210> SEQ ID NO 119 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 119 Gly Cys Asn Ile Trp Val Asn Gly Gly Asp
Cys Arg Ser Phe Val Tyr 1 5 10 15 Ser Gly <210> SEQ ID NO 120
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 120 Gly Cys Asn
Ile Trp Leu Asn Gly Gly Asp Cys Arg Gly Trp Glu Ala 1 5 10 15 Ser
Gly <210> SEQ ID NO 121 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 121 Gly Cys Asn Ile Trp Ala His Gly Gly Asp Cys Arg Gly
Phe Ile Glu 1 5 10 15 Pro Gly <210> SEQ ID NO 122 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 122 Gly Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg Thr Phe Val Ala 1 5 10 15 Ser Gly
<210> SEQ ID NO 123 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
123 Gly Cys Asn Ile Trp Ala His Gly Gly Asp Cys Arg Gly Phe Ile Glu
1 5 10 15 Pro Gly <210> SEQ ID NO 124 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 124 Gly Phe Leu Glu Asn Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Thr Gly <210> SEQ ID NO 125
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 125 Gly Ile Tyr
Glu Asn Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg 1 5 10 15 Met
Gly <210> SEQ ID NO 126 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 126 Gly Ile Pro Asp Asn Cys Asn Ile Trp Ile Asn Gly Gly
Asp Cys Arg 1 5 10 15 Tyr Gly <210> SEQ ID NO 127 <211>
LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 127 Gln Gly Gln Ser Gly Gln
Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met Asp
Cys 20 <210> SEQ ID NO 128 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 128 Gln Gly Gln Ser Gly Gln Gly Asp Phe Asp Ile Pro Phe
Pro Ala His 1 5 10 15 Trp Val Pro Ile Thr 20 <210> SEQ ID NO
129 <211> LENGTH: 24 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 129 Gln Gly
Gln Ser Gly Gln Met Gly Val Pro Ala Gly Cys Val Trp Asn 1 5 10 15
Tyr Ala His Ile Phe Met Asp Cys 20 <210> SEQ ID NO 130
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 130 Tyr Arg Ser
Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 131 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
131 Pro Gly Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr
1 5 10 15 <210> SEQ ID NO 132 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 132 Glu Ser Ser Cys Val Trp Asn Tyr Val His
Ile Tyr Met Asp Cys 1 5 10 15 <210> SEQ ID NO 133 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 133 Tyr Pro Gly Cys Lys Trp
Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
134 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 134 Tyr Arg
Thr Cys Ser Trp Asn Tyr Val Gly Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 135 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
135 Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Met Asp Cys 1 5
10 15 <210> SEQ ID NO 136 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 136
Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10
15 <210> SEQ ID NO 137 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 137 Tyr Gly Ser Cys Asn Trp Asn Tyr Val His Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 138 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 138 Tyr Thr Ser Cys Asn Trp Asn Tyr Val His
Ile Phe Met Asp Cys 1 5 10 15 <210> SEQ ID NO 139 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 139 Tyr Pro Gly Cys Lys Trp
Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
140 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 140 Trp Arg
Ser Cys Asn Trp Asn Tyr Ala His Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 141 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
141 Trp Ser Asn Cys His Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 142 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 142 Asp Arg Ser Cys Thr Trp Asn Tyr Val Arg Ile Ser Tyr
Asp Cys 1 5 10 15 <210> SEQ ID NO 143 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 143 Ser Gly Ser Cys Lys Trp Asp Tyr Val His
Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO 144 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 144 Ser Arg Ser Cys Ile Trp
Asn Tyr Ala His Ile His Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
145 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 145 Ser Met
Ser Cys Tyr Trp Gln Tyr Glu Arg Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 146 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
146 Tyr Arg Ser Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 147 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 147 Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Met
Asp Cys 1 5 10 15 <210> SEQ ID NO 148 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 148 Ser Gly Ser Cys Lys Trp Asp Tyr Val His
Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO 149 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 149 Tyr Lys Ser Cys His Trp
Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
150 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 150 Tyr Gly
Ser Cys Thr Trp Asn Tyr Val His Ile Phe Met Glu Cys 1 5 10 15
<210> SEQ ID NO 151 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
151 Phe Ser Ser Cys Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5
10 15 <210> SEQ ID NO 152 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 152 Trp Arg Ser Cys Asn Trp Asn Tyr Ala His Ile Phe Leu
Asp Cys 1 5 10 15 <210> SEQ ID NO 153 <211> LENGTH: 15
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 153 Tyr Gly Ser Cys Gln Trp Asn Tyr Val His
Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO 154 <211>
LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 154 Tyr Arg Ser Cys Asn Trp
Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15 <210> SEQ ID NO
155 <211> LENGTH: 15 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 155 Asn Met
Ser Cys His Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
<210> SEQ ID NO 156 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
156 Phe Gly Pro Cys Thr Trp Asn Tyr Ala Arg Ile Ser Trp Asp Cys 1 5
10 15 <210> SEQ ID NO 157 <211> LENGTH: 15 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <220>
FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:
(1)..(2) <223> OTHER INFORMATION: Xaa can be any naturally
occurring amino acid <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (5)..(5) <223> OTHER
INFORMATION: Xaa can be any naturally occurring amino acid
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (7)..(7) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <220> FEATURE: <221>
NAME/KEY: misc_feature <222> LOCATION: (13)..(13) <223>
OTHER INFORMATION: Xaa can be any naturally occurring amino acid
<400> SEQUENCE: 157 Xaa Xaa Ser Cys Xaa Trp Xaa Tyr Val His
Ile Phe Xaa Asp Cys 1 5 10 15 <210> SEQ ID NO 158 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 158 Met Gly Val Pro Ala Gly
Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Asp Cys
<210> SEQ ID NO 159 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
159 Arg Asp Thr Gly Gly Gln Cys Arg Trp Asp Tyr Val His Ile Phe Met
1 5 10 15 Asp Cys <210> SEQ ID NO 160 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 160 Ala Gly Val Pro Ala Gly Cys Thr Trp Asn
Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 161
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 161 Val Gly Val
Pro Asn Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu
Cys <210> SEQ ID NO 162 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 162 Asp Gly Gly Pro Ala Gly Cys Ser Trp Asn Tyr Val His
Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 163 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 163 Ala Val Gly Pro Ala Gly
Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
<210> SEQ ID NO 164 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
164 Cys Thr Trp Asn Tyr Val His Ile Phe Met Asp Cys Gly Glu Gly Glu
1 5 10 15 Gly Pro <210> SEQ ID NO 165 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 165 Gly Gly Val Pro Glu Gly Cys Thr Trp Asn
Tyr Ala His Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 166
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 166 Ala Glu Val
Pro Ala Gly Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu
Cys <210> SEQ ID NO 167 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: masking moiety <400>
SEQUENCE: 167 Ala Gly Val Pro Ala Gly Cys Thr Trp Asn Tyr Val His
Ile Phe Met 1 5 10 15 Glu Cys <210> SEQ ID NO 168 <211>
LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
masking moiety <400> SEQUENCE: 168 Ser Gly Ala Ser Gly Gly
Cys Lys Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Asp Cys
<210> SEQ ID NO 169 <211> LENGTH: 18 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
169 Met Gly Val Pro Ala Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met
1 5 10 15 Asp Cys <210> SEQ ID NO 170 <211> LENGTH: 18
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 170 Thr Pro Gly Cys Arg Trp Asn Tyr Val His
Ile Phe Met Glu Cys Glu 1 5 10 15 Ala Leu <210> SEQ ID NO 171
<211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 171 Val Gly Val
Pro Asn Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met
1 5 10 15 Glu Cys <210> SEQ ID NO 172 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 172 Pro Gly Ala Phe Asp Ile Pro Phe Pro Ala
His Trp Val Pro Asn Thr 1 5 10 15 <210> SEQ ID NO 173
<211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: masking moiety <400> SEQUENCE: 173 Arg Gly Ala
Cys Asp Ile Pro Phe Pro Ala His Trp Ile Pro Asn Thr 1 5 10 15
<210> SEQ ID NO 174 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
174 Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Ile Thr
1 5 10 15 <210> SEQ ID NO 175 <211> LENGTH: 16
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (1)..(1) <223> OTHER INFORMATION: Xaa can be any
naturally occurring amino acid <400> SEQUENCE: 175 Xaa Gly
Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr 1 5 10 15
<210> SEQ ID NO 176 <211> LENGTH: 19 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
176 Arg Gly Asp Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val
1 5 10 15 Pro Arg Thr <210> SEQ ID NO 177 <211> LENGTH:
19 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 177 Ser Gly Val Gly Arg Asp Arg Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr <210> SEQ ID NO
178 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 178 Trp Ala
Gly Gly Asn Asp Cys Asp Ile Pro Phe Pro Ala His Trp Ile 1 5 10 15
Pro Asn Thr <210> SEQ ID NO 179 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 179 Trp Gly Asp Gly Met Asp Val Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Val Thr <210> SEQ ID NO
180 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 180 Ala Gly
Ser Gly Asn Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15
Pro Arg Thr <210> SEQ ID NO 181 <211> LENGTH: 19
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: masking moiety
<400> SEQUENCE: 181 Glu Ser Arg Ser Gly Tyr Ala Asp Ile Pro
Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr <210> SEQ ID NO
182 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: masking moiety <400> SEQUENCE: 182 Arg Glu
Cys Gly Arg Cys Gly Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15
Pro Arg Thr <210> SEQ ID NO 183 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 183 Thr Gly Arg Gly Pro Ser Trp Val 1
5 <210> SEQ ID NO 184 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 184 Ser Ala Arg Gly Pro Ser Arg Trp 1 5 <210> SEQ
ID NO 185 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 185 Thr
Ala Arg Gly Pro Ser Phe Lys 1 5 <210> SEQ ID NO 186
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 186 Leu Ser Gly
Arg Ser Asp Asn His 1 5 <210> SEQ ID NO 187 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 187 Gly Gly Trp His Thr Gly
Arg Asn 1 5 <210> SEQ ID NO 188 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 188 His Thr Gly Arg Ser Gly Ala Leu 1
5 <210> SEQ ID NO 189 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety
<400> SEQUENCE: 189 Pro Leu Thr Gly Arg Ser Gly Gly 1 5
<210> SEQ ID NO 190 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 190 Ala Ala Arg Gly Pro Ala Ile His 1 5 <210> SEQ
ID NO 191 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 191 Arg
Gly Pro Ala Phe Asn Pro Met 1 5 <210> SEQ ID NO 192
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 192 Ser Ser Arg
Gly Pro Ala Tyr Leu 1 5 <210> SEQ ID NO 193 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 193 Arg Gly Pro Ala Thr Pro
Ile Met 1 5 <210> SEQ ID NO 194 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 194 Arg Gly Pro Ala 1 <210> SEQ
ID NO 195 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 195 Gly
Gly Gln Pro Ser Gly Met Trp Gly Trp 1 5 10 <210> SEQ ID NO
196 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 196 Phe
Pro Arg Pro Leu Gly Ile Thr Gly Leu 1 5 10 <210> SEQ ID NO
197 <211> LENGTH: 10 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 197 Val
His Met Pro Leu Gly Phe Leu Gly Pro 1 5 10 <210> SEQ ID NO
198 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 198 Ser
Pro Leu Thr Gly Arg Ser Gly 1 5 <210> SEQ ID NO 199
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 199 Ser Ala Gly
Phe Ser Leu Pro Ala 1 5 <210> SEQ ID NO 200 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 200 Leu Ala Pro Leu Gly Leu
Gln Arg Arg 1 5 <210> SEQ ID NO 201 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 201 Ser Gly Gly Pro Leu Gly Val Arg 1
5 <210> SEQ ID NO 202 <211> LENGTH: 4 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 202 Pro Leu Gly Leu 1 <210> SEQ ID NO 203
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 203 Pro Arg Phe
Lys Ile Ile Gly Gly 1 5 <210> SEQ ID NO 204 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 204 Pro Arg Phe Arg Ile Ile
Gly Gly 1 5 <210> SEQ ID NO 205 <211> LENGTH: 9
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 205 Ser Ser Arg His Arg Arg Ala Leu
Asp 1 5 <210> SEQ ID NO 206 <211> LENGTH: 14
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 206 Arg Lys Ser Ser Ile Ile Ile Arg
Met Arg Asp Val Val Leu 1 5 10 <210> SEQ ID NO 207
<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 207 Ser Ser Ser
Phe Asp Lys Gly Lys Tyr Lys Lys Gly Asp Asp Ala 1 5 10 15
<210> SEQ ID NO 208 <211> LENGTH: 15 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 208 Ser Ser Ser Phe Asp Lys Gly Lys Tyr Lys Arg Gly Asp
Asp Ala
1 5 10 15 <210> SEQ ID NO 209 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 209 Ile Glu Gly Arg 1 <210> SEQ
ID NO 210 <211> LENGTH: 4 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 210 Ile
Asp Gly Arg 1 <210> SEQ ID NO 211 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 211 Gly Gly Ser Ile Asp Gly Arg 1 5
<210> SEQ ID NO 212 <211> LENGTH: 6 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 212 Pro Leu Gly Leu Trp Ala 1 5 <210> SEQ ID NO 213
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 213 Gly Pro Gln
Gly Ile Ala Gly Gln 1 5 <210> SEQ ID NO 214 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 214 Gly Pro Gln Gly Leu Leu
Gly Ala 1 5 <210> SEQ ID NO 215 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 215 Gly Ile Ala Gly Gln 1 5
<210> SEQ ID NO 216 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 216 Gly Pro Leu Gly Ile Ala Gly Ile 1 5 <210> SEQ
ID NO 217 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 217 Gly
Pro Glu Gly Leu Arg Val Gly 1 5 <210> SEQ ID NO 218
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 218 Tyr Gly Ala
Gly Leu Gly Val Val 1 5 <210> SEQ ID NO 219 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 219 Ala Gly Leu Gly Val Val
Glu Arg 1 5 <210> SEQ ID NO 220 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 220 Ala Gly Leu Gly Ile Ser Ser Thr 1
5 <210> SEQ ID NO 221 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 221 Glu Pro Gln Ala Leu Ala Met Ser 1 5 <210> SEQ
ID NO 222 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 222 Gln
Ala Leu Ala Met Ser Ala Ile 1 5 <210> SEQ ID NO 223
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 223 Ala Ala Tyr
His Leu Val Ser Gln 1 5 <210> SEQ ID NO 224 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 224 Met Asp Ala Phe Leu Glu
Ser Ser 1 5 <210> SEQ ID NO 225 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 225 Glu Ser Leu Pro Val Val Ala Val 1
5 <210> SEQ ID NO 226 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable linker <400>
SEQUENCE: 226 Ser Ala Pro Ala Val Glu Ser Glu 1 5 <210> SEQ
ID NO 227 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable linker <400> SEQUENCE: 227 Asp
Val Ala Gln Phe Val Leu Thr 1 5 <210> SEQ ID NO 228
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable linker <400> SEQUENCE: 228 Val Ala Gln
Phe Val Leu Thr Glu 1 5 <210> SEQ ID NO 229 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable linker <400> SEQUENCE: 229 Ala Gln Phe Val Leu Thr
Glu Gly 1 5 <210> SEQ ID NO 230 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
linker <400> SEQUENCE: 230 Pro Val Gln Pro Ile Gly Pro Gln 1
5 <210> SEQ ID NO 231 <211> LENGTH: 1347 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: 5342-1204-4D11 Activatable
Antibody Heavy Chain <400> SEQUENCE: 231 gaggtgcagc
tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct
120 ccagggaagg ggctggagtg ggtgtcaagt attgacccgg aaggtcggca
gacatattac 180 gcagactccg tgaagggccg gttcaccatc tccagagaca
attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac
acggccgtat attactgtgc gaaagacatc 300 ggcggcaggt cggcctttga
ctactggggc cagggaaccc tggtcaccgt ctcctcagct 360 agcaccaagg
gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420
acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg
480 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 540 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 600 atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg acaagaaagt tgagcccaaa 660 tcttgtgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720 tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
840 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 900 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 960 tacaagtgca aggtctccaa caaagccctc
ccagccccca tcgagaaaac catctccaaa 1020 gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggaggagatg 1080 accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1140
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1200 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1260 caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 1320 aagagcctct ccctgtctcc gggtaaa 1347
<210> SEQ ID NO 232 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: masking moiety <400> SEQUENCE:
232 Gln Gly Gln Ser Gly Gln Cys Asn Ile Trp Leu Val Gly Gly Asp Cys
1 5 10 15 Arg Gly Trp Gln Gly 20 <210> SEQ ID NO 233
<211> LENGTH: 774 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 5342-1204-4D11 Activatable Antibody Light Chain
<400> SEQUENCE: 233 caaggccagt ctggccagtg caatatttgg
ctcgtaggtg gtgattgcag gggctggcag 60 gggggctcga gcggtggcag
cggtggctct ggtggtctga gcggccgttc cgataatcat 120 ggcggcggtt
ctgacatcca gatgacccag tctccatcct ccctgtctgc atctgtagga 180
gacagagtca ccatcacttg ccgggcaagt cagagcatta gcagctattt aaattggtat
240 cagcagaaac cagggaaagc ccctaagctc ctgatctatg cggcatccag
tttgcaaagt 300 ggggtcccat caaggttcag tggcagtgga tctgggacag
atttcactct caccatcagc 360 agtctgcaac ctgaagattt tgcaacttac
tactgtcaac agacggttgt ggcgcctccg 420 ttattcggcc aagggaccaa
ggtggaaatc aaacgtacgg tggctgcacc atctgtcttc 480 atcttcccgc
catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 540
aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
600 ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 660 agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 720 acccatcagg gcctgagctc gcccgtcaca
aagagcttca acaggggaga gtgt 774 <210> SEQ ID NO 234
<211> LENGTH: 258 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: 5342-1204-4D11 Activatable Antibody Light Chain
<400> SEQUENCE: 234 Gln Gly Gln Ser Gly Gln Cys Asn Ile Trp
Leu Val Gly Gly Asp Cys 1 5 10 15 Arg Gly Trp Gln Gly Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Leu Ser Gly Arg Ser Asp
Asn His Gly Gly Gly Ser Asp Ile Gln Met 35 40 45 Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr 50 55 60 Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr 65 70 75 80
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser 85
90 95 Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly 100 105 110 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu
Asp Phe Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln Thr Val Val Ala Pro
Pro Leu Phe Gly Gln 130 135 140 Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val Phe 145 150 155 160 Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 165 170 175 Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 180 185 190 Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 195 200 205
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 210
215 220 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
Val 225 230 235 240 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
Phe Asn Arg Gly 245 250 255 Glu Cys <210> SEQ ID NO 235
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: spacer peptide <400> SEQUENCE: 235 Gly Gln Ser
Gly Gln 1 5 <210> SEQ ID NO 236 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: spacer peptide
<400> SEQUENCE: 236 Gln Ser Gly Gln 1 <210> SEQ ID NO
237 <211> LENGTH: 3 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: spacer peptide <400> SEQUENCE: 237 Ser Gly
Gln 1 <210> SEQ ID NO 238 <211> LENGTH: 1350
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE:
<223> OTHER INFORMATION: C225v4 Antibody Heavy Chain
<400> SEQUENCE: 238 caggtgcagc tgaaacagag cggcccgggc
ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg tgagcggctt
tagcctgacc aactatggcg tgcattgggt gcgccagagc 120 ccgggcaaag
gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac 180
accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca ggtgtttttt
240 aaaatgaaca gcctgcaaag caacgatacc gcgatttatt attgcgcgcg
cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc cagggcaccc
tggtgaccgt gagcgcggct 360 agcaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540
ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac
600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt
tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
960 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 1020 gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggatgaactg 1080 accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320 aagagcctct ccctgtctcc gggtaaatga 1350 <210> SEQ ID NO
239 <211> LENGTH: 449 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: C225v4 Antibody Heavy Chain <400>
SEQUENCE: 239 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly
Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile
Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn
Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210>
SEQ ID NO 240 <211> LENGTH: 1350 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C225v6 Antibody Heavy Chain
<400> SEQUENCE: 240 caggtgcagc tgaaacagag cggcccgggc
ctggtgcagc cgagccagag cctgagcatt 60 acctgcaccg tgagcggctt
tagcctgacc aactatggcg tgcattgggt gcgccagagc 120 ccgggcaaag
gcctggaatg gctgggcgtg atttggagcg gcggcaacac cgattataac 180
accccgttta ccagccgcct gagcattaac aaagataaca gcaaaagcca ggtgtttttt
240 aaaatgaaca gcctgcaaag ccaggatacc gcgatttatt attgcgcgcg
cgcgctgacc 300 tattatgatt atgaatttgc gtattggggc cagggcaccc
tggtgaccgt gagcgcggct 360 agcaccaagg gcccatcggt cttccccctg
gcaccctcct ccaagagcac ctctgggggc 420 acagcggccc tgggctgcct
ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480 aactcaggcg
ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540
ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac
600 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt
tgagcccaaa 660 tcttgtgaca aaactcacac atgcccaccg tgcccagcac
ctgaactcct ggggggaccg 720 tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 780 gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 840 gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacgccagc 900
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag
960 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac
catctccaaa 1020 gccaaagggc agccccgaga accacaggtg tacaccctgc
ccccatcccg ggatgaactg 1080 accaagaacc aggtcagcct gacctgcctg
gtcaaaggct tctatcccag cgacatcgcc 1140 gtggagtggg agagcaatgg
gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200 gactccgacg
gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320 aagagcctct ccctgtctcc gggtaaatga 1350 <210> SEQ ID NO
241 <211> LENGTH: 449 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: C225v6 Antibody Heavy Chain <400>
SEQUENCE: 241 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln
Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe
Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro
Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Gly Gly
Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu Ser Ile
Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn
Ser Leu Gln Ser Gln Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg
Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210
215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330
335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
<210> SEQ ID NO 242 <211> LENGTH: 449 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Av1 Antibody Heavy Chain <400>
SEQUENCE: 242 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg
Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr
Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro
Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser Tyr Ser
Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly 100 105
110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230
235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355
360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210>
SEQ ID NO 243 <211> LENGTH: 214 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Av1 Antibody Light Chain <400>
SEQUENCE: 243 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg
Gly Glu Cys 210 <210> SEQ ID NO 244 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11 Light
Chain <400> SEQUENCE: 244 Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala
Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Val Val Ala Pro
Pro 85 90 95 Leu Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys 210 <210> SEQ ID NO 245
<211> LENGTH: 449 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11 Heavy
Chain <400> SEQUENCE: 245 Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile
Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315
320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 Lys <210> SEQ ID NO 246 <211> LENGTH: 449
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11v2 Heavy
Chain <400> SEQUENCE: 246 Glu Val His Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile
Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp
Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195
200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315
320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 Lys <210> SEQ ID NO 247 <211> LENGTH: 214
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: 4D11v2 Light
Chain <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (182)..(182) <223> OTHER INFORMATION:
Xaa can be any naturally occurring amino acid <400> SEQUENCE:
247 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Thr Val Val Ala Pro Pro 85 90 95 Leu Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Xaa Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 <210> SEQ ID NO 248 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Variable Light Chain
Lc4
<400> SEQUENCE: 248 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 249 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc4 <400>
SEQUENCE: 249 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly
Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 250
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Light Chain Lc5 <400> SEQUENCE: 250 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 251
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc5 <400> SEQUENCE: 251 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Tyr
His Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 <210> SEQ ID NO 252 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc7 <400> SEQUENCE: 252 Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 253 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc7 <400> SEQUENCE: 253 Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 254 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc8 <400>
SEQUENCE: 254 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ
ID NO 255 <211> LENGTH: 119 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Heavy Chain Hc8 <400> SEQUENCE:
255 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr
Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys His Ile
Gly Arg Thr Asn Pro Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115
<210> SEQ ID NO 256 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc13
<400> SEQUENCE: 256 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 257 <211> LENGTH: 116 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc13
<400> SEQUENCE: 257 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Glu Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID NO 258
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Light Chain Lc16 <400> SEQUENCE: 258
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 259
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc16 <400> SEQUENCE: 259
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro
Tyr Tyr Gly Gln Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 260 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc19 <400> SEQUENCE: 260 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 261 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc19 <400> SEQUENCE: 261 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 262 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc21
<400> SEQUENCE: 262 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 263 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc21
<400> SEQUENCE: 263 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75
80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 264 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc24 <400> SEQUENCE:
264 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 265
<211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc24 <400> SEQUENCE: 265
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Glu Met Gly Trp Gln Thr Leu
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Ala
Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 <210> SEQ ID NO 266 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc26 <400> SEQUENCE: 266 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 267 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc26 <400> SEQUENCE: 267 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 268 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc27
<400> SEQUENCE: 268 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 269 <211> LENGTH: 119 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc27
<400> SEQUENCE: 269 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ser Pro Pro Phe Tyr Gly Gln Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 270 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc28 <400> SEQUENCE:
270 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 271
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc28 <400> SEQUENCE: 271
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln
Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 <210> SEQ ID NO 272 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Variable Light Chain Lc30
<400> SEQUENCE: 272 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 273 <211> LENGTH: 115 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc30
<400> SEQUENCE: 273 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Glu Met Gly Trp Gln Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Ala 85
90 95 Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
Thr 100 105 110 Val Ser Ser 115 <210> SEQ ID NO 274
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Light Chain Lc31 <400> SEQUENCE: 274
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 275
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc31 <400> SEQUENCE: 275
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly
Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 276 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc32 <400> SEQUENCE: 276 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 277 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc32 <400> SEQUENCE: 277 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Pro Glu Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 278 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc37 <400> SEQUENCE:
278 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 279
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc37
<400> SEQUENCE: 279 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu
Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ser Pro Pro His Asn Gly Gln Phe Asp Tyr Trp Gly Gln
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID
NO 280 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable Light Chain Lc39 <400> SEQUENCE:
280 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 281
<211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable Heavy Chain Hc39 <400> SEQUENCE: 281
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly Trp Gln Thr Glu
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Ala
Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 <210> SEQ ID NO 282 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Light
Chain Lc40 <400> SEQUENCE: 282 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 283 <211> LENGTH: 119
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable Heavy
Chain Hc40 <400> SEQUENCE: 283 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Gln Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Pro Pro Phe Phe Gly Gln Phe Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
<210> SEQ ID NO 284 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Light Chain Lc47
<400> SEQUENCE: 284 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Val Val Ala Pro Leu 85
90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
<210> SEQ ID NO 285 <211> LENGTH: 116 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable Heavy Chain Hc47
<400> SEQUENCE: 285 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Asp
Glu Met Gly Trp Gln Thr Glu Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID NO 286
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 4B2 Light Chain <400> SEQUENCE: 286 Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Thr Leu Asp Ala Pro Pro 85 90 95
Gln Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210>
SEQ ID NO 287 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Variable 4B2 Heavy Chain <400>
SEQUENCE: 287 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Glu Gln Met Gly
Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Lys Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ ID NO 288
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 4D11 Light Chain <400> SEQUENCE: 288
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5
10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Thr Val Val Ala Pro Pro 85 90 95 Leu Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 289
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 4D11 Heavy Chain <400> SEQUENCE: 289
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Asp Pro Glu Gly Arg Gln Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly
Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 290 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4E7
Light Chain <400> SEQUENCE: 290 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr
Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Leu Val Ala
Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 <210> SEQ ID NO 291 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 4E7
Heavy Chain <400> SEQUENCE: 291 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Glu Glu Met Gly Trp Gln Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 292 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable 4E11 Light Chain <400> SEQUENCE:
292 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ala Leu Asp Ala Pro Leu 85 90 95 Met Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 293
<211> LENGTH: 119 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 4E11 Heavy Chain <400> SEQUENCE: 293
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ser Ile Glu Pro Met Gly Gln Leu Thr Glu
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Gly
Gly Arg Ser Ala Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 <210> SEQ ID NO 294 <211> LENGTH:
108 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 6B7
Light Chain <400> SEQUENCE: 294 Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Leu Val
Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 <210> SEQ ID NO 295 <211> LENGTH: 116
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Variable 6B7
Heavy Chain <400> SEQUENCE: 295 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ser Ile Asp Glu Met Gly Trp Gln Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> SEQ ID
NO 296 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Variable 6F8 Light Chain <400> SEQUENCE:
296 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr
Tyr Cys Gln Gln Ala Leu Val Ala Pro Leu 85 90 95 Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> SEQ ID NO 297
<211> LENGTH: 116 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Variable 6F8 Heavy Chain <400> SEQUENCE: 297 Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Ser Ile Asp Glu Met Gly Trp Gln Thr Tyr Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Ala Ala Ala
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser
115 <210> SEQ ID NO 298 <211> LENGTH: 8 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: cleavable moiety
<400> SEQUENCE: 298 Ile Ser Ser Gly Leu Leu Ser Ser 1 5
<210> SEQ ID NO 299 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 299 Gln Asn Gln Ala Leu Arg Met Ala 1 5 <210> SEQ
ID NO 300 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 300 Ala
Gln Asn Leu Leu Gly Met Val 1 5 <210> SEQ ID NO 301
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 301 Ser Thr Phe
Pro Phe Gly Met Phe 1 5 <210> SEQ ID NO 302 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 302 Pro Val Gly Tyr Thr Ser
Ser Leu 1 5 <210> SEQ ID NO 303 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 303 Asp Trp Leu Tyr Trp Pro Gly Ile 1
5 <210> SEQ ID NO 304 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 304 Met Ile Ala Pro Val Ala Tyr Arg 1 5 <210> SEQ
ID NO 305 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 305 Arg
Pro Ser Pro Met Trp Ala Tyr 1 5 <210> SEQ ID NO 306
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 306 Trp Ala Thr
Pro Arg Pro Met Arg 1 5 <210> SEQ ID NO 307 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 307 Phe Arg Leu Leu Asp Trp
Gln Trp 1 5 <210> SEQ ID NO 308 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 308
Leu Lys Ala Ala Pro Arg Trp Ala 1 5 <210> SEQ ID NO 309
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 309 Gly Pro Ser
His Leu Val Leu Thr 1 5 <210> SEQ ID NO 310 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 310 Leu Pro Gly Gly Leu Ser
Pro Trp 1 5 <210> SEQ ID NO 311 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 311 Met Gly Leu Phe Ser Glu Ala Gly 1
5 <210> SEQ ID NO 312 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 312 Ser Pro Leu Pro Leu Arg Val Pro 1 5 <210> SEQ
ID NO 313 <211> LENGTH: 8 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 313 Arg
Met His Leu Arg Ser Leu Gly 1 5 <210> SEQ ID NO 314
<211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: cleavable moiety <400> SEQUENCE: 314 Leu Ala Ala
Pro Leu Gly Leu Leu 1 5 <210> SEQ ID NO 315 <211>
LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
cleavable moiety <400> SEQUENCE: 315 Ala Val Gly Leu Leu Ala
Pro Pro 1 5 <210> SEQ ID NO 316 <211> LENGTH: 8
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: cleavable
moiety <400> SEQUENCE: 316 Leu Leu Ala Pro Ser His Arg Ala 1
5 <210> SEQ ID NO 317 <211> LENGTH: 8 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: cleavable moiety <400>
SEQUENCE: 317 Pro Ala Gly Leu Trp Leu Asp Pro 1 5 <210> SEQ
ID NO 318 <211> LENGTH: 7 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: cleavable moiety <400> SEQUENCE: 318 Ile
Ser Ser Gly Leu Ser Ser 1 5
* * * * *