U.S. patent application number 14/497089 was filed with the patent office on 2015-03-26 for matrix metalloproteinase substrates and other cleavable moieties and methods of use thereof.
The applicant listed for this patent is CytomX Therapeutics, Inc.. Invention is credited to Jeanne Grace Flandez, Daniel R. Hostetter, Stephen James Moore, Margaret Thy Luu Nguyen, Olga Vasiljeva.
Application Number | 20150087810 14/497089 |
Document ID | / |
Family ID | 51691175 |
Filed Date | 2015-03-26 |
United States Patent
Application |
20150087810 |
Kind Code |
A1 |
Moore; Stephen James ; et
al. |
March 26, 2015 |
Matrix Metalloproteinase Substrates And Other Cleavable Moieties
And Methods Of Use Thereof
Abstract
The invention relates generally to polypeptides that include a
cleavable moiety that is a substrate for at least one matrix
metalloprotease (MMP), to activatable antibodies and other larger
molecules that include the cleavable moiety that is a substrate for
at least one MMP protease, and to methods of making and using these
polypeptides that include a cleavable moiety that is a substrate
for at least one MMP protease in a variety of therapeutic,
diagnostic and prophylactic indications.
Inventors: |
Moore; Stephen James;
(Danville, CA) ; Nguyen; Margaret Thy Luu; (San
Jose, CA) ; Hostetter; Daniel R.; (Palo Alto, CA)
; Vasiljeva; Olga; (Cupertino, CA) ; Flandez;
Jeanne Grace; (Oakland, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CytomX Therapeutics, Inc. |
South San Francisco |
CA |
US |
|
|
Family ID: |
51691175 |
Appl. No.: |
14/497089 |
Filed: |
September 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61882377 |
Sep 25, 2013 |
|
|
|
61971332 |
Mar 27, 2014 |
|
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Current U.S.
Class: |
530/387.3 ;
530/300 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/73 20130101; C07K 7/06 20130101; C07K 16/2866 20130101;
C07K 2319/50 20130101; C07K 16/2863 20130101; C07K 16/44
20130101 |
Class at
Publication: |
530/387.3 ;
530/300 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 7/06 20060101 C07K007/06 |
Claims
1. An isolated polypeptide comprising a cleavable moiety (CM)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 353-363, 372-375, 376-378, 395-401,
411-419, 426-433, 437-449, 454-456, 459-469, 475-482, 487-495,
318-323, 325-327, 330-335, 341-347, 14-33, and 159, wherein the
cleavable moiety is a substrate for a protease.
2. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 353-363, 372-375, 376-378, 395-401, 411-419, 426-433, 437-449,
454-456, 459-469, 475-482, and 487-495.
3. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 318-323, 325-327, 330-335, and 341-347.
4. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 14-33, and 159.
5. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 353-363, wherein the cleavable moiety is a substrate for a
protease.
6. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 372-375, wherein the cleavable moiety is a substrate for a
protease.
7. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 376-378, wherein the cleavable moiety is a substrate for a
protease.
8. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 395-401, wherein the cleavable moiety is a substrate for a
protease.
9. The isolated polypeptide of claim 1, wherein the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 411-419, wherein the cleavable moiety is a substrate for a
protease.
10. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 426-433, wherein the cleavable moiety is a substrate for a
protease.
11. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 437-449, wherein the cleavable moiety is a substrate for a
protease.
12. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 454-456, wherein the cleavable moiety is a substrate for a
protease.
13. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 459-469, wherein the cleavable moiety is a substrate for a
protease.
14. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 475-482, wherein the cleavable moiety is a substrate for a
protease.
15. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 487-495, wherein the cleavable moiety is a substrate for a
protease.
16. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 318-323, wherein the cleavable moiety is a substrate for a
protease.
17. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 325-327, wherein the cleavable moiety is a substrate for a
protease.
18. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 330-335, wherein the cleavable moiety is a substrate for a
protease.
19. The isolated polypeptide of claim 1, wherein the CM comprises
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 341-347, wherein the cleavable moiety is a substrate for a
protease.
20. An isolated polypeptide comprising a cleavable moiety (CM)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 364-370, 379-393, 402-409, 420-424, 434,
435, 450-452, 457, 470-472, 474, and 483, wherein the cleavable
moiety is a substrate for a protease.
21. An isolated polypeptide comprising a cleavable moiety (CM)
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 328, 336-339, and 348-351, wherein the
cleavable moiety is a substrate for a protease.
22. The isolated polypeptide of claim 1, wherein the CM is cleaved
by at least one matrix metalloprotease (MMP).
23. The isolated polypeptide of claim 22, wherein the CM is cleaved
by at least one of MMP9 or MMP14.
24. The isolated polypeptide of claim 2, wherein the CM is cleaved
by at least MMP14.
25. The isolated polypeptide of claim 3, wherein the CM is cleaved
by at least MMP9.
26. The isolated polypeptide of claim 1, wherein the polypeptide
comprises an antibody or antigen binding fragment thereof (AB) that
binds a target.
27. The isolated polypeptide of claim 26, wherein the CM is a
substrate for a protease that is co-localized in a tissue with the
target.
28. The isolated polypeptide of claim 26, 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.
29. The isolated polypeptide of claim 26, wherein the AB is linked
to the CM.
30. The isolated polypeptide of claim 29, wherein the AB is linked
directly to the CM.
31. The isolated polypeptide of claim 29, wherein the AB is linked
to the CM via a linking peptide.
32. The isolated polypeptide of claim 26, wherein the isolated
polypeptide comprises a masking moiety (MM), wherein the MM has an
equilibrium dissociation constant for binding to the AB that is
greater than the equilibrium dissociation constant of the AB for
binding to the target.
33. The isolated polypeptide of claim 32, wherein the MM is a
polypeptide of no more than 40 amino acids in length.
34. The isolated polypeptide of claim 32, wherein the MM is linked
to the CM such that the isolated polypeptide in an uncleaved state
comprises the structural arrangement from N-terminus to C-terminus
as follows: MM-CM-AB or AB-CM-MM.
35. The isolated polypeptide of claim 34, wherein the isolated
polypeptide comprises a linking peptide between the MM and the
CM.
36. The isolated polypeptide of claim 34, wherein the isolated
polypeptide comprises a linking peptide between the CM and the
AB.
37. The isolated polypeptide of claim 34, wherein the isolated
polypeptide comprises a first linking peptide (LP1) and a second
linking peptide (LP2), and wherein the isolated polypeptide has the
structural arrangement from N-terminus to C-terminus as follows in
the uncleaved state: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
38. The isolated polypeptide of claim 37, wherein the two linking
peptides need not be identical to each other.
39. The isolated polypeptide of claim 37, wherein each of LP1 and
LP2 is a peptide of about 1 to 20 amino acids in length.
40. The isolated polypeptide of claim 32, wherein the amino acid
sequence of the MM is different from that of the target and is no
more than 50% identical to the amino acid sequence of a natural
binding partner of the AB.
41. The isolated polypeptide of claim 32, wherein the MM does not
interfere or compete with the AB for binding to the target in a
cleaved state.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/882,377, filed Sep. 25, 2013 and U.S.
Provisional Application No. 61/971,332, filed Mar. 27, 2014, the
contents of each of which are incorporated herein by reference in
their entireties.
FIELD OF THE INVENTION
[0002] The invention relates generally to polypeptides that include
a cleavable moiety that is a substrate for at least one matrix
metalloprotease (MMP), to activatable antibodies and other larger
molecules that include the cleavable moiety that is a substrate for
at least one MMP protease, and to methods of making and using these
polypeptides that include a cleavable moiety that is a substrate
for at least one MMP protease in a variety of therapeutic,
diagnostic and prophylactic indications.
BACKGROUND OF THE INVENTION
[0003] Proteases are enzymes that degrade proteins by cleaving the
peptide bonds between amino acid residues. Proteases occur
naturally in all organisms and are involved in a variety of
physiological reactions from simple degradation to highly regulated
pathways. Some proteases are known to break specific peptide bonds
based on the presence of a particular amino acid sequence within a
protein.
[0004] Accordingly, there exists a need to identify new substrates
for proteases and to use these substrates in a variety of
therapeutic, diagnostic and prophylactic indications.
SUMMARY OF THE INVENTION
[0005] The disclosure provides amino acid sequences that include a
cleavable moiety (CM) that is a substrate for at least one matrix
metalloprotease (MMP). These CMs are useful in a variety of
therapeutic, diagnostic and prophylactic indications.
[0006] 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, MMP11, and MMP19. 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.
[0007] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID
NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17);
PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ
ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22);
FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ
ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27);
SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ
ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32);
PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).
[0008] In some embodiments, the CM comprises the amino acid
sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM
comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some
embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ
ID NO: 16). In some embodiments, the CM comprises the amino acid
sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM
comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some
embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ
ID NO: 19). In some embodiments, the CM comprises the amino acid
sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM
comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some
embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ
ID NO: 22). In some embodiments, the CM comprises the amino acid
sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM
comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some
embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ
ID NO: 25). In some embodiments, the CM comprises the amino acid
sequence LPGGLSPW (SEQ ID NO: 26). In some embodiments, the CM
comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 27). In some
embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ
ID NO: 28). In some embodiments, the CM comprises the amino acid
sequence RMHLRSLG (SEQ ID NO: 29). In some embodiments, the CM
comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 30). In some
embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ
ID NO: 31). In some embodiments, the CM comprises the amino acid
sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments, the CM
comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). In some
embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ
ID NO: 159).
[0009] In some embodiments, the CM is linked or otherwise attached
to an antibody. For example, the CM is used to link one or more
agents to the antibody or antigen binding fragment thereof (AB)
that binds a given target, such that the CM is cleaved when exposed
to the MMP and the agent is released from the AB. Exemplary targets
include, but are not limited to the targets shown in Table 1.
Exemplary ABs include, but are not limited to, the targets shown in
Table 2. In some embodiments, the antibody in the uncleaved state
has the structural arrangement from N-terminus to C-terminus as
follows: Agent-CM-AB or AB-CM-Agent. In some embodiments, the
antibody comprises a linking peptide between the AB and the CM. In
some embodiments, the antibody comprises a linking peptide between
the CM and the conjugated agent.
[0010] In some embodiments, the antibody comprises a first linking
peptide (LP1) and a second linking peptide (LP2), and the antibody
in the uncleaved state has the structural arrangement from
N-terminus to C-terminus as follows: Agent-LP1-CM-LP2-AB or
AB-LP2-CM-LP1-Agent. In some embodiments, each of LP1 and LP2 is a
peptide of about 1 to 20 amino acids in length. In some
embodiments, the two linking peptides need not be identical to each
other.
[0011] In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least
one.
[0012] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of GGSG (SEQ
ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ
ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).
[0013] In some embodiments, LP1 comprises the amino acid sequence
GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10),
GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155),
GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157).
[0014] In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0015] In some embodiments, the AB has an equilibrium dissociation
constant of about 100 nM or less for binding to the target.
[0016] In some embodiments, the antibody includes an antibody or
antigen-binding fragment thereof that specifically binds a 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, or a single domain light chain antibody. In some
embodiments, such an antibody or immunologically active fragment
thereof that binds the target is a mouse, other rodent, chimeric,
humanized or fully human monoclonal antibody.
[0017] In some embodiments, the MMP protease is co-localized with
the target in a tissue, and the MMP protease cleaves the CM in the
antibody when the antibody is exposed to the protease.
[0018] In some embodiments, the CM is a polypeptide of up to 15
amino acids in length.
[0019] 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, MMP11, and MMP19. 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.
[0020] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID
NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17);
PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ
ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22);
FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ
ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27);
SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ
ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32);
PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).
[0021] In some embodiments, the CM comprises the amino acid
sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM
comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some
embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ
ID NO: 16). In some embodiments, the CM comprises the amino acid
sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM
comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some
embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ
ID NO: 19). In some embodiments, the CM comprises the amino acid
sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM
comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some
embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ
ID NO: 22). In some embodiments, the CM comprises the amino acid
sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM
comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some
embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ
ID NO: 25). In some embodiments, the CM comprises the amino acid
sequence LPGGLSPW (SEQ ID NO: 26). In some embodiments, the CM
comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 27). In some
embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ
ID NO: 28). In some embodiments, the CM comprises the amino acid
sequence RMHLRSLG (SEQ ID NO: 29). In some embodiments, the CM
comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 30). In some
embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ
ID NO: 31). In some embodiments, the CM comprises the amino acid
sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments, the CM
comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). In some
embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ
ID NO: 159).
[0022] In some embodiments, the CM is a substrate for at least one
matrix metalloprotease (MMP) and includes a motif sequence that is
recognized by MMP9. In some embodiments, the CM is a substrate for
at least one MMP and includes a motif sequence that is recognized
by MMP14.
[0023] In some embodiments, the CM is a substrate for at least one
MMP, and the CM polypeptide and/or the CM portion of any
polypeptide that comprises the CM comprises a polypeptide having a
length less than 50 amino acids, less than 40 amino acids, less
than 30 amino acids, less than 25 amino acids, less than 20 amino
acids, less than 19 amino acids, less than 18 amino acids, less
than 17 amino acids, less than 16 amino acids, less than 15 amino
acids long, less than 14 amino acids, less than 13 amino acids,
less than 12 amino acids, less than 11 amino acids, or less than 10
amino acids long.
[0024] In some embodiments, the CM is a substrate for at least one
MMP and comprises a polypeptide sequence that is not substantially
identical to any human polypeptide sequence that is naturally
cleaved by the same MMP protease. In some embodiments, the CM is a
substrate for at least one MMP and comprises a polypeptide sequence
that is no more than 90% or more identical to any human polypeptide
sequence that is naturally cleaved by the same MMP protease.
[0025] In some embodiments, the motif sequence is a substrate for
at least MMP and includes a core CM consensus sequence shown in
Tables 8A-8M below. In some embodiments, the motif sequence
includes a subgenus, i.e., a subset, of the core CM consensus
sequence shown in Tables 8A-8M below.
[0026] In some embodiments, the motif sequence is a substrate for
at least MMP9 and includes a core CM consensus sequence shown in
Tables 8A-8D. In some embodiments, the motif sequence is a
substrate for at least MMP9 and includes a subgenus, i.e., a
subset, of the core CM consensus sequence shown in Tables 8A-8D
below.
[0027] In some embodiments, the motif sequence is a substrate for
at least MMP14 and includes a core CM consensus sequence shown in
Tables 8E-8M. In some embodiments, the motif sequence is a
substrate for at least MMP14 and includes a subgenus, i.e., a
subset, of the core CM consensus sequence shown in Tables 8E-8M
below.
TABLE-US-00001 TABLE 8A MMP9 Cleavable Core CM Consensus Sequence 1
Core CM Consensus 1 Subgenus of Core CM Consensus 1
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
Subgenus 1.1:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 318), (SEQ ID NO: 317), wherein wherein X.sub.22 is G,
P, R, or S; X.sub.23 is P or S; X.sub.24 is L, M, P, or S; X.sub.22
is A, C, D, G, H, L, P, X.sub.25 is A, G, P, or S; X.sub.26 is L,
M, or R; X.sub.27 is G or W; X.sub.28 is A, R, or S; G, S, or Y;
and X.sub.29 is L, R, V, or Y. X.sub.23 is L, M, P, S, or T;
Subgenus 1.2:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 319), X.sub.24 is A, D, F, G, L, M, N, wherein X.sub.22
is G, P or R; X.sub.23 is P; X.sub.24 is L, M, or S; X.sub.25 is G,
P, R, S, T, or V; P, or S; X.sub.26 is L, M, or R; X.sub.27 is W;
X.sub.28 is A, G, or S; and X.sub.29 X.sub.25 is A, D, E, G, H, I,
M, is R, V, or Y. P, S, or V; Subgenus 1.3:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 320), X.sub.26 is A, C, D, G, L, M, N, wherein X.sub.22
is P or R; X.sub.23 is P; X.sub.24 is M or S; X.sub.25 is G or P;
X.sub.26 R, V, W, or Y; is L, M, or R; X.sub.27 is W; X.sub.28 is
A, G, or S; and X.sub.29 is R, V, or Y. X.sub.27 is C, F, G, H, P,
Q, Subgenus 1.4:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 321), R, T, V, or W; wherein X.sub.22 is P or R;
X.sub.23 is P; X.sub.24 is S; X.sub.25 is G or P; X.sub.26 is
X.sub.28 is A, D, G, L, M, S, T, M, or R; X.sub.27 is W; X.sub.28
is A or S; and X.sub.29 is Y. V, or Y; and Subgenus 1.5:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 322), X.sub.29 is C, H, L, R, S, V, W, wherein X.sub.22
is P or R; X.sub.23 is P; X.sub.24 is S; X.sub.25 is G or P;
X.sub.26 is or Y. M, or R; X.sub.27 is W; X.sub.28 is A or S; and
X.sub.29 is Y. Subgenus 1.6:
X.sub.22X.sub.23X.sub.24X.sub.25X.sub.26X.sub.27X.sub.28X.sub.29
(SEQ ID NO: 323), wherein X.sub.22 is C, G, H, L, or R; X.sub.23 is
P, S or T; X.sub.24 is N, R, S or T; X.sub.25 is P or S; X.sub.26
is C, M, R, V, or W; X.sub.27 is C, P, R, or W; X.sub.28 is A, D,
or G; and X.sub.29 is C or Y.
TABLE-US-00002 TABLE 8B MMP9 Cleavable Core CM Consensus Sequence 2
Core CM Consensus 2 Subgenus of Core CM Consensus 2
X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
Subgenus 2.1:
X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
(SEQ ID (SEQ ID NO: 324), wherein NO: 325), wherein X.sub.32 is W;
X.sub.33 is D, P, or T; X.sub.34 is H, Q, X.sub.32 is F, G, V, or
W; or W; X.sub.35 is D or P; X.sub.36 is I or R; X.sub.37 is S;
X.sub.38 is L, X.sub.33 is A, D, L, M, P, R, T, or M, or V; and
X.sub.39 is G, L, or S. V; Subgenus 2.2:
X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
(SEQ ID X.sub.34 is C, G, H, L, Q, S, T, W, NO: 326), wherein
X.sub.32 is W; X.sub.33 is D; X.sub.34 is H, Q, or W; X.sub.35
X.sub.35 is D, G, L, P; is D or P; X.sub.36 is I or R; X.sub.37 is
G, S, or V; X.sub.38 is L, M, X.sub.36 is E, G, I, L, N, P, R, or
V; or V; and X.sub.39 is G, L, or S. X.sub.37 is G, L, P, R, S, or
V; Subgenus 2.3:
X.sub.32X.sub.33X.sub.34X.sub.35X.sub.36X.sub.37X.sub.38X.sub.39
(SEQ ID X.sub.38 is A, I, L, M, T, or V; and NO: 327), wherein
X.sub.32 is W; X.sub.33 is D; X.sub.34 is H, Q, or W; X.sub.35
X.sub.39 is A, G, L, P, Q, R, S, or V. is P; X.sub.36 is I or R;
X.sub.37 is S; X.sub.38 is L, M, or V; and X.sub.39 is L.
TABLE-US-00003 TABLE 8C MMP9 Cleavable Core CM Consensus Sequence 3
Core CM Consensus 3 Subgenus of Core CM Consensus 3
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
Subgenus 3.1:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 330), (SEQ ID NO: 329), wherein X.sub.42 is I, L, M, or
S; X.sub.43 is D, P, S, or T; X.sub.44 is F, L, S, or wherein V;
X.sub.45 is L. P, or S; X.sub.46 is A, F, R, S, or T; X.sub.47 is
G, H, T or Y; X.sub.42 is G, I, L, M, P, R, S, X.sub.48 is G, I, M,
V, or W; and X.sub.49 is F, L, or S. T, or V; Subgenus 3.2:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 331), X.sub.43 is A, D, H, I, L, P, S, wherein X.sub.42
is L, M, or S; X.sub.43 is S or T; X.sub.44 is F or L; X.sub.45 is
P; X.sub.46 or T; is A, F, or T; X.sub.47 is G, H, T or Y; X.sub.48
is I, M, or W; and X.sub.49 is F. X.sub.44 is F, L, S, or V;
Subgenus 3.3:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 332), X.sub.45 is H, L, M, P, Q, R, wherein X.sub.42 is
L, M, or S; X.sub.43 is S or T; X.sub.44 is F; X.sub.45 is P;
X.sub.46 is S, or T; A, F, or T; X.sub.47 is G, H, or Y; X.sub.48
is I, M, or W; and X.sub.49 is F. X.sub.46 is A, D, F, G, L, M,
Subgenus 3.4:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 333), R, S, T, or V; wherein X.sub.42 is L or M;
X.sub.43 is S or T; X.sub.44 is F; X.sub.45 is P; X.sub.46 is A or
T; X.sub.47 is A, C, G, H, Q, T or X.sub.47 is H or Y; X.sub.48 is
I or W; and X.sub.49 is F. Y; Subgenus 3.5:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 334), X.sub.48 is C, G, I, M, R, S, T, wherein X.sub.42
is G, I, R, Or S; X.sub.43 is H Or T; X.sub.44 is F, L, S, or V;
X.sub.45 V, or W; and is L, P, or R; X.sub.46 is F, L, or S;
X.sub.47 is A, C, or G; X.sub.48 is I, M, or V; and X.sub.49 is F,
L, S, or Y. X.sub.49 is F or L. Subgenus 3.6:
X.sub.42X.sub.43X.sub.44X.sub.45X.sub.46X.sub.47X.sub.48X.sub.49
(SEQ ID NO: 335), wherein X.sub.42 is S; X.sub.43 is T; X.sub.44 is
F or V; X.sub.45 is L or P; X.sub.46 is F or L; X.sub.47 is G;
X.sub.48 is I or M; and X.sub.49 is F.
TABLE-US-00004 TABLE 8D MMP9 Cleavable Core CM Consensus Sequence 4
Core CM Consensus 4 Subgenus of Core CM Consensus 4
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
Subgenus 4.1:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 341), (SEQ ID NO: 340), wherein X.sub.52 is D, G, H, L,
P, Q, S or Y X.sub.53 is D, W, or Y; X.sub.54 is wherein H, L, or
R; X.sub.55 is H, L, M, P, or Y; X.sub.56 Is E, F, G, M, R, or W;
X.sub.57 X.sub.52 is D, G, H, L, N, P, is A, L, M, N, P, or R;
X.sub.58 is G, L, P, R, or S; and X.sub.59 is G, I, P, S, T, Q, R,
S, W, or Y or Y. X.sub.53 is A, C, D, G, L, R, Subgenus 4.2:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 342), V, W, or Y; wherein X.sub.52 is D or H; X.sub.53
is W or Y; X.sub.54 is H or L; X.sub.55 is H, L, X.sub.54 is D, H,
L, P, Q, R, S, or Y; X.sub.56 IS G or W; X.sub.57 is P or R;
X.sub.58 is G, L, or P; and X.sub.59 is G, or Y; I, S, or T.
X.sub.55 is D, F, H, I, L, M, P, Subgenus 4.3:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 343), S, or Y; wherein X.sub.52 is H; X.sub.53 is W;
X.sub.54 is H or L; X.sub.55 is H, L, or Y; X.sub.56 Is X.sub.56 is
A, C, E, F, G, K, G or W; X.sub.57 is P; X.sub.58 is L or P; and
X.sub.59 is G, I, S or T. M, R, S, V, or W; Subgenus 4.4:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 344), X.sub.57 is A, G, K L, M, N, wherein X.sub.52 is
H; X.sub.53 is W; X.sub.54 is H or L; X.sub.55 is L or Y; X.sub.56
Is G; P, R, S, or T; X.sub.57 is P; X.sub.58 is L or P; and
X.sub.59 is G, I, S, or T. X.sub.58 is A, F, G, H, L, P, R,
Subgenus 4.5:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 345), S, or T; and wherein X.sub.52 is H; X.sub.53 is
W; X.sub.54 is H or L X.sub.55 is L or Y X.sub.56 Is G; X.sub.59 is
A, G, H, I, N, P, S, X.sub.57 is P; X.sub.58 is P; and X.sub.59 is
T. T, or Y. Subgenus 4.6:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 346), wherein X.sub.52 is D, G, S, or Y; X.sub.53 is W;
X.sub.54 is L or P; X.sub.55 is D or Y; X.sub.56 is C, E, G, or W;
X.sub.57 is M or P; X.sub.58 is G, R, or S; and X.sub.59 is H, I,
or Y. Subgenus 4.7:
X.sub.52X.sub.53X.sub.54X.sub.55X.sub.56X.sub.57X.sub.58X.sub.59
(SEQ ID NO: 347), wherein X.sub.52 is D, G, or S; X.sub.53 is W;
X.sub.54 is L; X.sub.55 is Y; X.sub.56 is E or W; X.sub.57 is M or
P; X.sub.58 is G or S; and X.sub.59 is I or Y.
TABLE-US-00005 TABLE 8E MMP14 Cleavable Core CM Consensus Sequence
5 Core CM Consensus 5 Subgenus of Core CM Consensus 5
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
Subgenus 5.1:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 353), (SEQ ID NO: 352), wherein wherein X.sub.62 is A,
G, I, P, Q, S, T, or V; X.sub.63 is A, L, Q, S, or V; X.sub.62 is
A, I, G, L, M, P, Q, X.sub.64 is A, E, L, R, or S; X.sub.65 is D or
G; X.sub.66 is I or L; X.sub.67 is E, I, L, S, T, or V; M, Q, R, or
Y; X.sub.68 is F, H, L, M, R, or S; and X.sub.69 is A, G, H, L,
X.sub.63 is A, D, L, P, Q, S, T, N, P, Q, or S. V, or Y; Subgenus
5.2:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 354), X.sub.64 is A, C, E, F, G, H, K, wherein X.sub.62
is A, I, S or T; X.sub.63 is L, Q, S, or V; X.sub.64 is A, L, R, L,
P, Q, R, S, or V; or S; X.sub.65 is G; X.sub.66 is I or L; X.sub.67
is E, L, R, or Y; X.sub.68 is F, H, L, R, or X.sub.65 is D, E, G,
S, or V; S; and X.sub.69 is H, L, P, or S. X.sub.66 is A, I, L, M,
or V; Subgenus 5.3:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 355), X.sub.67 is C, E, G, I, K, L, M, wherein X.sub.62
is A, I, S or T; X.sub.63 is L, S, or V; X.sub.64 is A, R or S; N,
Q, R, or Y; X.sub.65 is G; X.sub.66 is L; X.sub.67 is E, L or R;
X.sub.68 is F, H, or S; and X.sub.69 is X.sub.68 is A, F, H, I, L,
M, N, L, P, or S. P, R, S, or T; and Subgenus 5.4:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 356), X.sub.69 is A, C, G, H, I, L, N, wherein X.sub.62
is A, I, S or T; X.sub.63 is L, S, or V; X.sub.64 is R or S;
X.sub.65 is P, Q, R, S, T, V, or W. G; X.sub.66 is L; X.sub.67 is L
or R; X.sub.68 is F, H, or S; and X.sub.69 is P or S. Subgenus 5.5:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 357), wherein X.sub.62 is A, I, S or T; X.sub.63 is L,
S, or V; X.sub.64 is R or S; X.sub.65 is G; X.sub.66 is L; X.sub.67
is L or R; X.sub.68 is S; and X.sub.69 is P or S. Subgenus 5.6:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 358), wherein X.sub.62 is T; X.sub.63 is L, S, or V;
X.sub.64 is S; X.sub.65 is G; X.sub.66 is L; X.sub.67 is R;
X.sub.68 is S; and X.sub.69 is P. Subgenus 5.7:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 359), wherein X.sub.62 is A, G, I, M, P, S, T, or V;
X.sub.63 is L, Q, S, or V; X.sub.64 is A, C, F, K, L, Q, R or S;
X.sub.65 is D, G, S, or V; X.sub.66 is L or M; X.sub.67 is G, I, L,
M, N, Q, or R; X.sub.68 is I, N, P, or S; and X.sub.69 is A, H, I,
N, Q, or S. Subgenus 5.8:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 360), wherein X.sub.62 is A, I, or S; X.sub.63 is L, Q,
S, or V; X.sub.64 is L, R or S; X.sub.65 is G; X.sub.66 is L;
X.sub.67 is L, M, or R; X.sub.68 is S; and X.sub.69 is A, H, N, Q,
or S. Subgenus 5.9:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 361), wherein X.sub.62 is A, I, or S; X.sub.63 is L, Q,
S, or V; X.sub.64 is L, R or S; X.sub.65 is G; X.sub.66 is L;
X.sub.67 is L, M, or R; X.sub.68 is S; and X.sub.69 is A, H, N, Q,
or S. Subgenus 5.10:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 362), wherein X.sub.62 is A or S; X.sub.63 is L or V;
X.sub.64 is L or S; X.sub.65 is G; X.sub.66 is L; X.sub.67 is L or
R; X.sub.68 is S; and X.sub.69 is H, or S. Subgenus 5.11:
X.sub.62X.sub.63X.sub.64X.sub.65X.sub.66X.sub.67X.sub.68X.sub.69
(SEQ ID NO: 363), wherein X.sub.62 is A or S; X.sub.63 is L or V;
X.sub.64 is S; X.sub.65 is G; X.sub.66 is L; X.sub.67 is L or R;
X.sub.68 is S; and X.sub.69 is H, or S.
TABLE-US-00006 TABLE 8F-1 MMP14 Cleavable Core CM Consensus
Sequence 6 Core CM Consensus 6 Subgenus of Core CM Consensus 6
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79
Subgenus 6.1:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79
(SEQ ID NO: 372), (SEQ ID NO: 371), wherein wherein X.sub.72 is A,
F, G, H, I, L, M, Q, R, or S; X.sub.73 is A, F, H, L, X.sub.72 is
A, C, D, E, F, G, H, or N; X.sub.74 is A, E, N, Q, or S; X.sub.75
is A, E, K, N, S, or T; X.sub.76 is L I, K, L, M, N, P, Q, R, S, or
M; X.sub.77 is A, I, K, L, P, R, or V; X.sub.78 is A, D, I, L, M,
R, T, or or V; V; and X.sub.79 is A, F, G, H, I, L, P, Q, R, or S.
X.sub.73 is A, C, E, F, H, L, N, Subgenus 6.2:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79
(SEQ ID NO: 373), R, S, or V; wherein X.sub.72 is G, L or R, or S;
X.sub.73 is A or L; X.sub.74 is A, E, N, Q, X.sub.74 is A, D, E, K,
N, P Q, or S; X.sub.75 is A, E, N, S, or T; X.sub.76 is L or M;
X.sub.77 is L or R; X.sub.78 is S, T, or Y; A, L, or T; and
X.sub.79 is F, G, L, R, or S. X.sub.75 is A, E, G, H, K, L, N,
Subgenus 6.3:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79
(SEQ ID NO: 374), P, R, S, or T; wherein X.sub.72 is L; X.sub.73 is
A or L; X.sub.74 is E, N, Q, or S; X.sub.75 is A or X.sub.76 is I,
K, L, M, N, R, T, S; X.sub.76 is L or M; X.sub.77 is R; X.sub.78 is
A or T; and X.sub.79 is F, L, or R. V or Y; Subgenus 6.4:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78X.sub.79
(SEQ ID NO: 375), X.sub.77 is A, D, E, I, K, L, P, wherein X.sub.72
is L; X.sub.73 is A or L; X.sub.74 is E, N, Q, or S; X.sub.75 is A
or Q, R, S, T, V, or Y; S; X.sub.76 is L or M; X.sub.77 is R;
X.sub.78 is A; and X.sub.79 is L or R. X.sub.78 is A, C, D, E, G,
I, L, M, Q, R, S, T, or V; and X.sub.79 is A, F, G, H, I, L, P, Q,
R, S, T, or Y.
TABLE-US-00007 TABLE 8F-2 MMP14 Cleavable Core CM Consensus
Sequence 6A Core CM Consensus 6A Subgenus of Core CM Consensus 6A
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 (SEQ ID
Subgenus 6A.1:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 (SEQ ID
NO: 485), wherein NO: 376), wherein X.sub.72 is A, E, L, N, P, or
Q; X.sub.73 is X.sub.72 is A, C, D, E, F, G, H, I, K, L, M, F, H,
L, N, or S; X.sub.74 is Q or Y; X.sub.75 is A; X.sub.76 is L, T, N,
P, Q, R, S, or V; V or Y; X.sub.77 is D, E, P, Q, or R; and
X.sub.78 is A, C, G, I, X.sub.73 is A, C, E, F, H, L, N, R, S, or
V; M, R, S, or T. X.sub.74 is A, D, E, K, N, P Q, S, T, or Y;
Subgenus 6A.2:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 (SEQ ID
X.sub.75 is A, E, G, H, K, L, N, P, R, S, or NO: 377), wherein
X.sub.72 is A, E, L, or Q; X.sub.73 is F, H, T; or N; X.sub.74 is
Q; X.sub.75 is A; X.sub.76 is L or T; X.sub.77 is Q or R; X.sub.76
is I, K, L, M, N, R, T, V or Y; and X.sub.78 is I or M. X.sub.77 is
A, D, E, I, K, L, P, Q, R, S, T, Subgenus 6A.3:
X.sub.72X.sub.73X.sub.74X.sub.75X.sub.76X.sub.77X.sub.78 (SEQ ID V,
or Y; and NO: 378), wherein X.sub.72 is A; X.sub.73 is F, H, or N;
X.sub.74 is X.sub.78 is A, C, D, E, G, I, L, M, Q, R, S, Q;
X.sub.75 is A; X.sub.76 is L; X.sub.77 is R; and X.sub.78 is M. T,
or V.
TABLE-US-00008 TABLE 8G MMP14 Cleavable Core CM Consensus Sequence
7 Core CM Consensus 7 Subgenus of Core CM Consensus 7
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
Subgenus 7.1:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 395), (SEQ ID NO: 394), wherein wherein X.sub.82 is L;
X.sub.83 is H, K, Q, R, or Y; X.sub.84 is A, L, M, S, T, X.sub.82
is A, F, L, Q, S, T, or V; or V; X.sub.85 is A, I, L, S, or V;
X.sub.86 is P; X.sub.87 is A, G, R, S, V, or X.sub.83 is A, E, G,
H, K, Q, R, V, W; X.sub.88 is I, R, T, or W; and X.sub.89 is A, F,
G, L, S, or V. or Y; Subgenus 7.2:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 396), X.sub.84 is A, G, I, K, L, M, N, S, wherein
X.sub.82 is L; X.sub.83 is H, K, R, or Y; X.sub.84 is A, L, or V;
X.sub.85 is T, or V; A, I, or L; X.sub.86 is P; X.sub.87 is G, R,
or V; X.sub.88 is T or W; and X.sub.89 is X.sub.85 is A, D, F, G,
I, L, N, P, A, F, L, or S. R, S, T, or V; Subgenus 7.3:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 397), X.sub.86 is A, P, or R; wherein X.sub.82 is L;
X.sub.83 is K, R, or Y; X.sub.84 is A; X.sub.85 is A or L; X.sub.87
is A, D, G, L, M, P, R, S, X.sub.86 is P; X.sub.87 is G, R, or V;
X.sub.88 is W; and X.sub.89 is A or L. T, V, W, or Y; Subgenus 7.4:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 398), X.sub.88 is A, C, E, F, H, I, L, N, wherein
X.sub.82 is A, F, L, Q, or S; X.sub.83 is A, E, G, H, K, Q, or Y;
R, S, T, W, or Y; and X.sub.84 is A, G, K, S, or V; X.sub.85 is A,
I, L, P, or T; X.sub.86 is A, P, or X.sub.89 is A, F, G, I, L, M,
R, S, T, or R; X.sub.87 is A, L, M, R, V, or Y; X.sub.88 is C, H,
R, T, or W; and X.sub.89 V. is A, F, L, R, S, or T. Subgenus 7.5:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 399), wherein X.sub.82 is F or L; X.sub.83 is G, K, Q,
or Y; X.sub.84 is A, G, S, or V; X.sub.85 is A, I, or L; X.sub.86
is P; X.sub.87 is A, R, or V; X.sub.88 is R or W; and X.sub.89 is
A, F, L, or R. Subgenus 7.6:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 400), wherein X.sub.82 is L; X.sub.83 is K or Y;
X.sub.84 is A or S; X.sub.85 is A, I, or L; X.sub.86 is P; X.sub.87
is A, R, or V; X.sub.88 is W; and X.sub.89 is A or F. Subgenus 7.7:
X.sub.82X.sub.83X.sub.84X.sub.85X.sub.86X.sub.87X.sub.88X.sub.89
(SEQ ID NO: 401), wherein X.sub.82 is L; X.sub.83 is K or Y;
X.sub.84 is A; X.sub.85 is A or I; X.sub.86 is P; X.sub.87 is R or
V; X.sub.88 is W; and X.sub.89 is A or F.
TABLE-US-00009 TABLE 8H-1 MMP14 Cleavable Core CM Consensus
Sequence 8 Core CM Consensus 8 Subgenus of Core CM Consensus 8
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 Subgenus
8.1: X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ
ID NO: 411), wherein (SEQ ID NO: 410), X.sub.92 is A, F, G, I, L,
M, N, S, T, V, or W; X.sub.93 is P; X.sub.94 is A, E, F, H, wherein
I, K, N, P, Q, R, S, T, or V; X.sub.95 is A, D, E, G, H, N, P, or
S; X.sub.96 is X.sub.92 is A, D, F, G, H, I, C, F, I, L, M, R, S,
or V; X.sub.97 is C, F, G, I, L, R, S, T, V, or Y; and L, M, N, P,
Q, R, S, X.sub.98 is A, F, L, M, P, Q, R, S, T, V, or Y. T, V, or
W; Subgenus 8.2:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 412), wherein X.sub.93 is A, P, R, or T; X.sub.92 is F, G, L,
S, T, or V; X.sub.93 is P; X.sub.94 is A, E, H, K, N, Q, R, S, T,
X.sub.94 is A, E, F, G, H, I, or V; X.sub.95 is A, G, H, N, P, or
S; X.sub.96 is I, L, M, or V; X.sub.97 is F, I, L, K, L, N, P, Q,
R, S, T, R, S, T, V, or Y; and X.sub.98 is A, F, L, R, T, V, or Y.
or V; Subgenus 8.3:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 413), wherein X.sub.95 is A, D, E, G, H, K, X.sub.92 is F, L,
or S; X.sub.93 is P; X.sub.94 is A, K, Q, R, or S; X.sub.95 is A,
G, H, or M, N, P, R, S, or T; S; X.sub.96 is I, L, M, or V;
X.sub.97 is F, L, R, S, T, V, or Y and; X.sub.98 is F, L, X.sub.96
is C, F, H, I, L, M, T, or V. P, R, S, V, W, or Y; Subgenus 8.4:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 414), wherein X.sub.97 is A, C, F, G, H, I, X.sub.92 is F, L,
or S; X.sub.93 is P; X.sub.94 is A, Q, or S; X.sub.95 is G or S;
X.sub.96 is I, K, L, M, R, S, T, V, L, or M; X.sub.97 is L, S, or
V; and X.sub.98 is F, L, or T. W, or Y; and Subgenus 8.5:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 415), wherein X.sub.98 is A, D, E, F, G, H, X.sub.92 is F, L,
or S; X.sub.93 is P; X.sub.94 is A, Q or S; X.sub.95 is G; X.sub.96
is I, L, or I, K, L, M, N, P, Q, R, S, T, M; X.sub.97 is L or V;
and X.sub.98 is L. V, or Y. Subgenus 8.6:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 416), wherein X.sub.92 is F, L, or S; X.sub.93 is P; X.sub.94
is A or S; X.sub.95 is G; X.sub.96 is I, L, or M; X.sub.97 is L or
V; and X.sub.98 is L. Subgenus 8.7:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 417), wherein X.sub.92 is F, G, L, M, P, S, V, or W; X.sub.93
is P; X.sub.94 is A, N, Q, or S; X.sub.95 is A, D, G, H, M, N, P,
or S; X.sub.96 is F, I, L, M, or V; X.sub.97 is A, I, L, M, S, or
V; and X.sub.98 is A, G, I, L, M, N, P, Q, R, S, T, or Y. Subgenus
8.8: X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ
ID NO: 418), wherein X.sub.92 is L, S, or V; X.sub.93 is P;
X.sub.94 is A, N, Q, or S; X.sub.95 is H, N, P, or S; X.sub.96 is
F, I, L, or M; X.sub.97 is I, L, S, or V; and X.sub.98 is A, L, or
Q. Subgenus 8.9:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98 (SEQ ID
NO: 419), wherein X.sub.92 is L; X.sub.93 is P; X.sub.94 is A, N,
Q, or S; X.sub.95 is H; X.sub.96 is I or L; X.sub.97 is V; and
X.sub.98 is L.
TABLE-US-00010 TABLE 8H-2 MMP14 Cleavable Extended Core CM
Consensus Sequence 8 Extended Core CM Subgenus of Extended Core CM
Consensus 8A Consensus 8A
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
Subgenus 8A.1:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 487), (SEQ ID NO: 486), wherein X.sub.92 is A, F, G, I,
L, M, N, S, T, V, or W; X.sub.93 is P; X.sub.94 is wherein A, E, F,
H, I, K, N, P, Q, R, S, T, or V; X.sub.95 is A, D, E, G, H, N, P,
X.sub.92 is A, D, F, G, H, I, L, or S; X.sub.96 is C, F, I, L, M,
R, S, or V; X.sub.97 is C, F, G, I, L, R, S, T, M, N, P, Q, R, S,
T, V, V, or Y; X.sub.98 is A, F, L, M, P, Q, R, S, T, V, or Y; and
X.sub.99 is A, D, or W; E, G, H, I, L, N, P, Q, R, S, T, V, W, or
Y. X.sub.93 is A, P, R, or T; Subgenus 8A.2:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 488), X.sub.94 is A, E, F, G, H, I, K, wherein X.sub.92
is F, G, L, S, T, or V; X.sub.93 is P; X.sub.94 is A, E, H, K, N,
L, N, P, Q, R, S, T, or Q, R, S, T, or V; X.sub.95 is A, G, H, N,
P, or S; X.sub.96 is I, L, M, or V; V; X.sub.97 is F, I, L, R, S,
T, V, or Y; X.sub.98 is A, F, L, R, T, V, or Y; and X.sub.95 is A,
D, E, G, H, K, X.sub.99 is A, D, G, L, P, R, S, T, V, or Y. M, N,
P, R, S, or T; Subgenus 8A.3:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 489), X.sub.96 is C, F, H, I, L, M, P, wherein X.sub.92
is F, L, or S; X.sub.93 is P; X.sub.94 is A, K, Q, R, or S;
X.sub.95 is R, S, V, W, or Y; A, G, H, or S; X.sub.96 is I, L, M,
or V; X.sub.97 is F, L, R, S, T, V, or Y; X.sub.97 is A, C, F, G,
H, I, K, X.sub.98 is F, L, T, or V; and X.sub.99 is A, D, G, L, R,
T, or V. L, M, R, S, T, V, W, or Subgenus 8A.4:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 490), Y; wherein X.sub.92 is F, L, or S; X.sub.93 is P;
X.sub.94 is A, Q, or S; X.sub.95 is G or S; X.sub.98 is A, D, E, F,
G, H, I, K, X.sub.96 is I, L, or M; X.sub.97 is L, S, or V;
X.sub.98 is F, L, or T; and X.sub.99 is A, L, M, N, P, Q, R, S, R,
or T. T, V, or Y; and Subgenus 8A.5:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 491), X.sub.99 is A, D, E, F, G, H, I, wherein X.sub.92
is F, L, or S; X.sub.93 is P; X.sub.94 is A, Q or S; X.sub.95 is G;
X.sub.96 K, L, N, P, Q, R, S, T, V, W, is I, L, or M; X.sub.97 is L
or V; X.sub.98 is L; and X.sub.99 is R. or Y. Subgenus 8A.6:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 492), wherein X.sub.92 is F, L, or S; X.sub.93 is P;
X.sub.94 is A or S; X.sub.95 is G; X.sub.96 is I, L, or M; X.sub.97
is L or V; X.sub.98 is L; and X.sub.99 is R. Subgenus 8A.7:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 493), wherein X.sub.92 is F, G, L, M, P, S, V, or W;
X.sub.93 is P; X.sub.94 is A, N, Q, or S; X.sub.95 is A, D, G, H,
M, N, P, or S; X.sub.96 is F, I, L, M, or V; X.sub.97 is A, I, L,
M, S, or V; X.sub.98 is A, G, I, L, M, N, P, Q, R, S, T, or Y; and
X.sub.99 is A, F, H, I, L, Q, R, T, V, W, or Y. Subgenus 8A.8:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 494), wherein X.sub.92 is L, S, or V; X.sub.93 is P;
X.sub.94 is A, N, Q, or S; X.sub.95 is H, N, P, or S; X.sub.96 is
F, I, L, or M; X.sub.97 is I, L, S, or V; X.sub.98 is A, L, or Q;
and X.sub.99 is L, T, V, or Y. Subgenus 8A.9:
X.sub.92X.sub.93X.sub.94X.sub.95X.sub.96X.sub.97X.sub.98X.sub.99
(SEQ ID NO: 495), wherein X.sub.92 is L; X.sub.93 is P; X.sub.94 is
A, N, Q, or S; X.sub.95 is H; X.sub.96 is I or L; X.sub.97 is V;
X.sub.98 is L; and X.sub.99 is L or V.
TABLE-US-00011 TABLE 8I MMP14 Cleavable Core CM Consensus Sequence
9 Core CM Consensus 9 Subgenus of Core CM Consensus 9
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
Subgenus 9.1:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID (SEQ ID NO: 425), wherein NO: 426), wherein X.sub.102 is A,
D, F, G, I, R, or S; X.sub.103 is D, X.sub.102 is A, D, F, G, H, I,
L, M, P, E, L, M, P, R, S, T, V, or Y; X.sub.104 is A, H, P, or S;
X.sub.105 is R, S, T, or V; A, D, E, H, L, M, N, R, T, or V;
X.sub.106 is A, G, or R; X.sub.107 is X.sub.103 is A, D, E, L, M,
P, Q, R, S, F, L, M, S, V, or W; X.sub.108 is A, E, H, L, M, R, S,
or V; and T, V, or Y; X.sub.109 is A, G, L, P, R, S, or V.
X.sub.104 is A, G, H, L, N, P, R, S, T, Subgenus 9.2:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID or V; NO: 427), wherein X.sub.102 is F, G, I, R, or S;
X.sub.103 is L, P, R, X.sub.105 is A, D, E, H, L, M, N, P, Q, or V;
X.sub.104 is A or H; X.sub.105 is A, D, or R; X.sub.106 is A or G;
R, S, T, or V; X.sub.107 is L or V; X.sub.108 is H, L, M, R, S, or
V; and X.sub.109 is A, X.sub.106 is A, G, R, S, or T; L, S, or V.
X.sub.107 is C, F, L, M, S, V, W, or Y; Subgenus 9.3:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID X.sub.108 is A, E, F, G, H, I, L, M, N, NO: 428), wherein
X.sub.102 is G, R or S; X.sub.103 is R or V; X.sub.104 is Q, R, S,
V, W, or Y; and A or H; X.sub.105 is A, D, or R; X.sub.106 is A or
G; X.sub.107 is L or V; X.sub.109 is A, E, G, L, P, R, S, or V.
X.sub.108 is H or R; and X.sub.109 is A, L, S, or V. Subgenus 9.4:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID NO: 429), wherein X.sub.102 is R; X.sub.103 is R; X.sub.104
is A or H; X.sub.105 is A or D; X.sub.106 is G; X.sub.107 is L or
V; X.sub.108 is R; and X.sub.109 is A, S, or V. Subgenus 9.5:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID NO: 430), wherein X.sub.102 is D, F, G, I, L, R, S, or T;
X.sub.103 is E, L, M, R, S, T, or V; X.sub.104 is H or N; X.sub.105
is A, D, L, M, R, or T; X.sub.106 is A, G, R, or T; X.sub.107 is C,
L, M, S, V, or W; X.sub.108 is A, E, F, G, L, R, S, or W; and
X.sub.109 is A, G, L, P, R, S, or V. Subgenus 9.6:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID NO: 431), wherein X.sub.102 is F, I, R, or S; X.sub.103 is
E, L, R, or V; X.sub.104 is H; X.sub.105 is D, M, R, or T;
X.sub.106 is A or G; X.sub.107 is L, M, S, or V; X.sub.108 is E, R,
or S; and X.sub.109 is A, P, S, or V. Subgenus 9.7:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID NO: 432), wherein X.sub.102 is I or R; X.sub.103 is E, R,
or V; X.sub.104 is H; X.sub.105 is D, M, R, or T; X.sub.106 is A or
G; X.sub.107 is L or V; X.sub.108 is R or S; and X.sub.109 is A, P,
S, or V. Subgenus 9.8:
X.sub.102X.sub.103X.sub.104X.sub.105X.sub.106X.sub.107X.sub.108X.sub.109
(SEQ ID NO: 433), wherein X.sub.102 is I or R; X.sub.103 is R;
X.sub.104 is H; X.sub.105 is D; X.sub.106 is A or G; X.sub.107 is L
or V; X.sub.108 is R or S; and X.sub.109 is A or S.
TABLE-US-00012 TABLE 8J MMP14 Cleavable Core CM Consensus Sequence
10 Core CM Consensus 10 Subgenus of Core CM Consensus 10
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
Subgenus 10.1:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ (SEQ ID NO: 436), wherein ID NO: 437), wherein X.sub.112 is A,
I, P, S, T, V, or Y; X.sub.113 is X.sub.112 is A, D, G, H, I, L, N,
P, R, A, D, G, L, M, Q, R, S, V, or Y; X.sub.114 is A, H, K, L, N,
S, S, T, V, W, or Y; or T; X.sub.115 is G, H, I, L, S, or V;
X.sub.116 is I, L, or V; X.sub.117 is X.sub.113 is A, D, G, H, L,
M, N, P, A, F, G, K, R, S, or W; X.sub.118 is D, H, L, M, N, Q, R,
or V; Q, R, S, V, or Y; and X.sub.119 is A, I, L, or V. X.sub.114
is A, H, K, L, N, P, Q, R, S, Subgenus 10.2:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ T, or V; ID NO: 438), wherein X.sub.112 is A, I, T, or V;
X.sub.113 is A, L, M, X.sub.115 is A, D, F, G, H, I, L, P, R, Q, R,
V, or Y; X.sub.114 is A, N, S, or T; X.sub.115 is G, L, S, or V; S,
V, or Y; X.sub.116 is L or V; X.sub.117 is A, F, G, K, or S;
X.sub.118 is M, N, Q, R, X.sub.116 is C, F, I, L, P, V, or Y; or V;
and X.sub.119 is I, L, or V. X.sub.117 is A, D, E, F, G, I, K, M,
N, Subgenus 10.3:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ R, S, T, V, or W; ID NO: 439), wherein X.sub.112 is A, I, T,
or V; X.sub.113 is M, Q, or X.sub.118 is A, D, E, F, H, K, L, M, N,
Y; X.sub.114 is A, N, or S; X.sub.115 is G, L, S, or V; X.sub.116
is L or V; Q, R, V, or Y; and X.sub.117 is A, F, G, or S; X.sub.118
is M, N, Q, or R; and X.sub.119 is I, L, X.sub.119 is A, F, I, L,
M, or V. or V. Subgenus 10.4:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 440), wherein X.sub.112 is A, I, or V; X.sub.113 is Y;
X.sub.114 is N or S; X.sub.115 is G, L, or V; X.sub.116 is L;
X.sub.117 is A, G, or S; X.sub.118 is M, Q, or R; and X.sub.119 is
L or V. Subgenus 10.5:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 441), wherein X.sub.112 is A, I, or V; X.sub.113 is Y;
X.sub.114 is N or S; X.sub.115 is G, L, or V; X.sub.116 is L;
X.sub.117 is G or S; X.sub.118 is M or R; and X.sub.119 is L or V.
Subgenus 10.6:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 442), wherein X.sub.112 is A, I, or V; X.sub.113 is Y;
X.sub.114 is N or S; X.sub.115 is G, L, or V; X.sub.116 is L;
X.sub.117 is S, X.sub.118 is M or R; and X.sub.119 is L or V.
Subgenus 10.7:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 443), wherein X.sub.112 is A; X.sub.113 is Y; X.sub.114
is N or S; X.sub.115 is G or L; X.sub.116 is L; X.sub.117 is S;
X.sub.118 is R; and X.sub.119 is L or V. Subgenus 10.8:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 444), wherein X.sub.112 is A, D, G, I, L, N, P, S, T,
V, W, or Y; X.sub.113 is A, D, G, L, M, Q, S, or V; X.sub.114 is H,
K, N, P, Q, R, S, or T; X.sub.115 is H, I, L, R, or V; X.sub.116 is
I, L, P, or V; X.sub.117 is A, D, E, G, I, K, M, N, S, or T;
X.sub.118 is D, F, L, M, Q, R, or V; and X.sub.119 is A, F, I, L,
or V. Subgenus 10.9:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 445), wherein X.sub.112 is A, I, T, or V; X.sub.113 is
A, D, G, L, M, Q, S, or V; X.sub.114 is H, K, N, S, or T; X.sub.115
is H, I, L, or V; X.sub.116 is L; X.sub.117 is A, G, K, or S;
X.sub.118 is L, M, Q, R, or V; and X.sub.119 is A, L, or V.
Subgenus 10.10:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 446), wherein X.sub.112 is A or I; X.sub.113 is A, L,
or Q; X.sub.114 is N, S, or T; X.sub.115 is L or V; X.sub.116 is L;
X.sub.117 is A, G, K, or S; X.sub.118 is M, R, or V; and X.sub.119
is L or V. Subgenus 10.11:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 447), wherein X.sub.112 is A or I; X.sub.113 is A, L,
or Q; X.sub.114 is N or S; X.sub.115 is L or V; X.sub.116 is L;
X.sub.117 is A or S; X.sub.118 is M or R; and X.sub.119 is L or V.
Subgenus 10.12:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 448), wherein X.sub.112 is I; X.sub.113 is A, L, or Q;
X.sub.114 is N; X.sub.115 is L or V; X.sub.116 is L; X.sub.117 is A
or S; X.sub.118 is M or R; and X.sub.119 is L or V. Subgenus 10.13:
X.sub.112X.sub.113X.sub.114X.sub.115X.sub.116X.sub.117X.sub.118X.sub.119
(SEQ ID NO: 449), wherein X.sub.112 is I; X.sub.113 is A, L, or Q;
X.sub.114 is N; X.sub.115 is L or V; X.sub.116 is L; X.sub.117 is
S, X.sub.118 is M; and X.sub.119 is L or V.
TABLE-US-00013 TABLE 8K MMP14 Cleavable Core CM Consensus Sequence
11 Core CM Consensus 11 Subgenus of Core CM Consensus 11
X.sub.122X.sub.123X.sub.124X.sub.125X.sub.126X.sub.127X.sub.128X.sub.129
Subgenus 11.1:
X.sub.122X.sub.123X.sub.124X.sub.125X.sub.126X.sub.127X.sub.128X.sub.129
(SEQ ID (SEQ ID NO: 453), wherein NO: 454), wherein X.sub.122 is A,
G, P, R, or S; X.sub.123 is A, R, or X.sub.122 is A, G, H, L, P, R,
S, or V; S; X.sub.124 is G, P, S, or T; X.sub.125 is L or V;
X.sub.126 is W; X.sub.127 is L, X.sub.123 is A, G, R, S, T or V; S,
V, or Y; X.sub.128 is D, E, P, or T; and X.sub.129 is P, Q or V.
X.sub.124 is A, G, P, R, S, or T; Subgenus 11.2:
X.sub.122X.sub.123X.sub.124X.sub.125X.sub.126X.sub.127X.sub.128X.sub.129
(SEQ ID X.sub.125 is H, I, L, P, R, or V; NO: 455), wherein
X.sub.122 is G, P, R, or S; X.sub.123 is A or R; X.sub.124
X.sub.126 is L or W; is G, P, or S; X.sub.125 is L or V; X.sub.126
is W; X.sub.127 is L or Y; X.sub.128 X.sub.127 is F, H, L, M, Q, S,
V, or Y; is E or T; and X.sub.129 is Q. X.sub.128 is A, D, E, I, K,
P, R, S, T, or Subgenus 11.3:
X.sub.122X.sub.123X.sub.124X.sub.125X.sub.126X.sub.127X.sub.128X.sub.129
(SEQ ID V; and NO: 456), wherein X.sub.122 is P; X.sub.123 is A;
X.sub.124 is P or S; X.sub.125 X.sub.129 is A, E, F, G, H, I, L, N,
P, Q, is L or V; X.sub.126 is W; X.sub.127 is Y; X.sub.128 is T;
and X.sub.129 is Q. R, or V.
TABLE-US-00014 TABLE 8L MMP14 Cleavable Core CM Consensus Sequence
12 Core CM Consensus 12 Subgenus of Core CM Consensus 12
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 Subgenus
12.1: X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ
ID NO: 459), wherein (SEQ ID NO: 458), X.sub.2 is A, G, L, P, or S;
X.sub.3 is A, E, G, H, L, P, Q, S, T, or V; X.sub.4 is G, wherein
N, R, or S; X.sub.5 is L, P, or S; X.sub.6 is I or L; X.sub.7 is A,
G, N, Q, R, or S; X.sub.2 is A, D, E, F, G, H, X.sub.8 is D, F, G,
I, L, M, P, S, or V; and X.sub.9 is F, G, L, P, Q, R, or S. I, L,
M, N, P, Q, R, S, Subgenus 12.2:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 460), wherein T, V, or Y; X.sub.2 is A, P, or S; X.sub.3 is L,
S or V; X.sub.4 is G, N, R, or S; X.sub.5 is L, P, or S; X.sub.3 is
A, E, F, G, H, I, X.sub.6 is L; X.sub.7 is A, G, R, or S; X.sub.8
is L, P, or V; and X.sub.9 is F, L, P, or S. K, L, M, P, Q, R, S,
Subgenus 12.3:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 461), wherein T, V, W, or Y; X.sub.2 is A, P, or S; X.sub.3 is
L, S, or V; X.sub.4 is G, N, R, or S; X.sub.5 is L, P, or X.sub.4
is A, E, G, H, K, N, S; X.sub.6 is L; X.sub.7 is A, G, R, or S;
X.sub.8 is L or P; and X.sub.9 is F, P, or S. P, R, S, T, V, or Y;
Subgenus 12.4:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 462), wherein X.sub.5 is A, G, H, I, L, N, X.sub.2 is A, P, or
S; X.sub.3 is L or V; X.sub.4 is G, N, or S; X.sub.5 is L or S;
X.sub.6 is L; P, R, S, T, or V; X.sub.7 is A, G, R, or S; X.sub.8
is L or P; and X.sub.9 is P or S. X.sub.6 is I, L, M, Q, T, V, W,
or Subgenus 12.5:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 463), wherein Y; X.sub.2 is A or S; X.sub.3 is L; X.sub.4 is G,
N, or S; X.sub.5 is L or S; X.sub.6 is L; X.sub.7 is R X.sub.7 is
A, D, G, H, K, L, or S; X.sub.8 is L; and X.sub.9 is P. N, P, Q, R,
S, T, or V; Subgenus 12.6:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 464), wherein X.sub.8 is A, D, E, F, G, I, X.sub.2 is A, E, G,
H, I, L, M, P, or S; X.sub.3 is A, E, G, H, I, K, L, P, Q, R, K, L,
M, P, Q, R, S, S, T, V, W, or Y; X.sub.4 is A, G, N, R, S, T, or V;
X.sub.5 is A, G, H, L, N, V, W, or Y; and P, R, S, T, or V; X.sub.6
is I, L, M, or Q; X.sub.7 is A, D, G, K, L, N, Q, R, S, X.sub.9 is
A, F, G, I, L, M, or V; X.sub.8 is A, D, E, F, G, I, K, L, M, P, R,
V, W, or Y; and X.sub.9 is A, N, P, Q, R, S, T, V or Y. F, G, M, P,
Q, R, S, V, or Y. Subgenus 12.7:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 465), wherein X.sub.2 is A, P, or S; X.sub.3 is A, H, Q, S, or
V; X.sub.4 is G, N, or S; X.sub.5 is L, P, or S; X.sub.6 is L;
X.sub.7 is A, D, G, R, or S; X.sub.8 is F, I, L, M, or P; and
X.sub.9 is F, P, Q, or R. Subgenus 12.8:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 466), wherein X.sub.2 is A, P, or S; X.sub.3 is H, S, or V;
X.sub.4 is G, N, or S; X.sub.5 is L, P, or S; X.sub.6 is L; X.sub.7
is A, G, R, or S; X.sub.8 is F, I, M, or P; and X.sub.9 is P or R.
Subgenus 12.9:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 467), wherein X.sub.2 is A, P, or S; X.sub.3 is S or V; X.sub.4
is G, N, or S; X.sub.5 is L; X.sub.6 is L; X.sub.7 is A, G or R;
X.sub.8 is F, I, or P; and X.sub.9 is P. Subgenus 12.10:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 468), wherein X.sub.2 is A, P, or S; X.sub.3 is S or V; X.sub.4
is G, N, or S; X.sub.5 is L; X.sub.6 is L; X.sub.7 is A or R;
X.sub.8 is F or P; and X.sub.9 is P. Subgenus 12.11:
X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9 (SEQ ID
NO: 469), wherein X.sub.2 is A or P; X.sub.3 is S; X.sub.4 is G or
N; X.sub.5 is L; X.sub.6 is L; X.sub.7 is R; X.sub.8 is F; and
X.sub.9 is P.
TABLE-US-00015 TABLE 8M MMP14 Cleavable Core CM Consensus Sequence
13 Core CM Consensus 13 Subgenus of Core CM Consensus 13
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
Subgenus 13.1:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 475), (SEQ ID NO: 473), wherein wherein X.sub.12 is F,
I, L, M, S, or V; X.sub.13 is A, E, H, K, L, M, N, X.sub.12 is F,
I, L, M, R, S, T, or Q, S, T, V, or Y; X.sub.14 is A, F, H, L, M,
Q, S, T, or V; X.sub.15 is A, V; G, or P; X.sub.16 is A, F, G, H,
I, L, M, N, R, S, V, or Y; X.sub.17 is A, E, X.sub.13 is A, E, G,
H, I, K, L, G, H, L, M, P, Q, R, S, T, or V; X.sub.18 is A, D, E,
F, G, H, L, M, M, N, P, Q, R, S, T, V, W, or Y; N, R, S, V, or Y;
and X.sub.19 is A, F, G, I, L, M, P, Q, R, S, W, or Y. X.sub.14 is
A, D, E, F, G, H, I, K, Subgenus 13.2:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 476), L, M, N, P, Q, R, S, T, V, wherein X.sub.12 is L,
M, or V; X.sub.13 is A, H, L, N, Q, S, or V; X.sub.14 is or Y; A,
L, M, Q, S, T, or V; X.sub.15 is P; X.sub.16 is A, F, G, I, L, R,
S, V, or X.sub.15 is A, E, G, N, P, Q, S, T, Y; X.sub.17 is H, L,
M, Q, or S; X.sub.18 is A, D, G, H, R, or S; and X.sub.19 is V, or
W; A, F, G, L, R, or S. X.sub.16 is A, F, G, H, I, K, L, Subgenus
13.3:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 477), M, N, P, Q, R, S, T, V, or wherein X.sub.12 is L,
M, or V; X.sub.13 is A or L; X.sub.14 is A, L, or S; X.sub.15 Y; is
P; X.sub.16 is L or V; X.sub.17 is H, L, or Q; X.sub.18 is G or S;
and X.sub.19 is X.sub.17 is A, D, E, F, G, H, I, L, G, R, or S. M,
N, P, Q, R, S, T, V, or Subgenus 13.4:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 478), Y; wherein X.sub.12 is L or V; X.sub.13 is A or
L; X.sub.14 is L or S; X.sub.15 is P; X.sub.16 X.sub.18 is A, C, D,
E, F, G, H, I, is L or V; X.sub.17 is H or L; X.sub.18 is G or S;
and X.sub.19 is R or S. L, M, N, P, Q, R, S, T, V, or Y; and
Subgenus 13.5:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 479), X.sub.19 is A, D, F, G, H, I, L, wherein X.sub.12
is L or V; X.sub.13 is A or L; X.sub.14 is L or S; X.sub.15 is P;
X.sub.16 M, N, P, Q, R, S, T, V, W, or Y. is L; X.sub.17 is H or L;
X.sub.18 is G; and X.sub.19 is S. Subgenus 13.6:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 480), wherein X.sub.12 is F, I, L, M, S, T, or V;
X.sub.13 is A, E, G, H, L, M, S, V, W, or Y; X.sub.14 is A, D, E,
G, K, L, M, N, Q, R, S, T, or V; X.sub.15 is E, G, N, P, S, T, or
V; X.sub.16 is A, F, G, L, N, P, Q, R, S, V, or Y; X.sub.17 is A,
E, H, P, Q, or R; X.sub.18 is D, E, G, N, R, S, or T; and X.sub.19
is A, D, G, Q, S, T, or V. Subgenus 13.:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 481), wherein X.sub.12 is L, M, or V; X.sub.13 is A or
L; X.sub.14 is A, L, Q, or S; X.sub.15 is G, P, or T; X.sub.16 is
A, S, or Y; X.sub.17 is H or P; X.sub.18 is D or G; and X.sub.19 is
A, G or S. Subgenus 13.7:
X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19
(SEQ ID NO: 482), wherein X.sub.12 is L or M; X.sub.13 is A or L;
X.sub.14 is L; X.sub.15 is G or P; X.sub.16 is A or S; X.sub.17 is
H; X.sub.18 is G; and X.sub.19 is A or G.
[0028] In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 352, 371, 394,
410, 425, 436, 453, 458, 473, 485, and 486. In some embodiments,
the CM comprises an amino acid sequence selected from the group
consisting of SEQ ID NO: 353-363, 372-375, 376-378, 395-401,
411-419, 426-433, 437-449, 454-456, 459-469, 475-482, and 487-495.
In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NO: 353-363. In some
embodiments, the CM comprises an amino acid sequence selected from
the group consisting of 372-375. In some embodiments, the CM
comprises an amino acid sequence selected from the group consisting
of 376-378. In some embodiments, the CM comprises an amino acid
sequence selected from the group consisting of 395-401. In some
embodiments, the CM comprises an amino acid sequence selected from
the group consisting of 411-419. In some embodiments, the CM
comprises an amino acid sequence selected from the group consisting
of 426-433. In some embodiments, the CM comprises an amino acid
sequence selected from the group consisting of 437-449. In some
embodiments, the CM comprises an amino acid sequence selected from
the group consisting of 454-456. In some embodiments, the CM
comprises an amino acid sequence selected from the group consisting
of 459-469. In some embodiments, the CM comprises an amino acid
sequence selected from the group consisting of 475-482. In some
embodiments, the CM comprises an amino acid sequence selected from
the group consisting of 487-495.
[0029] In some embodiments, the CM comprises an amino sequence
selected from the group consisting of SEQ ID NOs: 317, 324, 329 and
340. In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 318-323, 325-327,
330-335, and 341-347. In some embodiments, the CM comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 318-323. In some embodiments, the CM comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 325-327.
In some embodiments, the CM comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 330-335. In some
embodiments, the CM comprises an amino acid sequence selected from
the group consisting of SEQ ID NOs: 341-347.
[0030] In some embodiments, the CM comprises a core CM consensus 1
sequence comprising the amino acid sequence RPSPMWAY (SEQ ID NO:
21).
[0031] In some embodiments, the CM comprises a core CM consensus 2
sequence comprising the amino acid sequence WDHPISLL (SEQ ID NO:
328). In some embodiments, the CM comprises a core CM consensus 2
sequence comprising the amino acid sequence WATPRPMR (SEQ ID NO:
22).
[0032] In some embodiments, the CM comprises a core CM consensus 3
sequence comprising the amino acid sequence LTFPTYIF (SEQ ID NO:
336). In some embodiments, the CM comprises a core CM consensus 3
sequence comprising the amino acid sequence MTFPTYIF (SEQ ID NO:
337). In some embodiments, the CM comprises a core CM consensus 3
sequence comprising the amino acid sequence LTFPTYWF (SEQ ID NO:
338). In some embodiments, the CM comprises a core CM consensus 3
sequence comprising the amino acid sequence MTFPTYWF (SEQ ID NO:
339). In some embodiments, the CM comprises a core CM consensus 3
sequence comprising the amino acid sequence STFPFGMF (SEQ ID NO:
17).
[0033] In some embodiments, the CM comprises a core CM consensus 4
sequence comprising the amino acid sequence DWLYWMGI (SEQ ID NO:
348). In some embodiments, the CM comprises a core CM consensus 4
sequence comprising the amino acid sequence DWLYWPGI (SEQ ID NO:
19). In some embodiments, the CM comprises a core CM consensus 4
sequence comprising the amino acid sequence DWLYWMSI (SEQ ID NO:
349). In some embodiments, the CM comprises a core CM consensus 4
sequence comprising the amino acid sequence DWLYWPSI (SEQ ID NO:
350). In some embodiments, the CM comprises a core CM consensus 4
sequence comprising the amino acid sequence HWHLGPPT (SEQ ID NO:
351).
[0034] In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence ISSGLLSS (SEQ ID NO:
14). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence SVSGLLSH (SEQ ID NO:
364). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence SVSGLLSS (SEQ ID NO:
365). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence SVSGLRSH (SEQ ID NO:
366). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence SVSGLRSS (SEQ ID NO:
367). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence TLSGLRSP (SEQ ID NO:
368). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence TSSGLRSP (SEQ ID NO:
369). In some embodiments, the CM comprises a core CM consensus 5
sequence comprising the amino acid sequence TVSGLRSP (SEQ ID NO:
370).
[0035] In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence AFQALRM (SEQ ID NO:
379). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence AHQALRM (SEQ ID NO:
380). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence ANQALRM (SEQ ID NO:
381). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence ANQALRMA (SEQ ID NO:
382). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLEALRAL (SEQ ID NO:
383). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLNALRAL (SEQ ID NO:
384). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLQALRAL (SEQ ID NO:
385). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLSALRAL (SEQ ID NO:
386). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLESLRAL (SEQ ID NO:
387). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLNSLRAL (SEQ ID NO:
388). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLQSLRAL (SEQ ID NO:
389). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence LLSSLRAL (SEQ ID NO:
390). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence QFQALRM (SEQ ID NO:
391). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence QHQALRM (SEQ ID NO:
392). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence QNQALRM (SEQ ID NO:
393). In some embodiments, the CM comprises a core CM consensus 6
sequence comprising the amino acid sequence QNQALRMA (SEQ ID NO:
15).
[0036] In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LKAAPRWA (SEQ ID NO:
24). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LKAAPVWA (SEQ ID NO:
403). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LKAAPRWF (SEQ ID NO:
404). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LKAAPVWF (SEQ ID NO:
405). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LYAAPRWA (SEQ ID NO:
406). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LYAAPVWA (SEQ ID NO:
407). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LYAAPRWF (SEQ ID NO:
408). In some embodiments, the CM comprises a core CM consensus 7
sequence comprising the amino acid sequence LYAAPVWF (SEQ ID NO:
409).
[0037] In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence GPSHLVLT (SEQ ID NO:
25). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPAGLLL (SEQ ID NO:
402). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPAGLLLR (SEQ ID NO:
420). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPAHLVLL (SEQ ID NO:
421). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPSHLVLL (SEQ ID NO:
422). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPAHLVLV (SEQ ID NO:
423). In some embodiments, the CM comprises a core CM consensus 8
sequence comprising the amino acid sequence LPSHLVLV (SEQ ID NO:
424).
[0038] In some embodiments, the CM comprises a core CM consensus 9
sequence comprising the amino acid sequence RMHLRSLG (SEQ ID NO:
29). In some embodiments, the CM comprises a core CM consensus 9
sequence comprising the amino acid sequence RRHDGLRA (SEQ ID NO:
434). In some embodiments, the CM comprises a core CM consensus 9
sequence comprising the amino acid sequence RRHDGLRS (SEQ ID NO:
435).
[0039] In some embodiments, the CM comprises a core CM consensus 10
sequence comprising the amino acid sequence AQNLLGMV (SEQ ID NO:
16). In some embodiments, the CM comprises a core CM consensus 10
sequence comprising the amino acid sequence IANLLSMV (SEQ ID NO:
450). In some embodiments, the CM comprises a core CM consensus 10
sequence comprising the amino acid sequence ILNLLSMV (SEQ ID NO:
451). In some embodiments, the CM comprises a core CM consensus 10
sequence comprising the amino acid sequence IQNLLSMV (SEQ ID NO:
452).
[0040] In some embodiments, the CM comprises a core CM consensus 11
sequence comprising the amino acid sequence PAGLWLDP (SEQ ID NO:
33). In some embodiments, the CM comprises a core CM consensus 11
sequence comprising the amino acid sequence PASLWYTQ (SEQ ID NO:
457).
[0041] In some embodiments, the CM comprises a core CM consensus 12
sequence comprising the amino acid sequence ALGLLRLP (SEQ ID NO:
470). In some embodiments, the CM comprises a core CM consensus 12
sequence comprising the amino acid sequence ALGLLSLP (SEQ ID NO:
471). In some embodiments, the CM comprises a core CM consensus 12
sequence comprising the amino acid sequence ASGLLRFP (SEQ ID NO:
472). In some embodiments, the CM comprises a core CM consensus 12
sequence comprising the amino acid sequence AVGLLAPP (SEQ ID NO:
31).
[0042] In some embodiments, the CM comprises a core CM consensus 13
sequence comprising the amino acid sequence LAAPLGLL (SEQ ID NO:
30). In some embodiments, the CM comprises a core CM consensus 13
sequence comprising the amino acid sequence LLAPSHRA (SEQ ID NO:
32).
[0043] In some embodiments, the CM comprises a core CM consensus 13
sequence comprising the amino acid sequence LLLPAHGG (SEQ ID NO:
474). In some embodiments, the CM comprises a core CM consensus 13
sequence comprising the amino acid sequence LLLPLLGS (SEQ ID NO:
483).
[0044] In some embodiments, the CM is a substrate for at least two
proteases. In some embodiments, at least one protease is an MMP and
at least one protease is selected from the group consisting of
those shown in Table 7.
TABLE-US-00016 TABLE 7 Exemplary Proteases and/or Enzymes 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
[0045] In some embodiments, the antibody includes at least a first
CM and a second CM. In some embodiments, the first CM and the
second CM are each polypeptides of no more than 15 amino acids
long. In some embodiments, the first CM and the second CM in the
antibody in the uncleaved state have the structural arrangement
from N-terminus to C-terminus as follows: Agent-CM1-CM2-AB,
AB-CM2-CM1-Agent, Agent-CM2-CM1-AB, or AB-CM1-CM2-Agent. In some
embodiments, the activatable antibody includes a linking peptide
between the agent and CM1. In some embodiments, the activatable
antibody includes a linking peptide between CM1 and CM2. In some
embodiments, the activatable antibody includes a linking peptide
between CM2 and AB. In some embodiments, the activatable antibody
includes a linking peptide between the agent and CM1 and a linking
peptide between CM2 and AB. In some embodiments, the activatable
antibody includes a linking peptide between agent and CM1 and a
linking peptide between CM1 and CM2. In some embodiments, the
activatable antibody includes a linking peptide between CM1 and CM2
and a linking peptide between CM2 and AB. In some embodiments, the
activatable antibody includes a linking peptide between agent and
CM1, a linking peptide between CM1 and CM2, and a linking peptide
between CM2 and AB.
[0046] In some embodiments, the activatable antibody includes at
least a first CM that includes a substrate for at least one matrix
metalloprotease (MMP) and a second CM that includes a substrate
sequence. Exemplary substrates for the second CM (CM2) include but
are not limited to substrates cleavable by one or more of the
following enzymes or proteases listed in Table 7.
[0047] In some embodiments, the CM2 is selected for use with a
specific protease. In some embodiments, the CM2 is a substrate for
at least one protease selected from the group consisting of a
matrix metalloprotease (MMP), a neutrophil elastase, u-type
plasminogen activator (uPA, also referred to as urokinase),
legumain, matriptase (also referred to herein as MT-SP1 or MTSP1),
thrombin, a cysteine protease such as a cathepsin, ADAM17, BMP-1,
HtrA1, and a TMPRSS such as TMPRSS3 or TMPRSS4.
[0048] In some embodiments, the CM2 is a substrate for a neutrophil
elastase. In some embodiments, the CM2 is a substrate for uPA. In
some embodiments, the CM2 is a substrate for legumain. In some
embodiments, the CM2 is a substrate for matriptase. In some
embodiments, the CM2 is a substrate for thrombin. In some
embodiments, the CM2 is a substrate for a cysteine protease. In
some embodiments, the CM2 is a substrate for a cathepsin. In some
embodiments, the CM2 is a substrate for ADAM17. In some
embodiments, the CM2 is a substrate for BMP-1. In some embodiments,
the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is a
substrate for a TMPRSS. In some embodiments, the CM2 is a substrate
for TMPRSS3. In some embodiments, the CM2 is a substrate for
TMPRSS4.
[0049] For example, suitable CM2 are cleaved by at least one
protease and include the sequence TGRGPSWV (SEQ ID NO: 34);
SARGPSRW (SEQ ID NO: 35); TARGPSFK (SEQ ID NO: 36); LSGRSDNH (SEQ
ID NO: 37); GGWHTGRN (SEQ ID NO: 38); HTGRSGAL (SEQ ID NO: 39);
PLTGRSGG (SEQ ID NO: 40); AARGPAIH (SEQ ID NO: 41); RGPAFNPM (SEQ
ID NO: 42); SSRGPAYL (SEQ ID NO: 43); RGPATPIM (SEQ ID NO: 44);
RGPA (SEQ ID NO: 45); GGQPSGMWGW (SEQ ID NO: 46); FPRPLGITGL (SEQ
ID NO: 47); VHMPLGFLGP (SEQ ID NO: 48); SPLTGRSG (SEQ ID NO: 49);
SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 50); SGGPLGVR (SEQ
ID NO: 51); PLGL (SEQ ID NO: 52); GPRSFGL (SEQ ID NO: 315) and/or
GPRSFG (SEQ ID NO: 316).
[0050] In some embodiments, the CM2 comprises the amino acid
sequence TGRGPSWV (SEQ ID NO: 34). In some embodiments, the CM2
comprises the amino acid sequence SARGPSRW (SEQ ID NO: 35). In some
embodiments, the CM2 comprises the amino acid sequence TARGPSFK
(SEQ ID NO: 36). In some embodiments, the CM2 comprises the amino
acid sequence LSGRSDNH (SEQ ID NO: 37). In some embodiments, the
CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 38). In
some embodiments, the CM2 comprises the amino acid sequence
HTGRSGAL (SEQ ID NO: 39). In some embodiments, the CM2 comprises
the amino acid sequence PLTGRSGG (SEQ ID NO: 40). In some
embodiments, the CM2 comprises the amino acid sequence AARGPAIH
(SEQ ID NO: 41). In some embodiments, the CM2 comprises the amino
acid sequence RGPAFNPM (SEQ ID NO: 42). In some embodiments, the
CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 43). In
some embodiments, the CM2 comprises the amino acid sequence
RGPATPIM (SEQ ID NO: 44). In some embodiments, the CM2 comprises
the amino acid sequence RGPA (SEQ ID NO: 45). In some embodiments,
the CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO:
46). In some embodiments, the CM2 comprises the amino acid sequence
FPRPLGITGL (SEQ ID NO: 47). In some embodiments, the CM2 comprises
the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). In some
embodiments, the CM2 comprises the amino acid sequence SPLTGRSG
(SEQ ID NO: 49). In some embodiments, the CM2 comprises the amino
acid sequence LAPLGLQRR (SEQ ID NO: 50). In some embodiments, the
CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). In
some embodiments, the CM2 comprises the amino acid sequence PLGL
(SEQ ID NO: 52). In some embodiments, the CM2 comprises the amino
acid sequence GPRSFGL (SEQ ID NO: 315). In some embodiments, the
CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 316).
[0051] In some embodiments, the CM2 is a substrate for at least one
MMP. In some embodiments, the CM2 is a substrate for at least one
MMP listed in the Table 7. In some embodiments, the CM2 is a
substrate for MMP9. In some embodiments, the CM2 is a substrate for
MMP14. In some embodiments, CM1 is substrate for a first MMP, and
CM2 is a substrate for a second MMP, where the first MMP and the
second MMP are different MMPs. In some embodiments, CM1 is a first
substrate sequence for a MMP, and CM2 is a second substrate for the
same MMP, where the CM1 and CM2 have different substrate sequences.
In some embodiments, the CM2 is a substrate for two or more MMPs.
In some embodiments, the CM2 is a substrate for at least MMP9 or
MMP14. In some embodiments, the CM2 is a substrate for two or more
MMPs. In some embodiments, the CM2 is a substrate for at least MMP9
and MMP14. In some embodiments, CM1 and CM2 are both substrates for
MMP9. In some embodiments, CM1 and CM2 are both substrates for
MMP14. In some embodiments, CM1 is a substrate for MMP9 and CM2 is
a substrate for MMP14. In some embodiments, CM1 is a substrate for
MMP14 and CM2 is a substrate for MMP9.
[0052] In some embodiments, at least one of CM1 and/or CM2 is a
substrate for an MMP and includes the sequence ISSGLLSS (SEQ ID NO:
14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF
(SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO:
19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR
(SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO:
24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG
(SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO:
29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA
(SEQ ID NO: 32), PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID
NO: 159).
[0053] In some embodiments, the first cleaving agent and the second
cleaving agent are the same protease, and the first CM and the
second CM are different substrates for the enzyme. In some
embodiments, the first cleaving agent and the second cleaving agent
are different proteases. In some embodiments, the first cleaving
agent and the second cleaving agent are co-localized in the target
tissue. In some embodiments, the first CM and the second CM are
cleaved by at least one cleaving agent in the target tissue.
[0054] In some embodiments, the agent conjugated to the AB is a
therapeutic agent. 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 conjugated to the AB via a cleavable linker. In some
embodiments, the agent is conjugated to the AB via a linker that
includes at least one MMP-cleavable substrate sequence. In some
embodiments, the agent is conjugated to the AB via a noncleavable
linker. In some embodiments, the agent is a microtubule inhibitor.
In some embodiments, the agent is a nucleic acid damaging agent,
such as a DNA alkylator or DNA intercalator, or other DNA damaging
agent. In some embodiments, the agent is an agent selected from the
group listed in Table 3. 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
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is a pyrrolobenzodiazepine.
[0055] In some embodiments, the agent is an anti-inflammatory
agent.
[0056] In some embodiments, the antibody also includes a detectable
moiety. In some embodiments, the detectable moiety is a diagnostic
agent.
[0057] In some embodiments, the conjugated antibody and/or
conjugated activatable antibody includes a detectable label. In
some embodiments, 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, the imaging agent comprises a radioisotope. In some
embodiments, the radioisotope is indium or technetium. In some
embodiments, the contrasting agent comprises iodine, gadolinium or
iron oxide. In some embodiments, the enzyme comprises horseradish
peroxidase, alkaline phosphatase, or .beta.-galactosidase. In some
embodiments, 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, the luminescent label comprises an
N-methylacrydium derivative. In some embodiments, the label
comprises an Alexa Fluor.RTM. label, such as Alex Fluor.RTM. 680 or
Alexa Fluor.RTM. 750. In some embodiments, the ligand-based label
comprises biotin, avidin, streptavidin or one or more haptens.
[0058] In some embodiments, the AB of the antibody naturally
contains one or more disulfide bonds. In some embodiments, the AB
can be engineered to include one or more disulfide bonds.
[0059] In some embodiments, the antibody and/or conjugated antibody
is monospecific. In some embodiments, the antibody and/or
conjugated antibody is multispecific, referred to herein as
multispecific antibodies and/or conjugated multispecific
antibodies. In some embodiments, the multispecific antibody and/or
conjugated multispecific antibody is bispecific or trifunctional.
In some embodiments, the antibody and/or conjugated antibody is
formulated as part of a pro-Bispecific T Cell Engager (pro-BITE)
molecule. In some embodiments, the antibody and/or conjugated
antibody is formulated as part of a pro-Chimeric Antigen Receptor
(pro-CAR) modified T cell or other engineered receptor.
[0060] 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, referred to herein as multispecific
activatable antibodies and/or conjugated multispecific activatable
antibodies. As used herein, terms such as "activatable antibody"
and all grammatical variations thereof, unless otherwise noted, are
intended to encompass, but are not limited to embodiments where the
activatable antibody is a multispecific activatable antibody of the
disclosure. As used herein, terms such as "conjugated activatable
antibody" and all grammatical variations thereof, unless otherwise
noted, are intended to encompass, but are not limited to
embodiments where the conjugated activatable antibody is a
conjugated multispecific activatable antibody of the disclosure. In
some embodiments, the multispecific activatable antibody and/or
conjugated multispecific activatable antibody is 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 (pro-BITE) molecule. In some
embodiments, the activatable antibody and/or conjugated activatable
antibody is formulated as part of a pro-Chimeric Antigen Receptor
(pro-CAR) modified T cell or other engineered receptor.
[0061] In some embodiments, the activatable antibodies, conjugated
activatable antibodies, multispecific activatable antibodies,
and/or conjugated multispecific activatable antibodies described
herein are used in conjunction with one or more additional agents
or a combination of additional agents. Suitable additional agents
include current pharmaceutical and/or surgical therapies for an
intended application, such as, for example, cancer. For example,
the activatable antibodies, conjugated activatable antibodies,
multispecific activatable antibodies, and/or conjugated
multispecific activatable antibodies can be used in conjunction
with an additional chemotherapeutic or anti-neoplastic agent.
[0062] The activatable antibodies described herein in an activated
state bind a given target and include (i) an antibody or an antigen
binding fragment thereof (AB) that specifically binds to the
target; (ii) a masking moiety (MM) that inhibits the binding of the
AB to the target in an uncleaved state; and (c) a cleavable moiety
(CM) coupled to the AB, wherein the CM is a polypeptide that
functions as a substrate for a matrix metalloprotease.
[0063] 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.
[0064] In some embodiments, the activatable antibody comprises a
linking peptide between the MM and the CM.
[0065] In some embodiments, the activatable antibody comprises a
linking peptide between the CM and the AB.
[0066] In some embodiments, the activatable antibody comprises 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-LP 1-MM.
[0067] In some embodiments, each of LP1 and LP2 is a peptide of
about 1 to 20 amino acids in length.
[0068] In some embodiments, the two linking peptides need not be
identical to each other.
[0069] In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least
one.
[0070] In some embodiments, at least one of LP1 or LP2 comprises an
amino acid sequence selected from the group consisting of GGSG (SEQ
ID NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ
ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).
[0071] In some embodiments, LP1 comprises the amino acid sequence
GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10),
GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155),
GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO: 157).
[0072] In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0073] In some embodiments, the AB has an equilibrium dissociation
constant of about 100 nM or less for binding to the target.
[0074] In some embodiments, the activatable antibody includes an
antibody or antigen-binding fragment thereof that specifically
binds a 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, or a single domain light chain antibody. In some
embodiments, such an antibody or immunologically active fragment
thereof that binds the target is a mouse, other rodent, chimeric,
humanized or fully human monoclonal antibody.
[0075] In some embodiments, the activatable antibody is a
multispecific activatable antibody. The multispecific activatable
antibodies provided herein are multispecific antibodies that
recognize two or more different antigens or epitopes and that
include at least one masking moiety (MM) linked to at least one
antigen- or epitope-binding domain of the multispecific antibody
such that coupling of the MM reduces the ability of the antigen- or
epitope-binding domain to bind its target. In some embodiments, the
MM is coupled to the antigen- or epitope-binding domain of the
multispecific antibody via a cleavable moiety (CM) that functions
as a substrate for at least one MMP protease. The activatable
multispecific antibodies provided herein are stable in circulation,
activated at intended sites of therapy and/or diagnosis but not in
normal, i.e., healthy tissue, and, when activated, exhibit binding
to a target that is at least comparable to the corresponding,
unmodified multispecific antibody.
[0076] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VH CDR1 sequence, a
VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of
the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3
sequence is selected from a VH CDR1 that sequence includes at least
the amino acid sequence SYAMS (SEQ ID NO: 498); a VH CD2 sequence
that includes at least the amino acid sequence SIDPEGRQTYYADSVKG
(SEQ ID NO: 499); a VH CDR3 sequence that includes at least the
amino acid sequence DIGGRSAFDY (SEQ ID NO: 500), and combinations
thereof.
[0077] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VL CDR1 sequence, a
VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of
the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3
sequence is selected from a VL CDR1 sequence that includes at least
the amino acid sequence RASQSISSY (SEQ ID NO: 501); a VL CDR2
sequence that includes at least the amino acid sequence AASSLQS
(SEQ ID NO: 502); a VL CDR3 sequence that includes at least the
amino acid sequence QQTVVAPPL (SEQ ID NO: 503), and combinations
thereof.
[0078] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VH CDR1 sequence, a
VH CDR2 sequence, and a VH CDR3 sequence, wherein at least one of
the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3
sequence is selected from a VH CDR1 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence SYAMS (SEQ ID
NO: 498); a VH CD2 sequence that includes a sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence SIDPEGRQTYYADSVKG (SEQ ID NO:
499); a VH CDR3 sequence that includes a sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500), and
combinations thereof.
[0079] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VL CDR1 sequence, a
VL CDR2 sequence, and a VL CDR3 sequence, wherein at least one of
the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3
sequence is selected from a VL CDR1 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence RASQSISSY
(SEQ ID NO: 501); a VL CDR2 sequence that includes a sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more identical to the amino acid sequence AASSLQS (SEQ ID NO: 502);
and a VL CDR3 sequence that includes a sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to the amino acid sequence QQTVVAPPL (SEQ ID NO: 503), and
combinations thereof.
[0080] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VH CDR1 sequence, a
VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2
sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence
includes at least the amino acid sequence SYAMS (SEQ ID NO: 498);
the VH CD2 sequence includes at least the amino acid sequence
SIDPEGRQTYYADSVKG (SEQ ID NO: 499); the VH CDR3 sequence includes
at least the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500); the
VL CDR1 sequence includes at least the amino acid sequence
RASQSISSY (SEQ ID NO: 501); the VL CDR2 sequence includes at least
the amino acid sequence AASSLQS (SEQ ID NO: 502); and the VL CDR3
sequence includes at least the amino acid sequence QQTVVAPPL (SEQ
ID NO: 503).
[0081] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds a Jagged target, e.g., Jagged 1 and/or
Jagged 2, and that contains a combination of a VH CDR1 sequence, a
VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1 sequence, a VL CDR2
sequence, and a VL CDR3 sequence, wherein the VH CDR1 sequence
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
SYAMS (SEQ ID NO: 498); the VH CD2 sequence includes a sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identical to the amino acid sequence SIDPEGRQTYYADSVKG (SEQ
ID NO: 499); the VH CDR3 sequence includes a sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence DIGGRSAFDY (SEQ ID NO: 500);
the VL CDR1 sequence includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence RASQSISSY (SEQ ID NO: 501); the VL CDR2
sequence includes a sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence AASSLQS (SEQ ID NO: 502); and the VL CDR3 sequence
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
QQTVVAPPL (SEQ ID NO: 503).
[0082] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds Epidermal Growth Factor Receptor (EGFR) and
that contains a combination of a VH CDR1 sequence, a VH CDR2
sequence, and a VH CDR3 sequence, wherein at least one of the VH
CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence is
selected from a VH CDR1 sequence that includes at least the amino
acid sequence NYGVH (SEQ ID NO: 504); a VH CD2 sequence that
includes at least the amino acid sequence VIWSGGNTDYNTPFTS (SEQ ID
NO: 505); a VH CDR3 sequence that includes at least the amino acid
sequence ALTYYDYEFAY (SEQ ID NO: 506); and combinations
thereof.
[0083] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds EGFR and that contains a combination of a
VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence,
wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence,
and the VL CDR3 sequence is selected from a VL CDR1 sequence that
includes at least the amino acid sequence RASQSIGTNIH (SEQ ID NO:
507); a VL CDR2 sequence that includes at least the amino acid
sequence KYASESIS (SEQ ID NO: 508); and a VL CDR3 sequence that
includes at least the amino acid sequence QQNNNWPTT (SEQ ID NO:
509), and combinations thereof.
[0084] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds EGFR and that contains a combination of a
VH CDR1 sequence, a VH CDR2 sequence, and a VH CDR3 sequence,
wherein at least one of the VH CDR1 sequence, the VH CDR2 sequence,
and the VH CDR3 sequence is selected from a VH CDR1 sequence that
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
NYGVH (SEQ ID NO: 504); a VH CD2 sequence that includes a sequence
that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
or more identical to the amino acid sequence VIWSGGNTDYNTPFTS (SEQ
ID NO: 505); a VH CDR3 sequence that includes a sequence that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence ALTYYDYEFAY (SEQ ID NO: 506);
and combinations thereof.
[0085] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds EGFR and that contains a combination of a
VL CDR1 sequence, a VL CDR2 sequence, and a VL CDR3 sequence,
wherein at least one of the VL CDR1 sequence, the VL CDR2 sequence,
and the VL CDR3 sequence is selected from a VL CDR1 sequence that
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
RASQSIGTNIH (SEQ ID NO: 507); a VL CDR2 sequence that includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence KYASESIS (SEQ
ID NO: 508); and a VL CDR3 sequence that includes a sequence that
is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
more identical to the amino acid sequence QQNNNWPTT (SEQ ID NO:
509), and combinations thereof.
[0086] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds EGFR and that contains a combination of a
VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the
VH CDR1 sequence includes at least the amino acid sequence NYGVH
(SEQ ID NO: 504); the VH CD2 sequence includes at least the amino
acid sequence VIWSGGNTDYNTPFTS (SEQ ID NO: 505); the VH CDR3
sequence includes at least the amino acid sequence ALTYYDYEFAY (SEQ
ID NO: 506); the VL CDR1 sequence includes at least the amino acid
sequence RASQSIGTNIH (SEQ ID NO: 507); the VL CDR2 sequence
includes at least the amino acid sequence KYASESIS (SEQ ID NO:
508); and the VL CDR3 sequence includes at least the amino acid
sequence QQNNNWPTT (SEQ ID NO: 509).
[0087] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a first antibody or antigen binding fragment thereof (AB1)
that specifically binds EGFR and that contains a combination of a
VH CDR1 sequence, a VH CDR2 sequence, a VH CDR3 sequence, a VL CDR1
sequence, a VL CDR2 sequence, and a VL CDR3 sequence, wherein the
VH CDR1 sequence includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence NYGVH (SEQ ID NO: 504); the VH CD2 sequence
includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence
VIWSGGNTDYNTPFTS (SEQ ID NO: 505); the VH CDR3 sequence includes a
sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or more identical to the amino acid sequence ALTYYDYEFAY
(SEQ ID NO: 506); the VL CDR1 sequence includes a sequence that is
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more
identical to the amino acid sequence RASQSIGTNIH (SEQ ID NO: 507);
the VL CDR2 sequence includes a sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the
amino acid sequence KYASESIS (SEQ ID NO: 508); and the VL CDR3
sequence includes a sequence that is at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid
sequence QQNNNWPTT (SEQ ID NO: 509).
[0088] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a heavy chain amino acid sequence selected from the group
consisting of SEQ ID NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, and 114. In some embodiments, the activatable
antibody and/or conjugated activatable antibody provided herein,
including but not limited to a multispecific activatable antibody
and/or conjugated multispecific activatable antibody of the
disclosure, includes at least a light chain amino acid sequence
selected from the group consisting of SEQ ID NOs: 55, 59, 60, 62,
64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,
99, 101, 103, 105, 107, 109, 111, and 113. In some embodiments, the
activatable antibody and/or conjugated activatable antibody
provided herein, including but not limited to a multispecific
activatable antibody and/or conjugated multispecific activatable
antibody of the disclosure, includes at least a heavy chain amino
acid sequence selected from the group consisting of SEQ ID NOs: 54,
56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90,
92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114 and a
light chain amino acid sequence selected from the group consisting
of SEQ ID NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
and 113.
[0089] In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a heavy chain amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and
114. In some embodiments, the activatable antibody and/or
conjugated activatable antibody provided herein, including but not
limited to a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a light chain amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113.
In some embodiments, the activatable antibody and/or conjugated
activatable antibody provided herein, including but not limited to
a multispecific activatable antibody and/or conjugated
multispecific activatable antibody of the disclosure, includes at
least a heavy chain amino acid sequence that is at least 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 54, 56, 57, 58, 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and
114 and a light chain amino acid sequence that is at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 55, 59, 60, 62, 64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and
113.
[0090] 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.
[0091] 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.
[0092] In some embodiments, the MM does not interfere or compete
with the AB for binding to the target in a cleaved state.
[0093] In some embodiments, the MM is a polypeptide of about 2 to
40 amino acids in length. For example, the MM is a polypeptide of
up to about 40 amino acids in length.
[0094] In some embodiments, the MM polypeptide sequence is
different from that of any natural binding partner of the AB. In
some embodiments, the MM polypeptide sequence is no more than 50%
identical to any natural binding partner of the AB. In some
embodiments, the MM polypeptide sequence is no more than 40%, 30%,
25%, 20%, 15%, or 10% identical to any natural binding partner of
the AB.
[0095] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind its target such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards the target is at least two times greater than the K.sub.d
of the AB when not coupled to the MM towards the target.
[0096] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind its target such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards the target is at least three times greater than the K.sub.d
of the AB when not coupled to the MM towards the target.
[0097] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind its target such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards the target is at least five times greater than the K.sub.d
of the AB when not coupled to the MM towards the target.
[0098] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind its target such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM
towards the target is at least 10 times greater than the K.sub.d of
the AB when not coupled to the MM towards the target.
[0099] In some embodiments, the coupling of the MM to the AB
reduces the ability of the AB to bind its 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.
[0100] In some embodiments, the coupling of the MM to the AB
reduces 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.
[0101] In some embodiments, the coupling of the MM to the AB
reduces 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 100 times greater than the K.sub.d
of the AB when not coupled to the MM towards the target.
[0102] In some embodiments, the coupling of the MM to the AB
reduces 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 1000 times greater than the K.sub.d
of the AB when not coupled to the MM towards the target.
[0103] In some embodiments, the coupling of the MM to the AB
reduces 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 10,000 times greater than the
K.sub.d of the AB when not coupled to the MM towards the
target.
[0104] In some embodiments, the MMP protease is co-localized with
the target in a tissue, and the MMP cleaves the CM in the
activatable antibody when the activatable antibody is exposed to
the MMP.
[0105] In some embodiments, in the presence of the target, the MM
reduces 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.
[0106] 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 reduced to occur with an
equilibrium dissociation constant that is at least twofold greater
than the equilibrium dissociation constant of an unmodified AB
binding to the target, whereas in the cleaved state (i.e., when the
activatable antibody is in the cleaved state), the AB binds the
target.
[0107] 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 reduced to occur with an
equilibrium dissociation constant that is at least five-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to the target, whereas in the cleaved state (i.e., when
the activatable antibody is in the cleaved state), the AB binds the
target.
[0108] 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 reduced to occur with an
equilibrium dissociation constant that is at least ten-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to the target, whereas in the cleaved state (i.e., when the
activatable antibody is in the cleaved state), the AB binds the
target.
[0109] 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 reduced to occur 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, whereas in the cleaved state (i.e., when the
activatable antibody is in the cleaved state), the AB binds the
target.
[0110] 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 reduced to occur 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, whereas in the cleaved state, the AB binds
the target.
[0111] 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 reduced to occur 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, whereas in the cleaved state, the AB binds
the target.
[0112] 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 reduced to occur 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, whereas in the cleaved state, the AB binds
the target.
[0113] 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 reduced to occur 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, whereas in the cleaved state, the AB binds
the target.
[0114] In some embodiments, the CM is a polypeptide of up to 15
amino acids in length.
[0115] 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, MMP11, and MMP19. 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.
[0116] In some embodiments, the CM is a substrate for an MMP and
includes the sequence ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID
NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17);
PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ
ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22);
FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ
ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27);
SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ
ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32),
PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID NO: 159).
[0117] In some embodiments, the CM comprises the amino acid
sequence ISSGLLSS (SEQ ID NO: 14). In some embodiments, the CM
comprises the amino acid sequence QNQALRMA (SEQ ID NO: 15). In some
embodiments, the CM comprises the amino acid sequence AQNLLGMV (SEQ
ID NO: 16). In some embodiments, the CM comprises the amino acid
sequence STFPFGMF (SEQ ID NO: 17). In some embodiments, the CM
comprises the amino acid sequence PVGYTSSL (SEQ ID NO: 18). In some
embodiments, the CM comprises the amino acid sequence DWLYWPGI (SEQ
ID NO: 19). In some embodiments, the CM comprises the amino acid
sequence MIAPVAYR (SEQ ID NO: 20). In some embodiments, the CM
comprises the amino acid sequence RPSPMWAY (SEQ ID NO: 21). In some
embodiments, the CM comprises the amino acid sequence WATPRPMR (SEQ
ID NO: 22). In some embodiments, the CM comprises the amino acid
sequence FRLLDWQW (SEQ ID NO: 23). In some embodiments, the CM
comprises the amino acid sequence LKAAPRWA (SEQ ID NO: 24). In some
embodiments, the CM comprises the amino acid sequence GPSHLVLT (SEQ
ID NO: 25). In some embodiments, the CM comprises the amino acid
sequence LPGGLSPW (SEQ ID NO: 26). In some embodiments, the CM
comprises the amino acid sequence MGLFSEAG (SEQ ID NO: 27). In some
embodiments, the CM comprises the amino acid sequence SPLPLRVP (SEQ
ID NO: 28). In some embodiments, the CM comprises the amino acid
sequence RMHLRSLG (SEQ ID NO: 29). In some embodiments, the CM
comprises the amino acid sequence LAAPLGLL (SEQ ID NO: 30). In some
embodiments, the CM comprises the amino acid sequence AVGLLAPP (SEQ
ID NO: 31). In some embodiments, the CM comprises the amino acid
sequence LLAPSHRA (SEQ ID NO: 32). In some embodiments, the CM
comprises the amino acid sequence PAGLWLDP (SEQ ID NO: 33). In some
embodiments, the CM comprises the amino acid sequence ISSGLSS (SEQ
ID NO: 159).
[0118] In some embodiments, the CM is a substrate for at least two
proteases. In some embodiments, at least one protease is an MMP and
at least one protease is selected from the group consisting of
those shown in Table 7.
[0119] In some embodiments, the activatable antibody includes at
least a first CM and a second CM. In some embodiments, the first CM
and the second CM are each polypeptides of no more than 15 amino
acids long. In some embodiments, the first CM and the second CM in
the activatable antibody have the structural arrangement from
N-terminus to C-terminus as follows in the uncleaved state: MM-CM
1-CM2-AB, AB-CM2-CM 1-MM, MM-CM2-CM1-AB, or AB-CM1-CM2-MM. In some
embodiments, the activatable antibody includes a linking peptide
between MM and CM1. In some embodiments, the activatable antibody
includes a linking peptide between CM1 and CM2. In some
embodiments, the activatable antibody includes a linking peptide
between CM2 and AB. In some embodiments, the activatable antibody
includes a linking peptide between MM and CM1 and a linking peptide
between CM2 and AB. In some embodiments, the activatable antibody
includes a linking peptide between MM and CM1 and a linking peptide
between CM1 and CM2. In some embodiments, the activatable antibody
includes a linking peptide between CM1 and CM2 and a linking
peptide between CM2 and AB. In some embodiments, the activatable
antibody includes a linking peptide between MM and CM1, a linking
peptide between CM1 and CM2, and a linking peptide between CM2 and
AB.
[0120] In some embodiments, the activatable antibody includes at
least a first CM that includes a substrate for at least one matrix
metalloprotease (MMP) and a second CM that includes a substrate
sequence. Exemplary substrates for the second CM (CM2) include but
are not limited to substrates cleavable by one or more of the
following enzymes or proteases listed in Table 7.
[0121] In some embodiments, the CM2 is selected for use with a
specific protease. In some embodiments, the CM2 is a substrate for
at least one protease selected from the group consisting of a
matrix metalloprotease (MMP), a neutrophil elastase, u-type
plasminogen activator (uPA, also referred to as urokinase),
legumain, matriptase (MT-SP1), thrombin, a cysteine protease such
as a cathepsin, ADAM17, BMP-1, HtrA1, and a TMPRSS such as TMPRSS3
or TMPRSS4.
[0122] In some embodiments, the CM2 is a substrate for a neutrophil
elastase. In some embodiments, the CM2 is a substrate for uPA. In
some embodiments, the CM2 is a substrate for legumain. In some
embodiments, the CM2 is a substrate for matriptase. In some
embodiments, the CM2 is a substrate for thrombin. In some
embodiments, the CM2 is a substrate for a cysteine protease. In
some embodiments, the CM2 is a substrate for a cathepsin. In some
embodiments, the CM2 is a substrate for ADAM17. In some
embodiments, the CM2 is a substrate for BMP-1. In some embodiments,
the CM2 is a substrate for HtrA1. In some embodiments, the CM2 is a
substrate for a TMPRSS. In some embodiments, the CM2 is a substrate
for TMPRSS3. In some embodiments, the CM2 is a substrate for
TMPRSS4.
[0123] For example, suitable CM2 are cleaved by at least one
protease and include the sequence TGRGPSWV (SEQ ID NO: 34);
SARGPSRW (SEQ ID NO: 35); TARGPSFK (SEQ ID NO: 36); LSGRSDNH (SEQ
ID NO: 37); GGWHTGRN (SEQ ID NO: 38); HTGRSGAL (SEQ ID NO: 39);
PLTGRSGG (SEQ ID NO: 40); AARGPAIH (SEQ ID NO: 41); RGPAFNPM (SEQ
ID NO: 42); SSRGPAYL (SEQ ID NO: 43); RGPATPIM (SEQ ID NO: 44);
RGPA (SEQ ID NO: 45); GGQPSGMWGW (SEQ ID NO: 46); FPRPLGITGL (SEQ
ID NO: 47); VHMPLGFLGP (SEQ ID NO: 48); SPLTGRSG (SEQ ID NO: 49);
SAGFSLPA (SEQ ID NO: 126); LAPLGLQRR (SEQ ID NO: 50); SGGPLGVR (SEQ
ID NO: 51); PLGL (SEQ ID NO: 52); GPRSFGL (SEQ ID NO: 315) and/or
GPRSFG (SEQ ID NO: 316).
[0124] In some embodiments, the CM2 comprises the amino acid
sequence TGRGPSWV (SEQ ID NO: 34). In some embodiments, the CM2
comprises the amino acid sequence SARGPSRW (SEQ ID NO: 35). In some
embodiments, the CM2 comprises the amino acid sequence TARGPSFK
(SEQ ID NO: 36). In some embodiments, the CM2 comprises the amino
acid sequence LSGRSDNH (SEQ ID NO: 37). In some embodiments, the
CM2 comprises the amino acid sequence GGWHTGRN (SEQ ID NO: 38). In
some embodiments, the CM2 comprises the amino acid sequence
HTGRSGAL (SEQ ID NO: 39). In some embodiments, the CM2 comprises
the amino acid sequence PLTGRSGG (SEQ ID NO: 40). In some
embodiments, the CM2 comprises the amino acid sequence AARGPAIH
(SEQ ID NO: 41). In some embodiments, the CM2 comprises the amino
acid sequence RGPAFNPM (SEQ ID NO: 42). In some embodiments, the
CM2 comprises the amino acid sequence SSRGPAYL (SEQ ID NO: 43). In
some embodiments, the CM2 comprises the amino acid sequence
RGPATPIM (SEQ ID NO: 44). In some embodiments, the CM2 comprises
the amino acid sequence RGPA (SEQ ID NO: 45). In some embodiments,
the CM2 comprises the amino acid sequence GGQPSGMWGW (SEQ ID NO:
46). In some embodiments, the CM2 comprises the amino acid sequence
FPRPLGITGL (SEQ ID NO: 47). In some embodiments, the CM2 comprises
the amino acid sequence VHMPLGFLGP (SEQ ID NO: 48). In some
embodiments, the CM2 comprises the amino acid sequence SPLTGRSG
(SEQ ID NO: 49). In some embodiments, the CM2 comprises the amino
acid sequence LAPLGLQRR (SEQ ID NO: 50). In some embodiments, the
CM2 comprises the amino acid sequence SGGPLGVR (SEQ ID NO: 51). In
some embodiments, the CM2 comprises the amino acid sequence PLGL
(SEQ ID NO: 52). In some embodiments, the CM2 comprises the amino
acid sequence GPRSFGL (SEQ ID NO: 315). In some embodiments, the
CM2 comprises the amino acid sequence GPRSFG (SEQ ID NO: 316)
[0125] In some embodiments, the CM2 is a substrate for at least one
MMP. In some embodiments, the CM2 is a substrate for at least one
MMP listed in the Table 7. In some embodiments, the CM2 is a
substrate for MMP9. In some embodiments, the CM2 is a substrate for
MMP14. In some embodiments, CM1 is substrate for a first MMP, and
CM2 is a substrate for a second MMP, where the first MMP and the
second MMP are different MMPs. In some embodiments, CM1 is a first
substrate sequence for a MMP, and CM2 is a second substrate for the
same MMP, where the CM1 and CM2 have different substrate sequences.
In some embodiments, the CM2 is a substrate for two or more MMPs.
In some embodiments, the CM2 is a substrate for at least MMP9 or
MMP14. In some embodiments, the CM2 is a substrate for two or more
MMPs. In some embodiments, the CM2 is a substrate for at least MMP9
and MMP14. In some embodiments, CM1 and CM2 are both substrates for
MMP9. In some embodiments, CM1 and CM2 are both substrates for
MMP14. In some embodiments, CM1 is a substrate for MMP9 and CM2 is
a substrate for MMP14. In some embodiments, CM1 is a substrate for
MMP14 and CM2 is a substrate for MMP9.
[0126] In some embodiments, at least one of CM1 and/or CM2 is a
substrate for an MMP and includes the sequence ISSGLLSS (SEQ ID NO:
14); QNQALRMA (SEQ ID NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF
(SEQ ID NO: 17); PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO:
19); MIAPVAYR (SEQ ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR
(SEQ ID NO: 22); FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO:
24); GPSHLVLT (SEQ ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG
(SEQ ID NO: 27); SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO:
29); LAAPLGLL (SEQ ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA
(SEQ ID NO: 32), PAGLWLDP (SEQ ID NO: 33); and/or ISSGLSS (SEQ ID
NO: 159).
[0127] In some embodiments, the first cleaving agent and the second
cleaving agent are the same matrix metalloprotease, and the first
CM and the second CM are different substrates for the enzyme. In
some embodiments, the first cleaving agent and the second cleaving
agent are different proteases, where at least one protease is an
MMP. In some embodiments, the first cleaving agent and the second
cleaving agent are co-localized in the target tissue. In some
embodiments, the first CM and the second CM are cleaved by at least
one cleaving agent in the target tissue.
[0128] In some embodiments, the activatable antibody is exposed to
and cleaved by a MMP 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 MMP has cleaved the CM.
[0129] In some embodiments, the CM comprises the non-prime side of
the protease cleavage site; that is, the CM comprises at least the
P1 and P2 amino acids, and in some embodiments, comprises the P1,
P2 and P3 amino acids and in some embodiments, comprises the P1,
P2, P3, and P4 amino acids. In some embodiments, the CM comprises
the non-prime side and the prime side of the protease cleavage
site. In some embodiments, the CM comprises the non-prime side but
lacks at least part of the prime side of the protease cleavage
site. In some embodiments, the CM comprises the non-prime side but
lacks the prime side of the protease cleavage site. Such a CM can
be linked directly or through a linker to an antibody or other
molecule as disclosed herein, such as, but not limited to, a
detection moiety.
[0130] In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB that is
or is derived from cetuximab or panitumumab; a MM comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 160, 167-200, and 497; and a CM comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 14-33,
and 159. In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB that is
or is derived from cetuximab or panitumumab; a MM comprising an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 160, 167-200, and 497; and a CM comprising an amino acid
sequence selected from the group consisting of the sequences
presented in Tables 8A-8M. In some embodiments, the anti-EGFR
activatable antibody also 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, each of LP1 and LP2 is a peptide of about 1 to 20
amino acids in length. In some embodiments, the two linking
peptides need not be identical to each other. In some embodiments,
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: 1) and (GGGS).sub.n (SEQ ID NO: 2), where
n is an integer of at least one. In some embodiments, at least one
of LP1 or LP2 comprises an amino acid sequence selected from the
group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),
GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7),
and GSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the
amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ
ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID
NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO:
157). In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0131] In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB
comprising a heavy chain amino acid sequence comprising the VH CDR1
sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505,
the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ
ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR2
sequence of SEQ ID NO: 509; a MM comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 160, 167-200, and
497; and a CM comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14-33, and 159. In some embodiments,
the activatable antibody is an anti-EGFR activatable antibody that
includes at least an AB comprising a heavy chain amino acid
sequence comprising the VH CDR1 sequence of SEQ ID NO: 504, the VH
CDR2 sequence of SEQ ID NO: 505, the VH CDR3 sequence of SEQ ID NO:
506, the VL CDR1 sequence of SEQ ID NO: 507, the VL CDR2 sequence
of SEQ ID NO: 508, and the VL CDR2 sequence of SEQ ID NO: 509; a MM
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM
comprising an amino acid sequence selected from the group
consisting of the sequences presented in Tables 8A-8M. In some
embodiments, the anti-EGFR activatable antibody also 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, each of
LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least one.
In some embodiments, at least one of LP1 or LP2 comprises an amino
acid sequence selected from the group consisting of GGSG (SEQ ID
NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID
NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some
embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG
(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID
NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO:
156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2
comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158),
GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).
[0132] In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB
comprising the heavy chain amino acid sequence of SEQ ID NO: 56, 57
or 58 and the light chain amino acid sequence of SEQ ID NO: 59; a
MM comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 160, 167-200, and 497; and a CM
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 14-33, and 159. In some embodiments, the
activatable antibody is an anti-EGFR activatable antibody that
includes at least an AB comprising the heavy chain amino acid
sequence of SEQ ID NO: 56, 57 or 58 and the light chain amino acid
sequence of SEQ ID NO: 59; a MM comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 160, 167-200, and
497; and a CM comprising an amino acid sequence selected from the
group consisting of the sequences presented in Tables 8A-8M. In
some embodiments, the anti-EGFR activatable antibody also 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-LP 1-MM. In some embodiments, each of
LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least one.
In some embodiments, at least one of LP1 or LP2 comprises an amino
acid sequence selected from the group consisting of GGSG (SEQ ID
NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID
NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some
embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG
(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID
NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO:
156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2
comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158),
GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).
[0133] In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB
comprising the heavy chain amino acid sequence of SEQ ID NO: 56 and
the light chain amino acid sequence of SEQ ID NO: 59; a MM
comprising the amino acid sequence of SEQ ID NO: 160; and a CM
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 14-33, and 159. In some embodiments, the
activatable antibody is an anti-EGFR activatable antibody that
includes at least an AB comprising the heavy chain amino acid
sequence of SEQ ID NO: 56 and the light chain amino acid sequence
of SEQ ID NO: 59; a MM comprising the amino acid sequence of SEQ ID
NO: 160; and a CM comprising an amino acid sequence selected from
the group consisting of the sequences presented in Tables 8A-8M. In
some embodiments, the anti-EGFR activatable antibody also 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, each of
LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least one.
In some embodiments, at least one of LP1 or LP2 comprises an amino
acid sequence selected from the group consisting of GGSG (SEQ ID
NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID
NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some
embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG
(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID
NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO:
156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2
comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158),
GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).
[0134] In some embodiments, the activatable antibody is an
anti-EGFR activatable antibody that includes at least an AB
comprising a heavy chain amino acid sequence comprising the VH CDR1
sequence of SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505,
the VH CDR3 sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ
ID NO: 507, the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR2
sequence of SEQ ID NO: 509; a MM comprising the amino acid sequence
of SEQ ID NO: 160; and a CM comprising an amino acid sequence
selected from the group consisting of SEQ ID NO: 14-33, and 159. In
some embodiments, the activatable antibody is an anti-EGFR
activatable antibody that includes at least an AB comprising a
heavy chain amino acid sequence comprising the VH CDR1 sequence of
SEQ ID NO: 504, the VH CDR2 sequence of SEQ ID NO: 505, the VH CDR3
sequence of SEQ ID NO: 506, the VL CDR1 sequence of SEQ ID NO: 507,
the VL CDR2 sequence of SEQ ID NO: 508, and the VL CDR2 sequence of
SEQ ID NO: 509; a MM comprising the amino acid sequence of SEQ ID
NO: 160; and a CM comprising an amino acid sequence selected from
the group consisting of the sequences presented in Tables 8A-8M. In
some embodiments, the anti-EGFR activatable antibody also 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-LP 1-MM. In some embodiments, each of
LP1 and LP2 is a peptide of about 1 to 20 amino acids in length. In
some embodiments, the two linking peptides need not be identical to
each other. In some embodiments, 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), where n is an integer of at least one.
In some embodiments, at least one of LP1 or LP2 comprises an amino
acid sequence selected from the group consisting of GGSG (SEQ ID
NO: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID
NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8). In some
embodiments, LP1 comprises the amino acid sequence GSSGGSGGSGGSG
(SEQ ID NO: 9), GSSGGSGGSGG (SEQ ID NO: 10), GSSGGSGGSGGS (SEQ ID
NO: 11), GSSGGSGGSGGSGGGS (SEQ ID NO: 155), GSSGGSGGSG (SEQ ID NO:
156), or GSSGGSGGSGS (SEQ ID NO: 157). In some embodiments, LP2
comprises the amino acid sequence GSS, GGS, GGGS (SEQ ID NO: 158),
GSSGT (SEQ ID NO: 12) or GSSG (SEQ ID NO: 13).
[0135] In some embodiments, the activatable antibody is an
anti-Jagged activatable antibody that includes at least an AB
comprising the heavy chain amino acid sequence of SEQ ID NO: 61,
63, 65, 68, 70, 72, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,
100, 102, 104, 106, 108, 110, 112, or 114 and the light chain amino
acid sequence of SEQ ID NO: 60, 62, 64, 67, 69, 71, 73, 75, 77, 79,
81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,
111, or 113; a MM comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 201-263, and 496; and a CM
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 14-33, and 159. In some embodiments, the
activatable antibody is an anti-Jagged activatable antibody that
includes at least an AB comprising the heavy chain amino acid
sequence of SEQ ID NO: 61, 63, 65, 68, 70, 72, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, or
114 and the light chain amino acid sequence of SEQ ID NO: 60, 62,
64, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,
99, 101, 103, 105, 107, 109, 111, or 113; a MM comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
201-263, and 496; and a CM comprising an amino acid sequence
selected from the group consisting of the sequences presented in
Tables 8A-8M. In some embodiments, the anti-Jagged activatable
antibody also 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, each of LP1 and LP2 is a peptide of about 1 to 20
amino acids in length. In some embodiments, the two linking
peptides need not be identical to each other. In some embodiments,
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: 1) and (GGGS).sub.n (SEQ ID NO: 2), where
n is an integer of at least one. In some embodiments, at least one
of LP1 or LP2 comprises an amino acid sequence selected from the
group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),
GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7),
and GSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the
amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ
ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID
NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO:
157). In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0136] In some embodiments, the activatable antibody is an
anti-Jagged activatable antibody that includes at least an AB
comprising the heavy chain amino acid sequence of SEQ ID NO: 112
and the light chain amino acid sequence of SEQ ID NO: 111; a MM
comprising the amino acid sequence selected of SEQ ID NO: 217; and
a CM comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 14-33, and 159. In some embodiments, the
activatable antibody is an anti-Jagged activatable antibody that
includes at least an AB comprising the heavy chain amino acid
sequence of SEQ ID NO: 112 and the light chain amino acid sequence
of SEQ ID NO: 111; a MM comprising the amino acid sequence selected
of SEQ ID NO: 217; and a CM comprising an amino acid sequence
selected from the group consisting of the sequences presented in
Tables 8A-8M. In some embodiments, the anti-Jagged activatable
antibody also 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-LP 1-MM. In
some embodiments, each of LP1 and LP2 is a peptide of about 1 to 20
amino acids in length. In some embodiments, the two linking
peptides need not be identical to each other. In some embodiments,
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: 1) and (GGGS).sub.n (SEQ ID NO: 2), where
n is an integer of at least one. In some embodiments, at least one
of LP1 or LP2 comprises an amino acid sequence selected from the
group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),
GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7),
and GSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the
amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ
ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID
NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO:
157). In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0137] In some embodiments, the activatable antibody is an
anti-Jagged activatable antibody that includes at least an AB
comprising a heavy chain amino acid sequence comprising the VH CDR1
sequence of SEQ ID NO: 498, the VH CDR2 sequence of SEQ ID NO: 499,
the VH CDR3 sequence of SEQ ID NO: 500, the VL CDR1 sequence of SEQ
ID NO: 501, the VL CDR2 sequence of SEQ ID NO: 502, and the VL CDR2
sequence of SEQ ID NO: 503; a MM comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 217; and a CM
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 14-33, and 159. In some embodiments, the
activatable antibody is an anti-Jagged activatable antibody that
includes at least an AB comprising a heavy chain amino acid
sequence comprising the VH CDR1 sequence of SEQ ID NO: 498, the VH
CDR2 sequence of SEQ ID NO: 499, the VH CDR3 sequence of SEQ ID NO:
500, the VL CDR1 sequence of SEQ ID NO: 501, the VL CDR2 sequence
of SEQ ID NO: 502, and the VL CDR2 sequence of SEQ ID NO: 503; a MM
comprising the amino acid sequence selected from the group
consisting of SEQ ID NO: 217; and a CM comprising an amino acid
sequence selected from the group consisting of the sequences
presented in Tables 8A-8M. In some embodiments, the anti-Jagged
activatable antibody also 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, each of LP1 and LP2 is a peptide of about 1 to 20
amino acids in length. In some embodiments, the two linking
peptides need not be identical to each other. In some embodiments,
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: 1) and (GGGS).sub.n (SEQ ID NO: 2), where
n is an integer of at least one. In some embodiments, at least one
of LP1 or LP2 comprises an amino acid sequence selected from the
group consisting of GGSG (SEQ ID NO: 3), GGSGG (SEQ ID NO: 4),
GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7),
and GSSSG (SEQ ID NO: 8). In some embodiments, LP1 comprises the
amino acid sequence GSSGGSGGSGGSG (SEQ ID NO: 9), GSSGGSGGSGG (SEQ
ID NO: 10), GSSGGSGGSGGS (SEQ ID NO: 11), GSSGGSGGSGGSGGGS (SEQ ID
NO: 155), GSSGGSGGSG (SEQ ID NO: 156), or GSSGGSGGSGS (SEQ ID NO:
157). In some embodiments, LP2 comprises the amino acid sequence
GSS, GGS, GGGS (SEQ ID NO: 158), GSSGT (SEQ ID NO: 12) or GSSG (SEQ
ID NO: 13).
[0138] In some embodiments, the activatable antibody also includes
an agent conjugated to the AB. In some embodiments, the agent is a
therapeutic agent. In some embodiments, the agent is an
antineoplastic agent. In some embodiments, the agent is a toxin or
a fragment 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 microtubule inhibitor.
In some embodiments, the agent is a nucleic acid damaging agent,
such as a DNA alkylator or DNA intercalator, or other DNA damaging
agent. In some embodiments, the linker is a cleavable linker. In
some embodiments, the agent is conjugated to the AB via a linker
that includes at least one MMP-cleavable substrate sequence. In
some embodiments, the agent is an agent selected from the group
listed in Table 3. 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
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is a pyrrolobenzodiazepine.
[0139] In some embodiments, the agent is an anti-inflammatory
agent.
[0140] In some embodiments, the activatable antibody also includes
a detectable moiety. In some embodiments, the detectable moiety is
a diagnostic agent.
[0141] In some embodiments, the conjugated antibody includes a
detectable label. In some embodiments, 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, the imaging agent
comprises a radioisotope. In some embodiments, the radioisotope is
indium or technetium. In some embodiments, the contrasting agent
comprises iodine, gadolinium or iron oxide. In some embodiments,
the enzyme comprises horseradish peroxidase, alkaline phosphatase,
or .beta.-galactosidase. In some embodiments, 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, the
luminescent label comprises an N-methylacrydium derivative. In some
embodiments, the label comprises an Alexa Fluor.RTM. label, such as
Alex Fluor.RTM. 680 or Alexa Fluor.RTM. 750. In some embodiments,
the ligand-based label comprises biotin, avidin, streptavidin or
one or more haptens.
[0142] 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. In some
embodiments, the spacer is joined directly to the MM of the
activatable antibody in the structural arrangement from N-terminus
to C-terminus of spacer-MM-CM-AB. An example of a spacer joined
directly to the N-terminus of MM of the activatable antibody is
QGQSGQ (SEQ ID NO: 53). In some embodiments, the spacer includes at
least the amino acid sequence QGQSGQ (SEQ ID NO: 53).
[0143] 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.
[0144] 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
activatable antibody is at least 5 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 4 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 3 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 2 days when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 24 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 20 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 18 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 16 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 14 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 12 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 10 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 8 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 6 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 4 hours when administered to an
organism. In some embodiments, the serum half-life of the
activatable antibody is at least 3 hours when administered to an
organism.
[0145] 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 (pro-BITE) molecule. In
some embodiments, the activatable antibody and/or conjugated
activatable antibody is formulated as part of a pro-Chimeric
Antigen Receptor (pro-CAR) modified T cell or other engineered
receptor.
[0146] The disclosure also provides compositions and methods that
include an activatable antibody that includes an antibody or
antibody fragment (AB) that specifically binds a given 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 at least one MMP. 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, for
example, 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 MMP that
can cleave the CM.
[0147] 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.
[0148] 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%, 97%, 96%,
95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%,
30%, 25%, 20%, 15%, 10% or less than 5% of all possible sites of
conjugation are reduced.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] The disclosure 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 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
the 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, wherein the CM is a
polypeptide that functions as a substrate for at least one MMP. In
some embodiments, the MM is coupled to the AB via the CM. 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.
[0153] The disclosure also provides partially reduced activatable
antibodies, including but not limited to multispecific activatable
antibodies of the disclosure, 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.
[0154] The disclosure 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 cleavable moiety
(CM) coupled to the AB, and the CM is a polypeptide that functions
as a substrate for at least one MMP protease.
[0155] 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.
[0156] 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.
[0157] 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 MMP protease
is co-localized with the target in a tissue, and the MMP protease
cleaves the CM in the activatable antibody when the activatable
antibody is exposed to the MMP protease. In some embodiments, the
MMP protease is a MMP9 protease. In some embodiments, the MMP
protease is a MMP14 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-LP 1-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: 1) and (GGGS).sub.n (SEQ ID NO: 2), 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: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ
ID NO: 5), GSGGG (SEQ ID NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG
(SEQ ID NO: 8). In some embodiments, the activatable antibody
includes a second CM; in some embodiments, the second CM is a
substrate for an enzyme selected from the group consisting of those
shown in Table 7.
[0158] The disclosure also provides polypeptides and other larger
molecules that include one or more of the MMP-cleavable substrate
sequences presented herein. By way of non-limiting example, the
MMP-cleavable substrate sequences presented herein are useful in
prodrug compositions and methods of use thereof. These
MMP-cleavable substrate sequences presented herein are also useful
in probes and other detection agents and methods of use thereof.
For example, the MMP-cleavable substrate sequences presented herein
can be used in conjunction with fluors and other quenchers to
produce detection agents, such as imaging agents and/or other
diagnostic agents. Those of ordinary skill in the art will
appreciate that the MMP-cleavable substrate sequences presented
herein are useful in any composition and/or method in the art that
would use a substrate that is cleavable by one or more MMPs, such
as MMP9 and/or MMP14.
[0159] The disclosure also provides an isolated nucleic acid
molecule encoding an antibody and/or an activatable antibody
described herein, as well as vectors that include these isolated
nucleic acid sequences. The disclosure provides methods of
producing an antibody and/or activatable antibody by culturing a
cell under conditions that lead to expression of the antibody
and/or activatable antibody, wherein the cell comprises such a
vector.
[0160] The disclosure provides a method of manufacturing a
conjugated antibody of the disclosure that bind a given target by
(a) culturing a cell comprising a nucleic acid construct that
encodes the antibody under conditions that lead to expression of
the antibody, (i) wherein the antibody includes a cleavable moiety
(CM), and (ii) wherein the CM is a polypeptide that functions as a
substrate for a matrix metalloprotease; (b) recovering the
antibody; and (c) conjugating the recovered antibody to one or more
additional agents.
[0161] The disclosure also provides a method of manufacturing the
activatable antibodies of the disclosure that bind in an activated
state a given target by (a) culturing a cell comprising a nucleic
acid construct that encodes the activatable antibody under
conditions that lead to expression of the activatable antibody,
wherein the activatable antibody comprises a masking moiety (MM), a
cleavable moiety (CM), and an antibody or an antigen binding
fragment thereof (AB) that specifically binds the target, (i)
wherein the CM is a polypeptide that functions as a substrate for a
MMP; and (ii) wherein the CM is positioned in the activatable
antibody such that, in an uncleaved state, the MM interferes with
specific binding of the AB to the target and in a cleaved state the
MM does not interfere or compete with specific binding of the AB to
the target; and (b) recovering the activatable antibody.
[0162] The disclosure provides methods of preventing, delaying the
progression of, treating, alleviating a symptom of, or otherwise
ameliorating a target-related disease in a subject by administering
a therapeutically effective amount of a conjugated antibody, an
activatable antibody and/or a conjugated activatable antibody
described herein to a subject in need thereof.
[0163] The disclosure provides methods of preventing, delaying the
progression of, treating, alleviating a symptom of, or otherwise
ameliorating inflammation and/or an inflammatory disorder in a
subject by administering a therapeutically effective amount of a
conjugated antibody, an activatable antibody and/or a conjugated
activatable antibody described herein to a subject in need thereof.
The disclosure also provides methods of preventing, delaying the
progression of, treating, alleviating a symptom of, or otherwise
ameliorating cancer in a subject by administering a therapeutically
effective amount of a conjugated antibody, an activatable antibody
and/or a conjugated activatable antibody described herein to a
subject in need thereof. The disclosure also provides methods of
preventing, delaying the progression of, treating, alleviating a
symptom of, or otherwise ameliorating an autoimmune disease in a
subject by administering a therapeutically effective amount a
conjugated antibody, an activatable antibody and/or a conjugated
activatable antibody described herein to a subject in need
thereof.
[0164] A conjugated antibody, an activatable antibody and/or a
conjugated activatable antibody used in any of the embodiments of
these methods and uses can be administered at any stage of the
disease. For example, such a conjugated antibody, activatable
antibody and/or conjugated activatable antibody can be administered
to a patient suffering cancer of any stage, from early to
metastatic. The terms subject and patient are used interchangeably
herein.
[0165] In some embodiments, the subject is a mammal, such as a
human, 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.
[0166] The conjugated antibody, activatable antibody and/or
conjugated activatable antibody and therapeutic formulations
thereof are administered to a subject suffering from or susceptible
to a disease or disorder associated with aberrant target expression
and/or activity. A subject suffering from or susceptible to a
disease or disorder associated with aberrant target expression
and/or activity 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/or stool analysis to evaluate health status. For
example, subjects suffering from inflammation and/or an
inflammatory disorder are identified using any of a variety of
clinical and/or laboratory tests such as physical examination
and/or bodily fluid analysis, e.g., blood, urine and/or stool
analysis, to evaluate health status.
[0167] Administration of a conjugated antibody, an activatable
antibody and/or a conjugated activatable antibody to a patient
suffering from a disease or disorder associated with aberrant
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 antibody, an activatable
antibody and/or a conjugated activatable antibody to a patient
suffering from a disease or disorder associated with aberrant
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 antibody, an
activatable antibody and/or a conjugated activatable antibody to a
patient suffering from a disease or disorder associated with
aberrant target expression and/or activity is considered successful
if the disease or disorder enters remission or does not progress to
a further, i.e., worse, state.
[0168] In some embodiments, the conjugated antibody, activatable
antibody and/or conjugated activatable antibody is administered
during and/or after treatment in combination with one or more
additional agents such as, by way of non-limiting example, an
anti-inflammatory agent, an immunosuppressive agent, a
chemotherapeutic agent, such as an alkylating agent, an
anti-metabolite, an anti-microtubule agent, a topoisomerase
inhibitor, a cytotoxic antibiotic, and/or 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.
[0169] In some embodiments, the additional agent is an antibody,
another conjugated antibody, another activatable antibody and/or
another conjugated activatable antibody. In some embodiments, the
conjugated antibody, activatable antibody and/or conjugated
activatable antibody and the additional agent(s) are administered
simultaneously. For example, the conjugated antibody, activatable
antibody and/or conjugated activatable antibody and the additional
agent(s) can be formulated in a single composition or administered
as two or more separate compositions. In some embodiments, the
conjugated antibody, activatable antibody and/or conjugated
activatable antibody and the additional agent(s) are administered
sequentially, or the antibody and/or conjugated antibodies and the
additional agent are administered at different times during a
treatment regimen. For example, the antibody and/or conjugated
antibodies is administered prior to the administration of the
additional agent, the antibody and/or conjugated antibodies is
administered subsequent to the administration of the additional
agent, or the antibody and/or conjugated antibodies and the
additional agent are administered in an alternating fashion. As
described herein, the antibody and/or conjugated antibodies and
additional agent are administered in single doses or in multiple
doses.
[0170] In some embodiments, the CM is linked or otherwise attached
to an activatable antibody that includes an antibody or
antigen-binding fragment thereof that specifically binds a given
target coupled to a masking moiety (MM), such that coupling of the
MM to the AB reduces the ability of the antibody or antigen-binding
fragment thereof to bind the target. In some embodiments, the MM is
coupled via the CM. Exemplary targets include, but are not limited
to the targets shown in Table 1. Exemplary ABs include, but are not
limited to, the targets shown in Table 2. 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 or other tissue not targeted for treatment and/or
diagnosis, and, when activated, exhibit binding to the target that
is at least comparable to the corresponding, unmodified
antibody.
[0171] The disclosure also provides methods and kits for using the
conjugated antibodies, activatable antibodies and/or conjugated
activatable antibodies in a variety of diagnostic and/or
prophylactic indications.
[0172] In some embodiments, the disclosure provides methods and
kits for 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 an activatable antibody, wherein the
activatable antibody comprises a masking moiety (MM), a cleavable
moiety (CM) that is cleaved by the cleaving agent, and an antigen
binding domain or fragment thereof (AB) that specifically binds the
target of interest, wherein the activatable antibody in an
uncleaved, non-activated state comprises a structural arrangement
from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB and is not a
modified form of a natural binding partner of the AB; and (b)
wherein, in an uncleaved, non-activated state, the MM interferes
with specific binding of the AB to the target, and in a cleaved,
activated state the MM does not interfere or compete with specific
binding of the AB to the target; and (ii) measuring a level of
activated activatable antibody in the subject or sample, wherein a
detectable level of activated 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 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.
[0173] In some embodiments, the activatable antibody is an
activatable antibody to which a therapeutic agent is conjugated. In
some embodiments, the activatable antibody is not conjugated to an
agent. In some embodiments, the activatable antibody comprises a
detectable label. In some embodiments, the detectable label is
positioned on the AB. In some embodiments, measuring the level of
activatable antibody in the subject or sample is accomplished using
a secondary reagent that specifically binds to the activated
antibody, wherein the reagent comprises a detectable label. In some
embodiments, the secondary reagent is an antibody comprising a
detectable label.
[0174] In some embodiments of these methods and kits, the
activatable antibody includes a detectable label. In some
embodiments of these methods and 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
kits, the imaging agent comprises a radioisotope. In some
embodiments of these methods and kits, the radioisotope is indium
or technetium. In some embodiments of these methods and kits, the
contrasting agent comprises iodine, gadolinium or iron oxide. In
some embodiments of these methods and kits, the enzyme comprises
horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase. In some embodiments of these methods and
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 kits, 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 and kits, the ligand-based label comprises biotin,
avidin, streptavidin or one or more haptens.
[0175] In some embodiments of these methods and kits, 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.
[0176] In some embodiments of these methods and kits, 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.
[0177] In some embodiments of the methods and kits, the method is
used to identify or otherwise refine a patient population suitable
for treatment with an activatable antibody of the disclosure,
followed by treatment by administering that activatable antibody
and/or conjugated activatable antibody to a subject in need
thereof. For example, patients that test positive for both the
target and at least one MMP that cleaves the substrate in the
cleavable moiety (CM) of the activatable antibody being tested in
these methods are identified as suitable candidates for treatment
with such an activatable antibody comprising such a CM, and the
patient is then administered a therapeutically effective amount of
the activatable antibody and/or conjugated activatable antibody
that was tested. Likewise, patients that test negative for either
or both of the target and the MMP 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 can be tested with
other activatable antibodies 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 activatable antibody and/or
conjugated for which the patient tested positive.
[0178] Pharmaceutical compositions according to the disclosure can
include an antibody of the disclosure and a carrier. These
pharmaceutical compositions can be included in kits, such as, for
example, diagnostic kits.
BRIEF DESCRIPTION OF THE DRAWINGS
[0179] FIGS. 1A and 1B are a series of graphs depicting the ability
of the activatable anti-EGFR antibody containing a masking moiety
comprising amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a
cleavage moiety comprising the MMP14 substrate 520 (also referred
to herein as MN520) ISSGLLSS (SEQ ID NO: 14), and the heavy and
light chains of the anti-EGFR antibody C225v5, where the entire
activatable antibody construct is referred to herein as Pb-MN520,
to inhibit tumor growth in the H292 xenograft lung cancer
model.
[0180] FIGS. 2A and 2B are a series of graphs depicting cleavage of
the substrate pool referred to herein as SMP87 by 5 nM MMP9.
[0181] FIGS. 3A and 3B are a series of graphs depicting cleavage of
substrate sequence VAGRSMRP (SEQ ID NO: 484) by 5 nM MMP9.
[0182] FIG. 4 is a graph depicting correlation of substrate
sequence frequency and function.
[0183] FIGS. 5A and 5B are a series of graphs depicting cleavage of
the substrate pool SMP39 by 60 nM MMP14.
[0184] FIGS. 6A and 6B are a series of graphs depicting cleavage of
the substrate sequence QNQALRMA (SEQ ID NO: 15) by 30 nM MMP14.
[0185] FIGS. 7A and 7B are a series of schematic representations of
the peptide display platforms used in the working examples provided
herein. FIG. 7A is a schematic representation of the sequence of
the display platform referred to herein as "Display Platform
CYTX-DP-XXXXXXXX" or "CYTX-DP-XXXXXXXX" (SEQ ID NO: 512). FIG. 7B
is a schematic representation of the sequence of the display
platform referred to herein as "Display Platform
SP-CYTX-DP-XXXXXXXX" or "SP-CYTX-DP-XXXXXXXX" (SEQ ID NO: 513),
where SP-CYTX-DP-XXXXXXXX is the CYTX-DP-XXXXXXXX platform with a
signal peptide.
DETAILED DESCRIPTION OF THE INVENTION
[0186] The disclosure provides amino acid sequences that include a
cleavable moiety (CM) that is a substrate for at least one matrix
metalloprotease (MMP). These CMs are useful in a variety of
therapeutic, diagnostic and prophylactic indications.
[0187] The working examples provided herein demonstrate that these
CM, when displayed in a peptide display platform, exhibit a number
of desirable cleavage characteristics when exposed to an MMP
protease under specified conditions. For example, Table 9 depicts
(a) the percentage of MMP9-selected substrates tested in the
CYTX-DP display platform that exhibited at least 20% cleavage when
incubated with 50 nM human MMP9 for 1 hour at 37.degree. C. in 50
mM Tris-HCl, pH 7.4, supplemented with 150 mM NaCl, 10 mM
CaCl.sub.2, and 0.05% (w/v) Brij-35 (>20% Cleavage with 50 nM
MMP9); (b) the percentage of MMP14-selected substrates tested in
the CYTX-DP display platform that exhibited at least 20% cleavage
when incubated with 50 nM human MMP14 for 1 hour at 37.degree. C.
in 50 mM HEPES, pH 6.8, supplemented with 10 mM CaCl.sub.2, and 0.5
mM MgCl.sub.2 (>20% Cleavage with 50 nM MMP14); and (c) the
percentage of MMP9-selected or MMP-14-selected substrates tested in
the CYTX-DP display platform that exhibited less than 20% cleavage
when incubated with 500 pM human plasmin for 1 hour at 37.degree.
C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mM NaCl, 0.01%
Tween20 and 1 mM EDTA (<20% cleavage with 500 pM plasmin).
[0188] In some embodiments, a MMP9 substrate when displayed in the
CYTX-DP platform exhibits at least 20% cleavage when incubated with
50 nM human MMP9 for 1 hour at 37.degree. C. in 50 mM Tris-HCl, pH
7.4, supplemented with 150 mM NaCl, 10 mM CaCl.sub.2, and 0.05%
(w/v) Brij-35. In some embodiments, a MMP9 substrate when displayed
in the CYTX-DP platform exhibits less than 20% cleavage when
incubated with 500 pM human plasmin for 1 hour at 37.degree. C. in
50 mM Tris-HCl, pH 7.4, supplemented with 100 mM NaCl, 0.01%
Tween20 and 1 mM EDTA. In some embodiments, a MMP9 substrate when
displayed in the CYTX-DP platform exhibits at least 20% cleavage
when incubated with 50 nM human MMP9 for 1 hour at 37.degree. C. in
50 mM Tris-HCl, pH 7.4, supplemented with 150 mM NaCl, 10 mM
CaCl.sub.2, and 0.05% (w/v) Brij-35 and exhibits less than 20%
cleavage when incubated with 500 pM human plasmin for 1 hour at
37.degree. C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mM
NaCl, 0.01% Tween20 and 1 mM EDTA.
[0189] In some embodiments a MMP14 substrate exhibits at least 20%
cleavage when incubated with 50 nM human MMP14 for 1 hour at
37.degree. C. in 50 mM HEPES, pH 6.8, supplemented with 10 mM
CaCl.sub.2, and 0.5 mM MgCl.sub.2. In some embodiments, a MMP14
substrate when displayed in the CYTX-DP platform exhibits less than
20% cleavage when incubated with 500 pM human plasmin for 1 hour at
37.degree. C. in 50 mM Tris-HCl, pH 7.4, supplemented with 100 mM
NaCl, 0.01% Tween20 and 1 mM EDTA. In some embodiments a MMP14
substrate exhibits at least 20% cleavage when incubated with 50 nM
human MMP14 for 1 hour at 37.degree. C. in 50 mM HEPES, pH 6.8,
supplemented with 10 mM CaCl.sub.2, and 0.5 mM MgCl.sub.2 and
exhibits less than 20% cleavage when incubated with 500 pM human
plasmin for 1 hour at 37.degree. C. in 50 mM Tris-HCl, pH 7.4,
supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA.
[0190] In some embodiments, the observed k.sub.cat/K.sub.M value of
a substrate in an activatable antibody for MMP9 is greater than 100
M.sup.-1 s.sup.-1. In some embodiments, the observed
k.sub.cat/K.sub.M value of a substrate in an activatable antibody
for MMP9 is greater than 1,000 M.sup.-1 s.sup.-1. In some
embodiments, the observed k.sub.cat/K.sub.M value of a substrate in
an activatable antibody for MMP9 is greater than 10,000 M.sup.-1
s.sup.-1.
[0191] In some embodiments, the observed k.sub.cat/K.sub.M value of
a substrate in an activatable antibody for MMP14 is greater than
100 M.sup.-1 s.sup.-1. In some embodiments, the observed
k.sub.cat/K.sub.M value of a substrate in an activatable antibody
for MMP14 is greater than 1,000 M.sup.-1 s.sup.-1. In some
embodiments, the observed k.sub.cat/K.sub.M value of a substrate in
an activatable antibody for MMP14 is greater than 10,000 M.sup.-1
s.sup.-1.
[0192] The disclosure also provides antibodies that include one or
more of these MMP-cleavable substrates. For example, these
MMP-cleavable substrates are useful when conjugating antibodies to
one or more additional agents to produce conjugated antibodies.
These MMP-cleavable are useful in activatable antibody
constructs.
[0193] The conjugated antibodies and/or activatable antibodies
include an antibody or antigen-binding fragment thereof (AB) that
specifically binds a target. Exemplary classes of targets of an AB
include, but are not necessarily limited to, cell surface receptors
and secreted binding proteins (e.g., growth factors), soluble
enzymes, structural proteins (e.g. collagen, fibronectin) and the
like. In some embodiments, conjugated antibodies and/or activatable
antibodies have an AB that binds an extracellular target, usually
an extracellular protein target. In some embodiments, conjugated
antibodies and/or activatable antibodies are designed for cellular
uptake and are switchable inside a cell.
[0194] As a non-limiting example, the AB is a binding partner for
any target listed in Table 1.
TABLE-US-00017 TABLE 1 Exemplary Targets 1-92-LFA-3 CD52 DL44 HVEM
LIF-R STEAP1 Alpha-4 CD56 DLK1 Hyaluronidase Lewis X STEAP2
integrin Alpha-V CD64 DLL4 ICOS LIGHT TAG-72 integrin alpha4beta1
CD70 DPP-4 IFNalpha LRP4 TAPA1 integrin alpha4beta7 CD71 DSG1
IFNbeta LRRC26 TGFbeta integrin AGR2 CD74 EGFR IFNgamma MCSP TIGIT
Anti-Lewis-Y EGFRviii IgE Mesothelin TIM-3 Apelin J CD80 Endothelin
B IgE Receptor MRP4 TLR2 receptor receptor (FceRI) (ETBR) APRIL
CD81 ENPP3 IGF MUC1 TLR4 B7-H4 CD86 EpCAM IGF1R Mucin-16 TLR6
(MUC16, CA-125) BAFF CD95 EPHA2 IL1B Na/K ATPase TLR7 BTLA CD117
EPHB2 IL1R Neutrophil TLR8 elastase C5 CD125 ERBB3 IL2 NGF TLR9
complement C-242 CD132 F protein of IL11 Nicastrin TMEM31 (IL-2RG)
RSV CA9 CD133 FAP IL12 Notch TNFalpha Receptors CA19-9 CD137 FGF-2
IL12p40 Notch 1 TNFR (Lewis a) Carbonic CD138 FGF8 IL-12R, Notch 2
TNFRS12A anhydrase 9 IL-12Rbeta1 CD2 CD166 FGFR1 IL13 Notch 3
TRAIL-R1 CD3 CD172A FGFR2 IL13R Notch 4 TRAIL-R2 CD6 CD248 FGFR3
IL15 NOV Transferrin CD9 CDH6 FGFR4 IL17 OSM-R Transferrin receptor
CD11a CEACAM5 Folate IL18 OX-40 TRK-A (CEA) receptor CD19 CEACAM6
GAL3ST1 IL21 PAR2 TRK-B (NCA-90) CD20 CLAUDIN-3 G-CSF IL23 PDGF-AA
uPAR CD22 CLAUDIN-4 G-CSFR IL23R PDGF-BB VAP1 CD24 cMet GD2
IL27/IL27R PDGFRalpha VCAM-1 (wsx1) CD25 Collagen GITR IL29
PDGFRbeta VEGF CD27 Cripto GLUT1 IL-31R PD-1 VEGF-A CD28 CSFR GLUT4
IL31/IL31R PD-L1 VEGF-B CD30 CSFR-1 GM-CSF IL2R PD-L2 VEGF-C CD33
CTLA-4 GM-CSFR IL4 Phosphatidyl- VEGF-D serine CD38 CTGF GP
IIb/IIIa IL4R P1GF VEGFR1 receptors CD40 CXCL10 Gp130 IL6, IL6R
PSCA VEGFR2 CD40L CXCL13 GPIIB/IIIA Insulin PSMA VEGFR3 Receptor
CD41 CXCR1 GPNMB Jagged RAAG12 VISTA Ligands CD44 CXCR2 GRP78
Jagged 1 RAGE WISP-1 CD44v6 HER2/neu Jagged 2 SLC44A4 WISP-2 CD47
CXCR4 HGF LAG-3 Sphingosine 1 WISP-3 Phosphate CD51 CYR61 hGH
[0195] As a non-limiting example, the AB is or is derived from an
antibody listed in Table 2.
TABLE-US-00018 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
[0196] Exemplary conjugated antibodies and/or activatable
antibodies of the disclosure include, for example, antibodies that
bind interleukin 6 receptor (IL-6R) and that include a heavy chain
and a light chain that are, or are derived from, the antibody
referred to herein as the"Av1" antibody, which binds interleukin-6
receptor (IL-6R). The amino acid sequences for the Av1 heavy chain
and the Av1 light chain are shown below in SEQ ID NO: 54 and SEQ ID
NO: 55, respectively.
Av1 Antibody Heavy Chain Amino Acid Sequence:
TABLE-US-00019 [0197] (SEQ ID NO: 54)
QVQLQESGPGLVRPSQTLSLTCTVSGYSITSDHAWSWVRQPPGRGLEWIG
YISYSGITTYNPSLKSRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSL
ARTTAMDYWGQGSLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Av1 Antibody Light Chain Amino Acid Sequence:
TABLE-US-00020 [0198] (SEQ ID NO: 55)
DIQMTQSPSSLSASVGDRVTITCRASQDISSYLNWYQQKPGKAPKLLIYY
TSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPYTFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0199] Exemplary conjugated antibodies and/or activatable
antibodies of the disclosure include, for example, antibodies that
bind interleukin 6 receptor (IL-6R) and that include a heavy chain
and a light chain that are, or are derived from, the Av1 antibody
and a masking moiety. Exemplary conjugated antibodies and/or
activatable antibodies of the disclosure include an amino acid
sequence attached to the N-terminus of the AV1 light chain. These
N-terminal amino acid sequences include, for example,
YGSCSWNYVHIFMDC (SEQ ID NO: 161); QGDFDIPFPAHWVPIT (SEQ ID NO:
162); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163); QGQSGQYGSCSWNYVHIFMDC
(SEQ ID NO: 164); QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 165); or
QGQSGQMGVPAGCVWNYAHIFMDC (SEQ ID NO: 166). It is also to be
appreciated that such amino acid sequences can be attached to the
N-terminus of the AV1 heavy chain or to the C-terminus of the AV1
heavy or light chain.
[0200] Exemplary activatable antibodies of the disclosure include,
for example, antibodies that bind Epidermal Growth Factor Receptor
(EGFR) and that include a heavy chain and a light chain that are,
or are derived from, an antibody selected from the group consisting
of the antibody referred to herein as the"c225v5" antibody, the
antibody referred to herein as the"c225v4" antibody, and the
antibody referred to herein as the"c225v6" antibody, each of which
binds EGFR. The c225v5 antibody, the c225v4 antibody, and the
c225v6 antibody share the same light chain sequence, referred to
herein as "c225 light chain." The amino acid sequences for the
c225v5 heavy chain, the c225v4 antibody, the c225v6 antibody, and
the c225 light chain are shown below.
C225v5 Antibody Heavy Chain Amino Acid Sequence:
TABLE-US-00021 [0201] (SEQ ID NO: 56)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALT
YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
C225v4 Antibody Heavy Chain Amino Acid Sequence:
TABLE-US-00022 [0202] (SEQ ID NO: 57)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALT
YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
C225v6 Antibody Heavy Chain Amino Acid Sequence:
TABLE-US-00023 [0203] (SEQ ID NO: 58)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGV
IWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSQDTAIYYCARALT
YYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYAS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
C225 Antibody Light Chain Amino Acid Sequence:
TABLE-US-00024 [0204] (SEQ ID NO: 59)
QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKY
ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA
GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC*
[0205] Exemplary conjugated antibodies and/or activatable
antibodies of the disclosure include, for example, antibodies that
bind a Jagged target, e.g., Jagged-1, Jagged-2 and/or both Jagged-1
and Jagged-2, and that include a combination of a variable heavy
chain region and a variable light chain region that are, or are
derived from, the variable heavy chain and variable light chain
sequences shown below.
TABLE-US-00025 Variable Light Chain Amino Sequence Lc4 (SEQ ID NO:
60)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc4 (SEQ ID NO: 61)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc5 (SEQ ID NO: 62)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc5 (SEQ ID NO: 63)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYHGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc7 (SEQ ID NO: 64)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc7 (SEQ ID NO: 65)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc8 (SEQ ID NO: 67)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc8 (SEQ ID NO: 68)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHIGRTNPFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc13 (SEQ ID NO: 69)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc13 (SEQ ID NO: 70)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable Light
Chain Amino Sequence Lc16 (SEQ ID NO: 71)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc16 (SEQ ID NO: 72)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPYYGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc19 (SEQ ID NO: 73)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc19 (SEQ ID NO: 74)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc21 (SEQ ID NO: 75)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc21 (SEQ ID NO: 76)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc24 (SEQ ID NO: 77)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc24 (SEQ ID NO: 78)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable Light
Chain Amino Sequence Lc26 (SEQ ID NO: 79)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc26 (SEQ ID NO: 80)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc27 (SEQ ID NO: 81)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc27 (SEQ ID NO: 82)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFYGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc28 (SEQ ID NO: 83)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc28 (SEQ ID NO: 84)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc30 (SEQ ID NO: 85)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc30 (SEQ ID NO: 86)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTLYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYAKSAAAFDYWGQGTLVTVSS Variable Light
Chain Amino Sequence Lc31 (SEQ ID NO: 87)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc31 (SEQ ID NO: 88)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc32 (SEQ ID NO: 89)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc32 (SEQ ID NO: 90)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable Light
Chain Amino Sequence Lc37 (SEQ ID NO: 91)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc37 (SEQ ID NO: 92)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPHNGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc39 (SEQ ID NO: 93)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc39 (SEQ ID NO: 94)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTEYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable Light
Chain Amino Sequence Lc40 (SEQ ID NO: 95)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Heavy Chain Amino
Sequence Hc40 (SEQ ID NO: 96)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPPFFGQFDYWGQGTLVTVSS Variable
Light Chain Amino Sequence Lc47 (SEQ ID NO: 97)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSVVAPLTFGQGTKVEIKR Variable Heavy Chain
Amino Sequence Hc47 (SEQ ID NO: 98)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTEYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 4B2
Light Chain (SEQ ID NO: 99)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQTLDAPPQFGQGTKVEIKR Variable 4B2 Heavy
Chain (SEQ ID NO: 100)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEQMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
4D11 Light Chain (SEQ ID NO: 101)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQGTKVEIKR Variable 4D11 Heavy
Chain (SEQ ID NO: 102)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDPEGRQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
4E7 Light Chain (SEQ ID NO: 103)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQSLVAPLTFGQGTKVEIKR Variable 4E7 Heavy
Chain (SEQ ID NO: 104)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEEMGWQTKYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 4E11
Light Chain (SEQ ID NO: 105)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQALDAPLMFGQGTKVEIKR Variable 4E11 Heavy
Chain (SEQ ID NO: 106)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIEPMGQLTEYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIGGRSAFDYWGQGTLVTVSS Variable
6B7 Light Chain (SEQ ID NO: 107)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6B7 Heavy
Chain (SEQ ID NO: 108)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS Variable 6F8
Light Chain (SEQ ID NO: 109)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQALVAPLTFGQGTKVEIKR Variable 6F8 Heavy
Chain (SEQ ID NO: 110)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIDEMGWQTYYADSV
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSAAAFDYWGQGTLVTVSS
[0206] Exemplary conjugated antibodies and/or activatable
antibodies of the disclosure include, for example, antibodies that
bind a Jagged target, e.g., Jagged-1, Jagged-2 and/or both Jagged-1
and Jagged-2, and that include a combination of a heavy chain
region and a light chain region that are, or are derived from, the
heavy chain and light chain sequences shown below.
TABLE-US-00026 4D11 Light Chain sequence: (SEQ ID NO: 111)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC
4D11 Heavy Chain sequence: (SEQ ID NO: 112)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Heavy
Chain sequence (SEQ ID NO: 113)
EVHLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSS
IDPEGRQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDI
GGRSAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4D11v2 Light
Chain Sequence (SEQ ID NO: 114)
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA
ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTVVAPPLFGQ
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLXKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC
[0207] The activatable antibodies and activatable antibody
compositions provided herein contain at least an antibody or
antibody fragment thereof (collectively referred to as AB
throughout the disclosure) that specifically binds a target, e.g.,
a human target, wherein the AB is modified by a masking moiety
(MM).
[0208] 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: 167). By
way of non-limiting examples, the MM can include a sequence such as
CISPRGC (SEQ ID NO: 497); CISPRGCG (SEQ ID NO: 168);
CISPRGCPDGPYVMY (SEQ ID NO: 160); CISPRGCPDGPYVM (SEQ ID NO: 169),
CISPRGCEPGTYVPT (SEQ ID NO: 170) and CISPRGCPGQIWHPP (SEQ ID NO:
171). 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: 172); CISPRGCGGSSASQSGQGSHCLIPINMGAPSC
(SEQ ID NO: 173); CNHHYFYTCGCISPRGCPG (SEQ ID NO: 174);
ADHVFWGSYGCISPRGCPG (SEQ ID NO: 175); CHHVYWGHCGCISPRGCPG (SEQ ID
NO: 176); CPHFTTTSCGCISPRGCPG (SEQ ID NO: 177); CNHHYHYYCGCISPRGCPG
(SEQ ID NO: 178); CPHVSFGSCGCISPRGCPG (SEQ ID NO: 179);
CPYYTLSYCGCISPRGCPG (SEQ ID NO: 180); CNHVYFGTCGCISPRGCPG (SEQ ID
NO: 181); CNHFTLTTCGCISPRGCPG (SEQ ID NO: 182); CHHFTLTTCGCISPRGCPG
(SEQ ID NO: 183); YNPCATPMCCISPRGCPG (SEQ ID NO: 184);
CNHHYFYTCGCISPRGCG (SEQ ID NO: 185); CNHHYHYYCGCISPRGCG (SEQ ID NO:
186); CNHVYFGTCGCISPRGCG (SEQ ID NO: 187); CHHVYWGHCGCISPRGCG (SEQ
ID NO: 188); CPHFTTTSCGCISPRGCG (SEQ ID NO: 189);
CNHFTLTTCGCISPRGCG (SEQ ID NO: 190); CHHFTLTTCGCISPRGCG (SEQ ID NO:
191); CPYYTLSYCGCISPRGCG (SEQ ID NO: 192); CPHVSFGSCGCISPRGCG (SEQ
ID NO: 193); ADHVFWGSYGCISPRGCG (SEQ ID NO: 194); YNPCATPMCCISPRGCG
(SEQ ID NO: 195); CHHVYWGHCGCISPRGCG (SEQ ID NO: 196);
C(N/P)H(HN/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO:
197); CISPRGCGQPIPSVK (SEQ ID NO: 198); CISPRGCTQPYHVSR (SEQ ID NO:
199); and/or CISPRGCNAVSGLGS (SEQ ID NO: 200).
[0209] 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: 496);
QGQSGQGQQQWCNIWINGGDCRGWNG (SEQ ID NO: 201); PWCMQRQDFLRCPQP (SEQ
ID NO: 202); QLGLPAYMCTFECLR (SEQ ID NO: 203); CNLWVSGGDCGGLQG (SEQ
ID NO: 204); SCSLWTSGSCLPHSP (SEQ ID NO: 205); YCLQLPHYMQAMCGR (SEQ
ID NO: 206); CFLYSCTDVSYWNNT (SEQ ID NO: 207); PWCMQRQDYLRCPQP (SEQ
ID NO: 208); CNLWISGGDCRGLAG (SEQ ID NO: 209); CNLWVSGGDCRGVQG (SEQ
ID NO: 210); CNLWVSGGDCRGLRG (SEQ ID NO: 211); CNLWISGGDCRGLPG (SEQ
ID NO: 212); CNLWVSGGDCRDAPW (SEQ ID NO: 213); CNLWVSGGDCRDLLG (SEQ
ID NO: 214); CNLWVSGGDCRGLQG (SEQ ID NO: 215); CNLWLHGGDCRGWQG (SEQ
ID NO: 216); CNIWLVGGDCRGWQG (SEQ ID NO: 217); CTTWFCGGDCGVMRG (SEQ
ID NO: 218); CNIWGPSVDCGALLG (SEQ ID NO: 219); CNIWVNGGDCRSFEG (SEQ
ID NO: 220); YCLNLPRYMQDMCWA (SEQ ID NO: 221); YCLALPHYMQADCAR (SEQ
ID NO: 222); CFLYSCGDVSYWGSA (SEQ ID NO: 223); CYLYSCTDSAFWNNR (SEQ
ID NO: 224); CYLYSCNDVSYWSNT (SEQ ID NO: 225); CFLYSCTDVSYW (SEQ ID
NO: 226); CFLYSCTDVAYWNSA (SEQ ID NO: 227); CFLYSCTDVSYWGDT (SEQ ID
NO: 228); CFLYSCTDVSYWGNS (SEQ ID NO: 229); CFLYSCTDVAYWNNT (SEQ ID
NO: 230); CFLYSCGDVSYWGNPGLS (SEQ ID NO: 231); CFLYSCTDVAYWSGL (SEQ
ID NO: 232); CYLYSCTDGSYWNST (SEQ ID NO: 233); CFLYSCSDVSYWGNI (SEQ
ID NO: 234); CFLYSCTDVAYW (SEQ ID NO: 235); CFLYSCTDVSYWGST (SEQ ID
NO: 236); CFLYSCTDVAYWGDT (SEQ ID NO: 237); GCNIWLNGGDCRGWVDPLQG
(SEQ ID NO: 238); GCNIWLVGGDCRGWIGDTNG (SEQ ID NO: 239);
GCNIWLVGGDCRGWIEDSNG (SEQ ID NO: 240); GCNIWANGGDCRGWIDNIDG (SEQ ID
NO: 241); GCNIWLVGGDCRGWLGEAVG (SEQ ID NO: 242);
GCNIWLVGGDCRGWLEEAVG (SEQ ID NO: 243); GGPALCNIWLNGGDCRGWSG (SEQ ID
NO: 244); GAPVFCNIWLNGGDCRGWMG (SEQ ID NO: 245);
GQQQWCNIWINGGDCRGWNG (SEQ ID NO: 246); GKSEFCNIWLNGGDCRGWIG (SEQ ID
NO: 247); GTPGGCNIWANGGDCRGWEG (SEQ ID NO: 248);
GASQYCNLWINGGDCRGWRG (SEQ ID NO: 249); GCNIWLVGGDCRPWVEGG (SEQ ID
NO: 250); GCNIWAVGGDCRPFVDGG (SEQ ID NO: 251); GCNIWLNGGDCRAWVDTG
(SEQ ID NO: 252); GCNIWIVGGDCRPFINDG (SEQ ID NO: 253);
GCNIWLNGGDCRPVVFGG (SEQ ID NO: 254); GCNIWLSGGDCRMFMNEG (SEQ ID NO:
255); GCNIWVNGGDCRSFVYSG (SEQ ID NO: 256); GCNIWLNGGDCRGWEASG (SEQ
ID NO: 257); GCNIWAHGGDCRGFIEPG (SEQ ID NO: 258);
GCNIWLNGGDCRTFVASG (SEQ ID NO: 259); GCNIWAHGGDCRGFIEPG (SEQ ID NO:
260); GFLENCNIWLNGGDCRTG (SEQ ID NO: 261); GIYENCNIWLNGGDCRMG (SEQ
ID NO: 262); and/or GIPDNCNIWINGGDCRYG (SEQ ID NO: 263).
[0210] 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: 264);
QGQSGQGDFDIPFPAHWVPIT (SEQ ID NO: 265); QGQSGQMGVPAGCVWNYAHIFMDC
(SEQ ID NO: 266); YRSCNWNYVSIFLDC (SEQ ID NO: 267);
PGAFDIPFPAHWVPNT (SEQ ID NO: 268); ESSCVWNYVHIYMDC (SEQ ID NO:
269); YPGCKWNYDRIFLDC (SEQ ID NO: 270); YRTCSWNYVGIFLDC (SEQ ID NO:
271); YGSCSWNYVHIFMDC (SEQ ID NO: 161); YGSCSWNYVHIFLDC (SEQ ID NO:
272); YGSCNWNYVHIFLDC (SEQ ID NO: 273); YTSCNWNYVHIFMDC (SEQ ID NO:
274); YPGCKWNYDRIFLDC (SEQ ID NO: 275); WRSCNWNYAHIFLDC (SEQ ID NO:
276); WSNCHWNYVHIFLDC (SEQ ID NO: 277); DRSCTWNYVRISYDC (SEQ ID NO:
278); SGSCKWDYVHIFLDC (SEQ ID NO: 279); SRSCIWNYAHIHLDC (SEQ ID NO:
280); SMSCYWQYERIFLDC (SEQ ID NO: 281); YRSCNWNYVSIFLDC (SEQ ID NO:
282); SGSCKWDYVHIFLDC (SEQ ID NO: 283); YKSCHWDYVHIFLDC (SEQ ID NO:
284); YGSCTWNYVHIFMEC (SEQ ID NO: 285); FSSCNWNYVHIFLDC (SEQ ID NO:
286); WRSCNWNYAHIFLDC (SEQ ID NO: 287); YGSCQWNYVHIFLDC (SEQ ID NO:
288); YRSCNWNYVHIFLDC (SEQ ID NO: 289); NMSCHWDYVHIFLDC (SEQ ID NO:
290); FGPCTWNYARISWDC (SEQ ID NO: 291); XXsCXWXYvhIfXdC (SEQ ID NO:
292); MGVPAGCVWNYAHIFMDC (SEQ ID NO: 163); RDTGGQCRWDYVHIFMDC (SEQ
ID NO: 293); AGVPAGCTWNYVHIFMEC (SEQ ID NO: 294);
VGVPNGCVWNYAHIFMEC (SEQ ID NO: 295); DGGPAGCSWNYVHIFMEC (SEQ ID NO:
296); AVGPAGCWWNYVHIFMEC (SEQ ID NO: 297); CTWNYVHIFMDCGEGEGP (SEQ
ID NO: 298); GGVPEGCTWNYAHIFMEC (SEQ ID NO: 299);
AEVPAGCWWNYVHIFMEC (SEQ ID NO: 300); AGVPAGCTWNYVHIFMEC (SEQ ID NO:
301); SGASGGCKWNYVHIFMDC (SEQ ID NO: 302); TPGCRWNYVHIFMECEAL (SEQ
ID NO: 303); VGVPNGCVWNYAHIFMEC (SEQ ID NO: 304); PGAFDIPFPAHWVPNT
(SEQ ID NO: 305); RGACDIPFPAHWIPNT (SEQ ID NO: 306);
QGDFDIPFPAHWVPIT (SEQ ID NO: 162); XGafDIPFPAHWvPnT (SEQ ID NO:
307); RGDGNDSDIPFPAHWVPRT (SEQ ID NO: 308); SGVGRDRDIPFPAHWVPRT
(SEQ ID NO: 309); WAGGNDCDIPFPAHWIPNT (SEQ ID NO: 310);
WGDGMDVDIPFPAHWVPVT (SEQ ID NO: 311); AGSGNDSDIPFPAHWVPRT (SEQ ID
NO: 312); ESRSGYADIPFPAHWVPRT (SEQ ID NO: 313); and/or
RECGRCGDIPFPAHWVPRT (SEQ ID NO: 314).
[0211] When the AB is modified with a MM and is in the presence of
the target, specific binding of the AB to its target is reduced or
inhibited, as compared to the specific binding of the AB not
modified with an MM or the specific binding of the parental AB to
the target.
[0212] The K.sub.d of the AB modified with a MM towards the target
is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000,
10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000,
50,000,000 or greater, or between 5-10, 10-100, 10-1,000,
10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,
100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times greater than the
K.sub.d of the AB not modified with an MM or of the parental AB
towards the target. Conversely, the binding affinity of the AB
modified with a MM towards the target is at least 2, 3, 4, 5, 10,
20, 25, 40, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000,
100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or
greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,
10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,
100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,
1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
lower than the binding affinity of the AB not modified with an MM
or of the parental AB towards the target.
[0213] The dissociation constant (K.sub.d) of the MM towards the AB
is generally greater than the K.sub.d of the AB towards the target.
The K.sub.d of the MM towards the AB can be at least 5, 10, 25, 50,
100, 250, 500, 1,000, 2,500, 5,000, 10,000, 100,000, 1,000,000 or
even 10,000,000 times greater than the K.sub.d of the AB towards
the target. Conversely, the binding affinity of the MM towards the
AB is generally lower than the binding affinity of the AB towards
the target. The binding affinity of MM towards the AB can be at
least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,
100,000, 1,000,000 or even 10,000,000 times lower than the binding
affinity of the AB towards the target.
[0214] When the AB is modified with a MM and is in the presence of
the target specific binding of the AB to its target is reduced or
inhibited, as compared to the specific binding of the AB not
modified with an MM or the specific binding of the parental AB to
the target. When compared to the binding of the AB not modified
with an MM or the binding of the parental AB to the target the AB's
ability to bind the target when modified with an MM can be reduced
by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36,
48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120,
150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12
months or more when measured in vivo or in an in vitro assay.
[0215] The MM inhibits the binding of the AB to the target. The MM
binds the antigen binding domain of the AB and inhibits binding of
the AB to the target. The MM can sterically inhibit the binding of
the AB to the target. The MM can allosterically inhibit the binding
of the AB to its target. In these embodiments when the AB is
modified or coupled to a MM and in the presence of target there is
no binding or substantially no binding of the AB to the target, or
no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,
9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding of the AB to
the target, as compared to the binding of the AB not modified with
an MM, the parental AB, or the AB not coupled to an MM to the
target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72,
84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180
days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer
when measured in vivo or in an in vitro assay.
[0216] When an AB is coupled to or modified by a MM, the MM `masks`
or reduces or otherwise inhibits the specific binding of the AB to
the target. When an AB is coupled to or modified by a MM, such
coupling or modification can effect a structural change that
reduces or inhibits the ability of the AB to specifically bind its
target.
[0217] An AB coupled to or modified with an MM can be represented
by the following formulae (in order from an amino (N) terminal
region to carboxyl (C) terminal region:
(MM)-(AB)
(AB)-(MM)
(MM)-L-(AB)
(AB)-L-(MM)
where MM is a masking moiety, the AB is an antibody or antibody
fragment thereof, and the L is a linker. In many embodiments, it
may be desirable to insert one or more linkers, e.g., flexible
linkers, into the composition so as to provide for flexibility.
[0218] In certain embodiments, the MM is not a natural binding
partner of the AB. In some embodiments, the MM contains no or
substantially no homology to any natural binding partner of the AB.
In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to
any natural binding partner of the AB. In some embodiments, the MM
is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding
partner of the AB. In some embodiments, the MM is no more than 25%
identical to any natural binding partner of the AB. In some
embodiments, the MM is no more than 50% identical to any natural
binding partner of the AB. In some embodiments, the MM is no more
than 20% identical to any natural binding partner of the AB. In
some embodiments, the MM is no more than 10% identical to any
natural binding partner of the AB.
[0219] In some embodiments, the activatable antibodies include an
AB that is modified by an MM and also includes one or more
cleavable moieties (CM). Such activatable antibodies exhibit
activatable/switchable binding, to the AB's target. Activatable
antibodies generally include an antibody or antibody fragment (AB),
modified by or coupled to a masking moiety (MM) and a modifiable or
cleavable moiety (CM). In some embodiments, the CM contains an
amino acid sequence that serves as a substrate for at least one
matrix metalloprotease of interest.
[0220] The elements of the activatable antibodies are arranged so
that the MM and CM are positioned such that in a cleaved (or
relatively active) state and in the presence of a target, the AB
binds a target while in an uncleaved (or relatively inactive) state
in the presence of the target, specific binding of the AB to its
target is reduced or inhibited. The specific binding of the AB to
its target can be reduced due to the inhibition or masking of the
AB's ability to specifically bind its target by the MM.
[0221] The K.sub.d of the AB modified with a MM and a CM towards
the target is at least 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000,
2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,
5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10,
10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,
10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,
100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,
1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
greater than the K.sub.d of the AB not modified with an MM and a CM
or of the parental AB towards the target. Conversely, the binding
affinity of the AB modified with a MM and a CM towards the target
is at least 2, 3, 4, 5, 10, 20, 25, 40, 50, 100, 250, 500, 1,000,
2,500, 5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000,
5,000,000, 10,000,000, 50,000,000 or greater, or between 5-10,
10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,
10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,
100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,
1000-10,000,000, 10,000-100,000, 10,000-1,000,000,
10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 times
lower than the binding affinity of the AB not modified with an MM
and a CM or of the parental AB towards the target.
[0222] When the AB is modified with a MM and a CM and is in the
presence of the target but not in the presence of a modifying agent
(for example a MMP), specific binding of the AB to its target is
reduced or inhibited, as compared to the specific binding of the AB
not modified with an MM and a CM or of the parental AB to the
target. When compared to the binding of the parental AB or the
binding of an AB not modified with an MM and a CM to its target,
the AB's ability to bind the target when modified with an MM and a
CM can be reduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4, 6, 8,
12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours or 5, 10, 15, 30,
45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months or longer when measured in vivo or in an in
vitro assay.
[0223] As used herein, the term cleaved state refers to the
condition of the activatable antibodies following modification of
the CM by at least one matrix metalloprotease. The term uncleaved
state, as used herein, refers to the condition of the activatable
antibodies in the absence of cleavage of the CM by a MMP. As
discussed above, the term "activatable antibodies" is used herein
to refer to an activatable antibody in both its uncleaved (native)
state, as well as in its cleaved state. It will be apparent to the
ordinarily skilled artisan that in some embodiments, a cleaved
activatable antibody may lack an MM due to cleavage of the CM by
protease, resulting in release of at least the MM (e.g., where the
MM is not joined to the activatable antibodies by a covalent bond
(e.g., a disulfide bond between cysteine residues).
[0224] By activatable or switchable is meant that the activatable
antibody exhibits a first level of binding to a target when in a
inhibited, masked or uncleaved state (i.e., a first conformation),
and a second level of binding to the target in the uninhibited,
unmasked and/or cleaved state (i.e., a second conformation), where
the second level of target binding is greater than the first level
of binding. In general, the access of target to the AB of the
activatable antibody is greater in the presence of a cleaving agent
capable of cleaving the CM than in the absence of such a cleaving
agent. Thus, when the activatable antibody is in the uncleaved
state, the AB is inhibited from target binding and can be masked
from target binding (i.e., the first conformation is such the AB
cannot bind the target), and in the cleaved state the AB is not
inhibited or is unmasked to target binding.
[0225] The CM and AB of the activatable antibodies are selected so
that the AB represents a binding moiety for a given target, and the
CM represents a substrate for a MMP that is co-localized with the
target at a treatment site or diagnostic site in a subject. The
activatable antibodies disclosed herein find particular use where,
for example, a MMP capable of cleaving a site in the CM is present
at relatively higher levels in target-containing tissue of a
treatment site or diagnostic site than in tissue of non-treatment
sites (for example in healthy tissue).
[0226] In some embodiments, activatable antibodies provide for
reduced toxicity and/or adverse side effects that could otherwise
result from binding of the AB at non-treatment sites if the AB were
not masked or otherwise inhibited from binding to the target.
[0227] In general, an activatable antibody can be designed by
selecting an AB of interest and constructing the remainder of the
activatable antibody so that, when conformationally constrained,
the MM provides for masking of the AB or reduction of binding of
the AB to its target. Structural design criteria can be to be taken
into account to provide for this functional feature.
[0228] Activatable antibodies exhibiting a switchable phenotype of
a desired dynamic range for target binding in an inhibited versus
an uninhibited conformation are provided. Dynamic range generally
refers to a ratio of (a) a maximum detected level of a parameter
under a first set of conditions to (b) a minimum detected value of
that parameter under a second set of conditions. For example, in
the context of an activatable antibody, the dynamic range refers to
the ratio of (a) a maximum detected level of target protein binding
to an activatable antibody in the presence of a MMP capable of
cleaving the CM of the activatable antibodies to (b) a minimum
detected level of target protein binding to an activatable antibody
in the absence of the protease. The dynamic range of an activatable
antibody can be calculated as the ratio of the equilibrium
dissociation constant of an activatable antibody cleaving agent
(e.g., enzyme) treatment to the equilibrium dissociation constant
of the activatable antibodies cleaving agent treatment. The greater
the dynamic range of an activatable antibody, the better the
switchable phenotype of the activatable antibody. Activatable
antibodies having relatively higher dynamic range values (e.g.,
greater than 1) exhibit more desirable switching phenotypes such
that target protein binding by the activatable antibodies occurs to
a greater extent (e.g., predominantly occurs) in the presence of a
cleaving agent (e.g., enzyme) capable of cleaving the CM of the
activatable antibodies than in the absence of a cleaving agent.
[0229] Activatable antibodies can be provided in a variety of
structural configurations. Exemplary formulae for activatable
antibodies are provided below. It is specifically contemplated that
the N- to C-terminal order of the AB, MM and CM may be reversed
within an activatable antibody. It is also specifically
contemplated that the CM and MM may overlap in amino acid sequence,
e.g., such that the CM is contained within the MM.
[0230] For example, activatable antibodies can be represented by
the following formula (in order from an amino (N) terminal region
to carboxyl (C) terminal region:
(MM)-(CM)-(AB)
(AB)-(CM)-(MM)
where MM is a masking moiety, CM is a cleavable moiety, and AB is
an antibody or fragment thereof. It should be noted that although
MM and CM are indicated as distinct components in the formulae
above, in all exemplary embodiments (including formulae) disclosed
herein it is contemplated that the amino acid sequences of the MM
and the CM could overlap, e.g., such that the CM is completely or
partially contained within the MM. In addition, the formulae above
provide for additional amino acid sequences that may be positioned
N-terminal or C-terminal to the activatable antibodies
elements.
[0231] In certain embodiments, the MM is not a natural binding
partner of the AB. In some embodiments, the MM contains no or
substantially no homology to any natural binding partner of the AB.
In some embodiments, the MM is no more than 5%, 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to
any natural binding partner of the AB. In some embodiments, the MM
is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, or 80% identical to any natural binding
partner of the AB. In some embodiments, the MM is no more than 50%
identical to any natural binding partner of the AB. In some
embodiments, the MM is no more than 25% identical to any natural
binding partner of the AB. In some embodiments, the MM is no more
than 20% identical to any natural binding partner of the AB. In
some embodiments, the MM is no more than 10% identical to any
natural binding partner of the AB.
[0232] In many embodiments, it may be desirable to insert one or
more linkers, e.g., flexible linkers, into the activatable antibody
construct so as to provide for flexibility at one or more of the
MM-CM junction, the CM-AB junction, or both. For example, the AB,
MM, and/or CM may not contain a sufficient number of residues
(e.g., Gly, Ser, Asp, Asn, especially Gly and Ser, particularly
Gly) to provide the desired flexibility. As such, the switchable
phenotype of such activatable antibody constructs may benefit from
introduction of one or more amino acids to provide for a flexible
linker. In addition, as described below, where the activatable
antibody is provided as a conformationally constrained construct, a
flexible linker can be operably inserted to facilitate formation
and maintenance of a cyclic structure in the uncleaved activatable
antibody.
[0233] For example, in certain embodiments, an activatable antibody
comprises one of the following formulae (where the formula below
represent an amino acid sequence in either N- to C-terminal
direction or C- to N-terminal direction):
(MM)-L1-(CM)-(AB)
(MM)-(CM)-L2-(AB)
(MM)-L1-(CM)-L2-(AB)
wherein MM, CM, and AB are as defined above; wherein L1 and L2 are
each independently and optionally present or absent, are the same
or different flexible linkers that include at least 1 flexible
amino acid (e.g., Gly). In addition, the formulae above provide for
additional amino acid sequences that may be positioned N-terminal
or C-terminal to the activatable antibodies elements. Examples
include, but are not limited to, targeting moieties (e.g., a ligand
for a receptor of a cell present in a target tissue) and serum
half-life extending moieties (e.g., polypeptides that bind serum
proteins, such as immunoglobulin (e.g., IgG) or serum albumin
(e.g., human serum albumin (HAS)).
[0234] The CM is specifically cleaved by at least one MMP at a rate
of about 0.001-1500.times.10.sup.4M.sup.-1 S.sup.-1 or at least
0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25,
50, 75, 100, 125, 150, 200, 250, 500, 750, 1000, 1250, or
1500.times.10.sup.4 M.sup.-1 S.sup.-1.
[0235] For specific cleavage by an enzyme, contact between the
enzyme and CM is made. When the activatable antibody comprising an
AB coupled to a MM and a CM is in the presence of target and
sufficient enzyme activity, the CM can be cleaved. Sufficient
enzyme activity can refer to the ability of the enzyme to make
contact with the CM and effect cleavage. It can readily be
envisioned that an enzyme may be in the vicinity of the CM but
unable to cleave because of other cellular factors or protein
modification of the enzyme.
[0236] Linkers suitable for use in compositions described herein
are generally ones that provide flexibility of the modified AB or
the activatable antibodies to facilitate the inhibition of the
binding of the AB to the target. Such linkers are generally
referred to as flexible linkers. Suitable linkers can be readily
selected and can be of any of a suitable of different lengths, such
as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino
acids to 15 amino acids, from 3 amino acids to 12 amino acids,
including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino
acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino
acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20 amino acids in length.
[0237] Exemplary flexible linkers include glycine polymers (G)n,
glycine-serine polymers (including, for example, (GS)n, (GSGGS)n
(SEQ ID NO: 1) and (GGGS)n (SEQ ID NO: 2), where n is an integer of
at least one), glycine-alanine polymers, alanine-serine polymers,
and other flexible linkers known in the art. Glycine and
glycine-serine polymers are relatively unstructured, and therefore
may be able to serve as a neutral tether between components.
Glycine accesses significantly more phi-psi space than even
alanine, and is much less restricted than residues with longer side
chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
Exemplary flexible linkers include, but are not limited to
Gly-Gly-Ser-Gly (SEQ ID NO: 3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4),
Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO:
6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 7), Gly-Ser-Ser-Ser-Gly (SEQ ID
NO: 8), and the like. The ordinarily skilled artisan will recognize
that design of an activatable antibodies can include linkers that
are all or partially flexible, such that the linker can include a
flexible linker as well as one or more portions that confer less
flexible structure to provide for a desired activatable antibodies
structure.
[0238] In some embodiments, the 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 anti-inflammatory and/or an
antineoplastic agent. In such embodiments, the agent is conjugated
to a carbohydrate moiety of the activatable antibody, for example,
in some embodiments, where the carbohydrate moiety is located
outside the antigen-binding region of the antibody or
antigen-binding fragment in the activatable antibody. In some
embodiments, the agent is conjugated to a sulfhydryl group of the
antibody or antigen-binding fragment in the activatable
antibody.
[0239] 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).
[0240] 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.
[0241] The disclosure also pertains to immunoconjugates comprising
an antibody conjugated to 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, dolastatins and derivatives thereof (e.g. auristatin E,
AFP, MMAF, MMAE, MMAD, DMAF, DMAE). For example, the agent is
monomethyl auristatin E (MMAE) or monomethyl auristatin D (MMAD).
In some embodiments, the agent is an agent selected from the group
listed in Table 3. 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
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is a pyrrolobenzodiazepine.
[0242] 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##
[0243] 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.
[0244] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. (See WO94/11026).
[0245] Table 3 lists some of the exemplary pharmaceutical agents
that may be employed in the herein described disclosure but in no
way is meant to be an exhaustive list.
TABLE-US-00027 TABLE 3 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 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)
Functionalized pyrrolobenzodiazepenes Calicheamicins
Podophyllotoxins Taxanes Vinca alkaloids CONJUGATABLE DETECTION
REAGENTS Fluorescein and derivatives thereof Fluorescein
isothiocyanate (FITC) RADIOPHARMACEUTICALS .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
[0246] 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 disclosure. (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).
[0247] Coupling may be accomplished by 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, however, 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 antibodies of the present
disclosure, 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).
[0248] 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.
[0249] 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. In some embodiments, suitable
linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)
carbodiimide hydrochloride; (ii) SMPT
(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene
(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6
[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat
#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.
#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce
Chem. Co., Cat. #24510) conjugated to EDC. Additional linkers
include, but are not limited to, SMCC, sulfo-SMCC, SPDB, or
sulfo-SPDB.
[0250] The linkers described above contain components that have
different attributes, thus leading to conjugates with differing
physio-chemical properties. For example, sulfo-NHS esters of alkyl
carboxylates are more stable than sulfo-NHS esters of aromatic
carboxylates. NHS-ester containing linkers are less soluble than
sulfo-NHS esters. Further, 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. Sulfo-NHS, in particular,
can enhance the stability of carbodimide couplings. Carbodimide
couplings (such as EDC) when used in conjunction with sulfo-NHS,
forms esters that are more resistant to hydrolysis than the
carbodimide coupling reaction alone.
[0251] 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, i.e., cleavable or non-cleavable, or the two or more
linkers are different, i.e., at least one cleavable and at least
one non-cleavable.
[0252] The present disclosure utilizes 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 disclosure, 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.
[0253] According to the present disclosure, 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).
[0254] According to the present disclosure, 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.
[0255] According to the present disclosure, 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.
[0256] 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.
[0257] Branched Linkers:
[0258] 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.
[0259] 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.
[0260] Cleavable Linkers:
[0261] 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 disclosure. According to one method of the present
disclosure, 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 disclosure,
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 3.
[0262] Non-limiting examples of cleavable linker sequences are
provided in Table 4.
TABLE-US-00028 TABLE 4 Exemplary Linker Sequences for Conjugation
Types of Cleavable Sequences Amino Acid Sequence Plasmin cleavable
sequences Pro-urokinase PRFKIIGG (SEQ ID NO: 127) PRFRIIGG (SEQ ID
NO: 128) TGF.beta. SSRHRRALD (SEQ ID NO: 129) Plasminogen
RKSSIIIRMRDVVL (SEQ ID NO: 130) Staphylokinase SSSFDKGKYKKGDDA (SEQ
ID NO: 131) SSSFDKGKYKRGDDA (SEQ ID NO: 132) Factor Xa cleavable
sequences IEGR (SEQ ID NO: 133) IDGR (SEQ ID NO: 134) GGSIDGR (SEQ
ID NO: 135) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO:
136) Collagenase cleavable sequences Calf skin collagen
(.alpha.1(I) chain) GPQGIAGQ (SEQ ID NO: 137) Calf skin collagen
(.alpha.2(I) chain) GPQGLLGA (SEQ ID NO: 138) Bovine cartilage
collagen (.alpha.1(II) chain) GIAGQ (SEQ ID NO: 139) Human liver
collagen (.alpha.1(III) chain) GPLGIAGI (SEQ ID NO: 140) Human
.alpha..sub.2M GPEGLRVG (SEQ ID NO: 141) Human PZP YGAGLGVV (SEQ ID
NO: 142) AGLGVVER (SEQ ID NO: 143) AGLGISST (SEQ ID NO: 144) Rat
.alpha..sub.1M EPQALAMS (SEQ ID NO: 145) QALAMSAI (SEQ ID NO: 146)
Rat .alpha..sub.2M AAYHLVSQ (SEQ ID NO: 147) MDAFLESS (SEQ ID NO:
148) Rat .alpha..sub.1I.sub.3(2J) ESLPVVAV (SEQ ID NO: 149) Rat
.alpha..sub.1I.sub.3(27J) SAPAVESE (SEQ ID NO: 150) Human
fibroblast collagenase DVAQFVLT (SEQ ID NO: 151) (autolytic
cleavages) VAQFVLTE (SEQ ID NO: 152) AQFVLTEG (SEQ ID NO: 153)
PVQPIGPQ (SEQ ID NO: 154)
[0263] 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.
[0264] Spacers and Cleavable Elements:
[0265] 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.
[0266] 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.
[0267] 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 particular mode
of attachment or reaction. Any reaction providing a product of
suitable stability and biological compatibility is acceptable.
[0268] Serum Complement and Selection of Linkers:
[0269] According to one method of the present disclosure, 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 disclosure, 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.
[0270] 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.
[0271] 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 3.
[0272] Linkers for Release without Complement Activation:
[0273] 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.
[0274] Biochemical Cross Linkers:
[0275] 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.
[0276] 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.
[0277] Exemplary hetero-bifunctional cross-linkers are referenced
in Table 5.
TABLE-US-00029 TABLE 5 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
reactive 11.6 .ANG. group Sulfhydryls Enzyme-antibody conjugation
Hapten-carrier proteinconjugation Sulfo-SMCC Primary amines Stable
maleimide reactive 11.6 .ANG. group Sulfhydryls Water-soluble
Enzyme-antibody conjugation MBS Primary amines Enzyme-antibody 9.9
.ANG. Sulfhydryls conjugation Hapten-carrier protein conjugation
Sulfo-MBS Primary amines Water-soluble 9.9 .ANG. Sulfhydryls SIAB
Primary amines Enzyme-antibody 10.6 .ANG. Sulfhydryls conjugation
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 conjugation 0 NHS Carboxyl groups ABH
Carbohydrates Reacts with sugar groups 11.9 .ANG. Nonselective
[0278] Non-Cleavable Linkers or Direct Attachment:
[0279] In some embodiments of the disclosure, 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.
[0280] 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.
[0281] Non-Cleavable Conjugates:
[0282] 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.
[0283] The antibodies disclosed herein can also be formulated as
immunoliposomes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.
Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0284] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present disclosure
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction.
DEFINITIONS
[0285] Unless otherwise defined, scientific and technical terms
used in connection with the present disclosure shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The term "a" entity or "an" entity refers to one or more
of that entity. For example, a compound refers to one or more
compounds. As such, the terms "a", "an", "one or more" and "at
least one" can be used interchangeably. 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.
[0286] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0287] 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.
[0288] 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.
[0289] 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.
[0290] 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).
[0291] 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, .ltoreq.100 nM and in some
embodiments, .ltoreq.10 nM.
[0292] As used herein, the terms "specific binding," "immunological
binding," and "immunological binding properties" refer to the
non-covalent interactions of the type which 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
disclosure is said to specifically bind to the target, when the
equilibrium binding constant (K.sub.d) is .ltoreq.1 .mu.M, in some
embodiments 100 nM, in some embodiments .ltoreq.10 nM, and in some
embodiments .ltoreq.100 pM to about 1 pM, as measured by assays
such as radioligand binding assays or similar assays known to those
skilled in the art.
[0293] 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 which 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 disclosure
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.
[0294] The term "isolated protein" referred to herein means a
protein of cDNA, recombinant RNA, or 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, e.g., free of murine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0295] 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
disclosure 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.
[0296] 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.
[0297] 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.
[0298] 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, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence in eukaryotes, generally, such
control sequences include promoters 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.
[0299] 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 and in some
embodiments, 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
disclosure are either sense or antisense oligonucleotides.
[0300] 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.
[0301] 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
disclosure. Examples of unconventional amino acids include: 4
hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .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.
[0302] 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".
[0303] 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, at least
90 percent sequence identity, in some embodiments, at least 95
percent sequence identity, and in some embodiments, at least 99
percent sequence identity.
[0304] In some embodiments, residue positions that are not
identical differ by conservative amino acid substitutions.
[0305] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present disclosure,
providing that the variations in the amino acid sequence maintain
at least 75%, in some embodiments, at least 80%, 90%, 95%, and in
some embodiments, 99%. 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 properties of the resulting
molecule, especially if the replacement does not involve an amino
acid within a framework site. 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. Suitable 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
disclosure.
[0306] Suitable amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (5) 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 (for example, conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(for example, 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).
[0307] 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
5, 6, 8 or 10 amino acids long, in some embodiments, at least 14
amino acids long, in some embodiments, at least 20 amino acids
long, usually at least 50 amino acids long, and in some
embodiments, 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 the 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.
[0308] 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.
[0309] 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 reduce 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.
[0310] 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)).
[0311] 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.
[0312] 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.
[0313] The term patient includes human and veterinary subjects.
[0314] Activatable antibodies of the disclosure specifically bind a
given target, e.g., a human target protein. Also included in the
disclosure are activatable antibodies that bind to the same epitope
as the activatable antibodies described herein.
[0315] Those skilled in the art will recognize that it is possible
to determine, without undue experimentation, if a monoclonal
antibody (e.g., a murine monoclonal or humanized antibody) has the
same specificity as a monoclonal antibody used in the methods
described herein by ascertaining whether the former prevents the
latter from binding to the target. If the monoclonal antibody being
tested competes with the monoclonal antibody of the disclosure, as
shown by a decrease in binding by the monoclonal antibody of the
disclosure, then the two monoclonal antibodies bind to the same, or
a closely related, epitope. A method for determining whether a
monoclonal antibody has the specificity of a monoclonal antibody of
the disclosure is to pre-incubate the monoclonal antibody of the
disclosure with the target and then add the monoclonal antibody
being tested to determine if the monoclonal antibody being tested
is inhibited in its ability to bind the target. If the monoclonal
antibody being tested is inhibited then, in all likelihood, it has
the same, or functionally equivalent, epitopic specificity as the
monoclonal antibody of the disclosure.
[0316] Multispecific Activatable Antibodies
[0317] The disclosure also provides multispecific activatable
antibodies. The multispecific activatable antibodies provided
herein are multispecific antibodies that recognize two or more
different antigens or epitopes and that include at least one
masking moiety (MM) linked to at least one antigen- or
epitope-binding domain of the multispecific antibody such that
coupling of the MM reduces the ability of the antigen- or
epitope-binding domain to bind its target. In some embodiments, the
MM is coupled to the antigen- or epitope-binding domain of the
multispecific antibody via a cleavable moiety (CM) that functions
as a substrate for at least one MMP protease. The activatable
multispecific antibodies provided herein are stable in circulation,
activated at intended sites of therapy and/or diagnosis but not in
normal, i.e., healthy tissue, and, when activated, exhibit binding
to a target that is at least comparable to the corresponding,
unmodified multispecific antibody.
[0318] In some embodiments, the multispecific activatable
antibodies are designed to engage immune effector cells, also
referred to herein as immune-effector cell engaging multispecific
activatable antibodies. In some embodiments, the multispecific
activatable antibodies are designed to engage leukocytes, also
referred to herein as leukocyte engaging multispecific activatable
antibodies. In some embodiments, the multispecific activatable
antibodies are designed to engage T cells, also referred to herein
as T-cell engaging multispecific activatable antibodies. In some
embodiments, the multispecific activatable antibodies engage a
surface antigen on a leukocyte, such as on a T cell, on a natural
killer (NK) cell, on a myeloid mononuclear cell, on a macrophage,
and/or on another immune effector cell. In some embodiments, the
immune effector cell is a leukocyte. In some embodiments, the
immune effector cell is a T cell. In some embodiments, the immune
effector cell is a NK cell. In some embodiments, the immune
effector cell is a mononuclear cell, such as a myeloid mononuclear
cell. In some embodiments, the multispecific activatable antibodies
are designed to bind or otherwise interact with more than one
target and/or more than one epitope, also referred to herein as
multi-antigen targeting activatable antibodies. As used herein, the
terms "target" and "antigen" are used interchangeably.
[0319] In some embodiments, immune effector cell engaging
multispecific activatable antibodies of the disclosure include a
targeting antibody or antigen-binding fragment thereof and an
immune effector cell engaging antibody or antigen-binding portion
thereof, where at least one of the targeting antibody or
antigen-binding fragment thereof and/or the immune effector cell
engaging antibody or antigen-binding portion thereof is masked. In
some embodiments, the immune effector cell engaging antibody or
antigen binding fragment thereof includes a first antibody or
antigen-binding fragment thereof (AB1) that binds a first, immune
effector cell engaging target, where the AB1 is attached to a
masking moiety (MM1) such that coupling of the MM1 reduces the
ability of the AB1 to bind the first target. In some embodiments,
the targeting antibody or antigen-binding fragment thereof includes
a second antibody or fragment thereof that includes a second
antibody or antigen-binding fragment thereof (AB2) that binds a
second target, where the AB2 is attached to a masking moiety (MM2)
such that coupling of the MM2 reduces the ability of the AB2 to
bind the second target. In some embodiments, the immune effector
cell engaging antibody or antigen binding fragment thereof includes
a first antibody or antigen-binding fragment thereof (AB1) that
binds a first, immune effector cell engaging target, where the AB1
is attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind the first target, and the
targeting antibody or antigen-binding fragment thereof includes a
second antibody or fragment thereof that includes a second antibody
or antigen-binding fragment thereof (AB2) that binds a second
target, where the AB2 is attached to a masking moiety (MM2) such
that coupling of the MM2 reduces the ability of the AB2 to bind the
second target. In some embodiments, the non-immune effector cell
engaging antibody is a cancer targeting antibody. In some
embodiments the non-immune cell effector antibody is an IgG. In
some embodiments the immune effector cell engaging antibody is a
scFv. In some embodiments the targeting antibody (e.g., non-immune
cell effector antibody) is an IgG and the immune effector cell
engaging antibody is a scFv. In some embodiments, the immune
effector cell is a leukocyte. In some embodiments, the immune
effector cell is a T cell. In some embodiments, the immune effector
cell is a NK cell. In some embodiments, the immune effector cell is
a myeloid mononuclear cell.
[0320] In some embodiments, T-cell engaging multispecific
activatable antibodies of the disclosure include a targeting
antibody or antigen-binding fragment thereof and a T-cell engaging
antibody or antigen-binding portion thereof, where at least one of
the targeting antibody or antigen-binding fragment thereof and/or
the T-cell engaging antibody or antigen-binding portion thereof is
masked. In some embodiments, the T-cell engaging antibody or
antigen binding fragment thereof includes a first antibody or
antigen-binding fragment thereof (AB1) that binds a first, T-cell
engaging target, where the AB1 is attached to a masking moiety
(MM1) such that coupling of the MM1 reduces the ability of the AB1
to bind the first target. In some embodiments, the targeting
antibody or antigen-binding fragment thereof includes a second
antibody or fragment thereof that includes a second antibody or
antigen-binding fragment thereof (AB2) that binds a second target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second target. In some embodiments, the T-cell engaging antibody or
antigen binding fragment thereof includes a first antibody or
antigen-binding fragment thereof (AB1) that binds a first, T-cell
engaging target, where the AB1 is attached to a masking moiety
(MM1) such that coupling of the MM1 reduces the ability of the AB1
to bind the first target, and the targeting antibody or
antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second target, where the AB2 is
attached to a masking moiety (MM2) such that coupling of the MM2
reduces the ability of the AB2 to bind the second target.
[0321] In some embodiments, the T-cell engaging multispecific
activatable antibodies include a cancer targeting antibody or
antigen-binding fragment thereof and a T-cell engaging antibody or
antigen-binding portion thereof, where at least one of the cancer
targeting antibody or antigen-binding fragment thereof and/or the
T-cell engaging antibody or antigen-binding portion thereof is
masked. In some embodiments, the T-cell engaging antibody or
antigen binding fragment thereof includes a first antibody or
antigen-binding fragment thereof (AB1) that binds a first, T-cell
engaging target, where the AB1 is attached to a masking moiety
(MM1) such that coupling of the MM1 reduces the ability of the AB1
to bind the first target. In some embodiments, the cancer targeting
antibody or antigen-binding fragment thereof includes a second
antibody or fragment thereof that includes a second antibody or
antigen-binding fragment thereof (AB2) that binds a second,
cancer-related target, where the AB2 is attached to a masking
moiety (MM2) such that coupling of the MM2 reduces the ability of
the AB2 to bind the second, cancer-related target. In some
embodiments, the T-cell engaging antibody or antigen binding
fragment thereof includes a first antibody or antigen-binding
fragment thereof (AB1) that binds a first, T-cell engaging target,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind the
first target, and the cancer targeting antibody or antigen-binding
fragment thereof includes a second antibody or fragment thereof
that includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second, cancer-related target, where the AB2 is
attached to a masking moiety (MM2) such that coupling of the MM2
reduces the ability of the AB2 to bind the second, cancer-related
target.
[0322] In some embodiments, the T-cell engaging multispecific
activatable antibodies include a cancer targeting IgG antibody or
antigen-binding fragment thereof and a T-cell engaging scFv, where
at least one of the cancer targeting IgG antibody or
antigen-binding fragment thereof and/or the T-cell engaging
antibody or antigen-binding portion thereof is masked. In some
embodiments, the T-cell engaging antibody or antigen binding
fragment thereof includes a first antibody or antigen-binding
fragment thereof (AB1) that binds a first, T-cell engaging target,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind the
first target. In some embodiments, the cancer targeting IgG
antibody or antigen-binding fragment thereof includes a second
antibody or fragment thereof that includes a second antibody or
antigen-binding fragment thereof (AB2) that binds a second,
cancer-related target, where the AB2 is attached to a masking
moiety (MM2) such that coupling of the MM2 reduces the ability of
the AB2 to bind the second, cancer-related target. In some
embodiments, the T-cell engaging antibody or antigen binding
fragment thereof includes a first antibody or antigen-binding
fragment thereof (AB1) that binds a first, T-cell engaging target,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind the
first target, and the cancer targeting IgG antibody or
antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second, cancer-related target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second, cancer-related target.
[0323] In some embodiments of an immune effector cell engaging
multispecific activatable antibody, one antigen is typically an
antigen present on the surface of a tumor cell or other cell type
associated with disease, such as, but not limited to, any target
listed in Table 1, such as, but not limited to, EGFR, erbB2, EpCAM,
Jagged, PD-L1, B7H3, or CD71 (transferrin receptor), and another
antigen is typically a stimulatory or inhibitory receptor present
on the surface of a T-cell, natural killer (NK) cell, myeloid
mononuclear cell, macrophage, and/or other immune effector cell,
such as, but not limited to, B7-H4, BTLA, CD3, CD4, CD8, CD16a,
CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM, ICOS,
LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. In some
embodiments, the antigen is a stimulatory receptor present on the
surface of a T cell or NK cell; examples of such stimulatory
receptors include, but are not limited to, CD3, CD27, CD28, CD137
(also referred to as 4-1BB), GITR, HVEM, ICOS, NKG2D, and OX40. In
some embodiments, the antigen is an inhibitory receptor present on
the surface of a T-cell; examples of such inhibitory receptors
include, but are not limited to, BTLA, CTLA-4, LAG3, PD-1, TIGIT,
TIM3, and NK-expressed KIRs. The antibody domain conferring
specificity to the T-cell surface antigen may also be substituted
by a ligand or ligand domain that binds to a T-cell receptor, a
NK-cell receptor, a macrophage receptor, and/or other immune
effector cell receptor, such as, but not limited to, B7-1, B7-2,
B7H3, PD-L1, PD-L2, or TNFSF9.
[0324] One embodiment of the disclosure is a multispecific
activatable antibody that is activatable in a cancer
microenvironment and that includes an antibody, for example a IgG
or scFv, directed to a tumor target and an agonist antibody, for
example an IgG or scFv, directed to a co-stimulatory receptor
expressed on the surface of an activated T cell or NK cell, wherein
at least one of the cancer target antibody and/or agonist antibody
is masked. Examples of co-stimulatory receptors include, but are
not limited to, CD27, CD137, GITR, HVEM, NKG2D, and OX40. In this
embodiment, the multispecific activatable antibody, once activated
by tumor-associated proteases, would effectively crosslink and
activate the T cell or NK cell expressed co-stimulatory receptors
in a tumor-dependent manner to enhance the activity of T cells that
are responding to any tumor antigen via their endogenous T cell
antigen or NK-activating receptors. The activation-dependent nature
of these T cell or NK cell costimulatory receptors would focus the
activity of the activated multispecific activatable antibody to
tumor-specific T cells, without activating all T cells independent
of their antigen specificity. In one embodiment, at least the
co-stimulatory receptor antibody of the multispecific activatable
antibody is masked to prevent activation of auto-reactive T cells
that may be present in tissues that also express the antigen
recognized by the tumor target-directed antibody in the
multispecific activatable antibody, but whose activity is
restricted by lack of co-receptor engagement.
[0325] One embodiment of the disclosure is a multispecific
activatable antibody that is activatable in a disease characterized
by T cell overstimulation, such as, but not limited to, an
autoimmune disease or inflammatory disease microenvironment. Such a
multispecific activatable antibody includes an antibody, for
example a IgG or scFv, directed to a target comprising a surface
antigen expressed in a tissue targeted by a T cell in autoimmune or
inflammatory disease and an antibody, for example a IgG or scFv,
directed to an inhibitory receptor expressed on the surface of a T
cell or NK cell, wherein at least one of the disease tissue target
antibody and/or T cell inhibitory receptor antibody is masked.
Examples of inhibitory receptors include, but are not limited to,
BTLA, CTLA-4, LAG3, PD-1, TIGIT, TIM3, and NK-expressed KIRs.
Examples of a tissue antigen targeted by T cells in autoimmune
disease include, but are not limited to, a surface antigen
expressed on myelin or nerve cells in multiple sclerosis or a
surface antigen expressed on pancreatic islet cells in Type 1
diabetes. In this embodiment, the multispecific activatable
antibody when localized in the tissue under autoimmune attack or
inflammation is activated and co-engages the T cell or NK cell
inhibitory receptor to suppress the activity of autoreactive T
cells responding to any disease tissue-targeted antigens via their
endogenous TCR or activating receptors. In one embodiment, at least
one or multiple antibodies are masked to prevent suppression of T
cell responses in non-disease tissues where the target antigen may
also be expressed.
[0326] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CD3 epsilon (CD3.epsilon.,
also referred to herein as CD3e and CD3) scFv and a targeting
antibody or antigen-binding fragment thereof, where at least one of
the anti-CD3.epsilon. scFv and/or the targeting antibody or
antigen-binding portion thereof is masked. In some embodiments, the
CD3.epsilon. scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CD3.epsilon., where the AB1 is
attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind CD3.epsilon.. In some
embodiments, the targeting antibody or antigen-binding fragment
thereof includes a second antibody or fragment thereof that
includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second target, where the AB2 is attached to a
masking moiety (MM2) such that coupling of the MM2 reduces the
ability of the AB2 to bind the second target. In some embodiments,
the CD3.epsilon.scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CD3.epsilon., where the AB1 is
attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind CD3.epsilon., and the
targeting antibody or antigen-binding fragment thereof includes a
second antibody or fragment thereof that includes a second antibody
or antigen-binding fragment thereof (AB2) that binds a second
target, where the AB2 is attached to a masking moiety (MM2) such
that coupling of the MM2 reduces the ability of the AB2 to bind the
second target.
[0327] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CD3.epsilon. scFv and a
cancer targeting antibody or antigen-binding fragment thereof,
where at least one of the anti-CD3.epsilon. scFv and/or the cancer
targeting antibody or antigen-binding portion thereof is masked. In
some embodiments, the CD3.epsilon. scFv includes a first antibody
or antigen-binding fragment thereof (AB1) that binds CD3.epsilon.,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind
CD3.epsilon.. In some embodiments, the cancer targeting antibody or
antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second, cancer-related target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second, cancer-related target. In some embodiments, the
CD3.epsilon. scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CD3.epsilon., where the AB1 is
attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind CD3.epsilon., and the cancer
targeting antibody or antigen-binding fragment thereof includes a
second antibody or fragment thereof that includes a second antibody
or antigen-binding fragment thereof (AB2) that binds a second,
cancer-related target, where the AB2 is attached to a masking
moiety (MM2) such that coupling of the MM2 reduces the ability of
the AB2 to bind the second, cancer-related target.
[0328] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CD3.epsilon. scFv and a
cancer targeting IgG antibody or antigen-binding fragment thereof,
where at least one of the anti-CD3.epsilon. scFv and/or the cancer
targeting IgG antibody or antigen-binding portion thereof is
masked. In some embodiments, the CD3.epsilon. scFv includes a first
antibody or antigen-binding fragment thereof (AB1) that binds
CD3.epsilon., where the AB1 is attached to a masking moiety (MM1)
such that coupling of the MM1 reduces the ability of the AB1 to
bind CD3.epsilon.. In some embodiments, the cancer targeting IgG
antibody or antigen-binding fragment thereof includes a second
antibody or fragment thereof that includes a second antibody or
antigen-binding fragment thereof (AB2) that binds a second,
cancer-related target, where the AB2 is attached to a masking
moiety (MM2) such that coupling of the MM2 reduces the ability of
the AB2 to bind the second, cancer-related target. In some
embodiments, the CD3.epsilon. scFv includes a first antibody or
antigen-binding fragment thereof (AB1) that binds CD3.epsilon.,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind
CD3.epsilon., and the cancer targeting antibody IgG or
antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second, cancer-related target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second, cancer-related target.
[0329] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CD3 epsilon (CD3.epsilon.)
scFv that is derived from OKT3, where at least one of the targeting
antibody or antigen-binding fragment thereof and/or the OKT3 scFv
or OKT3-derived scFv is masked. In some embodiments, the OKT3 scFv
or OKT3-derived scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CD3.epsilon., where the AB1 is
attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind CD3.epsilon.. In some
embodiments, the targeting antibody or antigen-binding fragment
thereof includes a second antibody or fragment thereof that
includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second target, where the AB2 is attached to a
masking moiety (MM2) such that coupling of the MM2 reduces the
ability of the AB2 to bind the second target. In some embodiments,
the OKT3 scFv or OKT3-derived scFv includes a first antibody or
antigen-binding fragment thereof (AB1) that binds CD3.epsilon.,
where the AB1 is attached to a masking moiety (MM1) such that
coupling of the MM1 reduces the ability of the AB1 to bind
CD3.epsilon., and the targeting antibody or antigen-binding
fragment thereof includes a second antibody or fragment thereof
that includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second target, where the AB2 is attached to a
masking moiety (MM2) such that coupling of the MM2 reduces the
ability of the AB2 to bind the second target.
[0330] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an OKT3 scFv or OKT3-derived scFv and
a cancer targeting antibody or antigen-binding fragment thereof,
where at least one of the OKT3 scFv or OKT3-derived scFv and/or the
cancer targeting antibody or antigen-binding portion thereof is
masked. In some embodiments, the OKT3 scFv or OKT3-derived scFv
includes a first antibody or antigen-binding fragment thereof (AB1)
that binds CD3.epsilon., where the AB1 is attached to a masking
moiety (MM1) such that coupling of the MM1 reduces the ability of
the AB1 to bind CD3.epsilon.. In some embodiments, the cancer
targeting antibody or antigen-binding fragment thereof includes a
second antibody or fragment thereof that includes a second antibody
or antigen-binding fragment thereof (AB2) that binds a second,
cancer-related target, where the AB2 is attached to a masking
moiety (MM2) such that coupling of the MM2 reduces the ability of
the AB2 to bind the second, cancer-related target. In some
embodiments, the OKT3 scFv or OKT3-derived scFv includes a first
antibody or antigen-binding fragment thereof (AB1) that binds
CD3.epsilon., where the AB1 is attached to a masking moiety (MM1)
such that coupling of the MM1 reduces the ability of the AB1 to
bind CD3.epsilon., and the cancer targeting antibody or
antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second, cancer-related target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second, cancer-related target.
[0331] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an OKT3 scFv or OKT3-derived scFv and
a cancer targeting IgG antibody or antigen-binding fragment
thereof, where at least one of the OKT3 scFv or OKT3-derived scFv
and/or the cancer targeting IgG antibody or antigen-binding portion
thereof is masked. In some embodiments, the OKT3 scFv or
OKT3-derived scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CD3.epsilon., where the AB1 is
attached to a masking moiety (MM1) such that coupling of the MM1
reduces the ability of the AB1 to bind CD3.epsilon.. In some
embodiments, the cancer targeting IgG antibody or antigen-binding
fragment thereof includes a second antibody or fragment thereof
that includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second, cancer-related target, where the AB2 is
attached to a masking moiety (MM2) such that coupling of the MM2
reduces the ability of the AB2 to bind the second, cancer-related
target. In some embodiments, the OKT3 scFv or OKT3-derived scFv
includes a first antibody or antigen-binding fragment thereof (AB1)
that binds CD3.epsilon., where the AB1 is attached to a masking
moiety (MM1) such that coupling of the MM1 reduces the ability of
the AB1 to bind CD3.epsilon., and the cancer targeting antibody IgG
or antigen-binding fragment thereof includes a second antibody or
fragment thereof that includes a second antibody or antigen-binding
fragment thereof (AB2) that binds a second, cancer-related target,
where the AB2 is attached to a masking moiety (MM2) such that
coupling of the MM2 reduces the ability of the AB2 to bind the
second, cancer-related target.
[0332] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CTLA-4 scFv, where at least
one of the targeting antibody or antigen-binding fragment thereof
and/or the anti-CTLA-4 scFv is masked. In some embodiments, the
anti-CTLA-4 scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CTLA-4, where the AB1 is attached
to a masking moiety (MM1) such that coupling of the MM1 reduces the
ability of the AB1 to bind CTLA-4. In some embodiments, the
targeting antibody or antigen-binding fragment thereof includes a
second antibody or fragment thereof that includes a second antibody
or antigen-binding fragment thereof (AB2) that binds a second
target, where the AB2 is attached to a masking moiety (MM2) such
that coupling of the MM2 reduces the ability of the AB2 to bind the
second target. In some embodiments, the anti-CTLA-4 scFv includes a
first antibody or antigen-binding fragment thereof (AB1) that binds
CTLA-4, where the AB1 is attached to a masking moiety (MM1) such
that coupling of the MM1 reduces the ability of the AB1 to bind
CTLA-4, and the targeting antibody or antigen-binding fragment
thereof includes a second antibody or fragment thereof that
includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second target, where the AB2 is attached to a
masking moiety (MM2) such that coupling of the MM2 reduces the
ability of the AB2 to bind the second target.
[0333] In some embodiments, the T-cell engaging multispecific
activatable antibody includes an anti-CTLA-4 scFv and a targeting
IgG antibody or antigen-binding fragment thereof, where at least
one of the anti-CTLA-4 scFv and/or the targeting IgG antibody or
antigen-binding portion thereof is masked. In some embodiments, the
anti-CTLA-4 scFv includes a first antibody or antigen-binding
fragment thereof (AB1) that binds CTLA-4, where the AB1 is attached
to a masking moiety (MM1) such that coupling of the MM1 reduces the
ability of the AB1 to bind CTLA-4. In some embodiments, the
targeting IgG antibody or antigen-binding fragment thereof includes
a second antibody or fragment thereof that includes a second
antibody or antigen-binding fragment thereof (AB2) that binds a
second target, where the AB2 is attached to a masking moiety (MM2)
such that coupling of the MM2 reduces the ability of the AB2 to
bind the second target. In some embodiments, the anti-CTLA-4 scFv
includes a first antibody or antigen-binding fragment thereof (AB1)
that binds CTLA-4, where the AB1 is attached to a masking moiety
(MM1) such that coupling of the MM1 reduces the ability of the AB1
to bind CTLA-4, and the targeting antibody IgG or antigen-binding
fragment thereof includes a second antibody or fragment thereof
that includes a second antibody or antigen-binding fragment thereof
(AB2) that binds a second target, where the AB2 is attached to a
masking moiety (MM2) such that coupling of the MM2 reduces the
ability of the AB2 to bind the second target.
[0334] In some embodiments, the multi-antigen targeting antibodies
and/or multi-antigen targeting activatable antibodies include at
least a first antibody or antigen-binding fragment thereof that
binds a first target and/or first epitope and a second antibody or
antigen-binding fragment thereof that binds a second target and/or
a second epitope. In some embodiments, the multi-antigen targeting
antibodies and/or multi-antigen targeting activatable antibodies
bind two or more different targets. In some embodiments, the
multi-antigen targeting antibodies and/or multi-antigen targeting
activatable antibodies bind two or more different epitopes on the
same target. In some embodiments, the multi-antigen targeting
antibodies and/or multi-antigen targeting activatable antibodies
bind a combination of two or more different targets and two or more
different epitopes on the same target.
[0335] In some embodiments, a multispecific activatable antibody
comprising an IgG has the IgG variable domains masked. In some
embodiments, a multispecific activatable antibody comprising a scFv
has the scFv domains masked. In some embodiments, a multispecific
activatable antibody has both IgG variable domains and scFv
domains, where at least one of the IgG variable domains is coupled
to a masking moiety. In some embodiments, a multispecific
activatable antibody has both IgG variable domains and scFv
domains, where at least one of the scFv domains is coupled to a
masking moiety. In some embodiments, a multispecific activatable
antibody has both IgG variable domains and scFv domains, where at
least one of the IgG variable domains is coupled to a masking
moiety and at least one of the scFv domains is coupled to a masking
moiety. In some embodiments, a multispecific activatable antibody
has both IgG variable domains and scFv domains, where each of the
IgG variable domains and the scFv domains is coupled to its own
masking moiety. In some embodiments, one antibody domain of a
multispecific activatable antibody has specificity for a target
antigen and another antibody domain has specificity for a T-cell
surface antigen. In some embodiments, one antibody domain of a
multispecific activatable antibody has specificity for a target
antigen and another antibody domain has specificity for another
target antigen. In some embodiments, one antibody domain of a
multispecific activatable antibody has specificity for an epitope
of a target antigen and another antibody domain has specificity for
another epitope of the target antigen.
[0336] In a multispecific activatable antibody, a scFv can be fused
to the carboxyl terminus of the heavy chain of an IgG activatable
antibody, to the carboxyl terminus of the light chain of an IgG
activatable antibody, or to the carboxyl termini of both the heavy
and light chains of an IgG activatable antibody. In a multispecific
activatable antibody, a scFv can be fused to the amino terminus of
the heavy chain of an IgG activatable antibody, to the amino
terminus of the light chain of an IgG activatable antibody, or to
the amino termini of both the heavy and light chains of an IgG
activatable antibody. In a multispecific activatable antibody, a
scFv can be fused to any combination of one or more carboxyl
termini and one or more amino termini of an IgG activatable
antibody. In some embodiments, a masking moiety (MM) linked to a
cleavable moiety (CM) is attached to and masks an antigen binding
domain of the IgG. In some embodiments, a masking moiety (MM)
linked to a cleavable moiety (CM) is attached to and masks an
antigen binding domain of at least one scFv. In some embodiments, a
masking moiety (MM) linked to a cleavable moiety (CM) is attached
to and masks an antigen binding domain of an IgG and a masking
moiety (MM) linked to a cleavable moiety (CM) is attached to and
masks an antigen binding domain of at least one scFv.
[0337] The disclosure provides examples of multispecific
activatable antibody structures which include, but are not limited
to, the following:
(VL-CL).sub.2:(VH-CH1-CH2-CH3-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2;
(VL-CL).sub.2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2;
(MM-L1-CM-L2-VL-CL).sub.2: (VH-CH1-CH2-CH3-L4-VH*-L3-VL*).sub.2;
(MM-L1-CM-L2-VL-CL).sub.2:(VH-CH1-CH2-CH3-L4-VL*-L3-VH*).sub.2;
(VL-CL).sub.2:(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL).sub.2:(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(MM-L1-CM-L2-VL-CL).sub.2: (VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(MM-L1-CM-L2-VL-CL).sub.2:(VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2: (VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2: (VH-CH1-CH2-CH3).sub.2;
(MM-L1-CM-L2-VL*-L3-VH*-L4-VL-CL).sub.2:(VH-CH1-CH2-CH3).sub.2;
(MM-L1-CM-L2-VH*-L3-VL*-L4-VL-CL).sub.2: (VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2:
(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2:
(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2:
(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2:
(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*).sub.2:
(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*).sub.2:
(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*).sub.2:
(MM-L1-CM-L2-VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*).sub.2:
(MM-L1-CM-L2-VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2:
(VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VH*-L3-VL*-L2-CM-L1-MM).sub.2:
(VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2;
(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2:
(VL*-L3-VH*-L4-VH-CH1-CH2-CH3).sub.2; or
(VL-CL-L4-VL*-L3-VH*-L2-CM-L1-MM).sub.2:
(VH*-L3-VL*-L4-VH-CH1-CH2-CH3).sub.2, wherein: VL and VH represent
the light and heavy variable domains of the first specificity,
contained in the IgG; VL* and VH* represent the variable domains of
the second specificity, contained in the scFv; L1 is a linker
peptide connecting the masking moiety (MM) and the cleavable moiety
(CM); L2 is a linker peptide connecting the cleavable moiety (CM),
and the antibody; L3 is a linker peptide connecting the variable
domains of the scFv; L4 is a linker peptide connecting the antibody
of the first specificity to the antibody of the second specificity;
CL is the light-chain constant domain; and CH1, CH2, CH3 are the
heavy chain constant domains. The first and second specificities
may be toward any antigen or epitope.
[0338] In some embodiments of a T-cell engaging multispecific
activatable antibody, one antigen is typically an antigen present
on the surface of a tumor cell or other cell type associated with
disease, such as, but not limited to, any target listed in Table 1,
such as, but not limited to, EGFR, erbB2, EpCAM, Jagged, PD-L1,
B7H3, or CD71 (transferrin receptor), and another antigen is
typically a stimulatory (also referred to herein as activating) or
inhibitory receptor present on the surface of a T-cell, natural
killer (NK) cell, myeloid mononuclear cell, macrophage, and/or
other immune effector cell, such as, but not limited to, B7-H4,
BTLA, CD3, CD4, CD8, CD16a, CD25, CD27, CD28, CD32, CD56, CD137
(also referred to as TNFRSF9), CTLA-4, GITR, HVEM, ICOS, LAG3,
NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA. The antibody domain
conferring specificity to the T-cell surface antigen may also be
substituted by a ligand or ligand domain that binds to a T-cell
receptor, a NK-cell receptor, a macrophage receptor, and/or other
immune effector cell receptor, such as, but not limited to, B7-1,
B7-2, B7H3, PD-L1, PD-L2, or TNFSF9. In some embodiments of a
multi-antigen targeting activatable antibody, one antigen is
selected from the group of targets listed in Table 1, and another
antigen is selected from the group of targets listed in Table
1.
[0339] In some embodiments, the targeting antibody is an anti-EGFR
antibody. In some embodiments, the targeting antibody is C225v5,
which is specific for binding to EGFR. In some embodiments, the
targeting antibody is C225, which is specific for binding to EGFR.
In some embodiments, the targeting antibody is C225v4, which is
specific for binding to EGFR. In some embodiments, the targeting
antibody is C225v6, which is specific for binding to EGFR. In some
embodiments, the targeting antibody is an anti-Jagged antibody. In
some embodiments, the targeting antibody is 4D11, which is specific
for binding to human and mouse Jagged 1 and Jagged 2. In some
embodiments, the targeting antibody is 4D11v2, which is specific
for binding to human and mouse Jagged 1 and Jagged 2.
[0340] In some embodiments, the targeting antibody can be in the
form an activatable antibody. In some embodiments, the scFv(s) can
be in the form of a Pro-scFv (see, e.g., WO 2009/025846, WO
2010/081173).
[0341] In some embodiments, the scFv is specific for binding
CD3.epsilon., and is or is derived from an antibody or fragment
thereof that binds CD3.epsilon., e.g., CH2527, FN18, H2C, OKT3,
2C11, UCHT1, or V9. In some embodiments, the scFv is specific for
binding CTLA-4 (also referred to herein as CTLA and CTLA4).
[0342] In some embodiments, the anti-CTLA-4 scFv includes the amino
acid sequence:
TABLE-US-00030 (SEQ ID NO: 510)
GGGSGGGGSGSGGGSGGGGSGGGEIVLTQSPGTLSLSPGERATLSCRASQ
SVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTIS
RLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRSGGSTITSYNVYYTKLSS
SGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEW
VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA
TNSLYWYFDLWGRGTLVTVSSAS
[0343] In some embodiments, the anti-CTLA-4 scFv includes the amino
acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or more identical to the amino acid sequence of SEQ
ID NO: 510.
[0344] In some embodiments, the anti-CD3.epsilon. scFv includes the
amino acid sequence:
TABLE-US-00031 (SEQ ID NO: 511)
GGGSGGGGSGSGGGSGGGGSGGGQVQLQQSGAELARPGASVKMSCKASGY
TFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS
TAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGG
GSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSP
KRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSN
PFTFGSGTKLEINR
[0345] In some embodiments, the anti-CD3.epsilon. scFv includes the
amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or more identical to the amino acid sequence of
SEQ ID NO: 511.
[0346] In some embodiments, the scFv is specific for binding one or
more T-cells, one or more NK-cells and/or one or more macrophages.
In some embodiments, the scFv is specific for binding a target
selected from the group consisting of B7-H4, BTLA, CD3, CD4, CD8,
CD16a, CD25, CD27, CD28, CD32, CD56, CD137, CTLA-4, GITR, HVEM,
ICOS, LAG3, NKG2D, OX40, PD-1, TIGIT, TIM3, or VISTA.
[0347] In some embodiments, the multispecific activatable antibody
also includes an agent conjugated to the AB. In some embodiments,
the agent is a therapeutic agent. In some embodiments, the agent is
an antineoplastic agent. In some embodiments, the agent is a toxin
or fragment thereof. In some embodiments, the agent is conjugated
to the multispecific activatable antibody via a linker. In some
embodiments, the agent is conjugated to the AB via a cleavable
linker. In some embodiments, the agent is conjugated to the AB via
a linker that includes at least one MMP-cleavable substrate
sequence. In some embodiments, the linker is a non-cleavable
linker. In some embodiments, the agent is a microtubule inhibitor.
In some embodiments, the agent is a nucleic acid damaging agent,
such as a DNA alkylator or DNA intercalator, or other DNA damaging
agent. In some embodiments, the linker is a cleavable linker. In
some embodiments, the agent is an agent selected from the group
listed in Table 4. 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
duocarmycin or derivative thereof. In some embodiments, the agent
is a calicheamicin or derivative thereof. In some embodiments, the
agent is a pyrrolobenzodiazepine.
[0348] In some embodiments, the multispecific activatable antibody
also includes a detectable moiety. In some embodiments, the
detectable moiety is a diagnostic agent.
[0349] In some embodiments, the multispecific activatable antibody
naturally contains one or more disulfide bonds. In some
embodiments, the multispecific activatable antibody can be
engineered to include one or more disulfide bonds.
[0350] The disclosure also provides an isolated nucleic acid
molecule encoding a multispecific activatable antibody described
herein, as well as vectors that include these isolated nucleic acid
sequences. The disclosure provides methods of producing a
multispecific activatable antibody by culturing a cell under
conditions that lead to expression of the activatable antibody,
wherein the cell comprises such a nucleic acid molecule. In some
embodiments, the cell comprises such a vector.
[0351] The disclosure also provides a method of manufacturing
multispecific activatable antibodies of the disclosure by (a)
culturing a cell comprising a nucleic acid construct that encodes
the multispecific activatable antibody under conditions that lead
to expression of the multispecific activatable, and (b) recovering
the multispecific activatable antibody.
[0352] The disclosure also provides multispecific activatable
antibodies and/or multispecific activatable antibody compositions
that include at least a first antibody or antigen-binding fragment
thereof (AB1) that specifically binds a first target or first
epitope and a second antibody or antigen-biding fragment thereof
(AB2) that binds a second target or a second epitope, where at
least AB1 is coupled or otherwise attached to a masking moiety
(MM1), such that coupling of the MM1 reduces the ability of AB1 to
bind its target. In some embodiments, the MM1 is coupled to AB1 via
a first cleavable moiety (CM1) sequence that includes a substrate
for a protease, for example, a protease that is co-localized with
the target of AB1 at a treatment site or a diagnostic site in a
subject. The multispecific activatable antibodies provided herein
are stable in circulation, activated at intended sites of therapy
and/or diagnosis but not in normal, i.e., healthy tissue, and, when
activated, exhibit binding to the target of AB1 that is at least
comparable to the corresponding, unmodified multispecific
antibody.
[0353] In some embodiments, the multispecific activatable antibody
comprises a linking peptide between the MM1 and the CM1.
[0354] In some embodiments, the multispecific activatable antibody
comprises a linking peptide between the CM1 and the AB1.
[0355] In some embodiments, the activatable antibody comprises a
first linking peptide (LP1) and a second linking peptide (LP2), and
at least a portion of the multispecific activatable antibody has
the structural arrangement from N-terminus to C-terminus as follows
in the uncleaved state: MM1-LP1-CM1-LP2-AB1 or AB1-LP2-CM1-LP1-MM1.
In some embodiments, the two linking peptides need not be identical
to each other.
[0356] 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: 1) and
(GGGS).sub.n (SEQ ID NO: 2), 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: 3), GGSGG (SEQ ID NO: 4), GSGSG (SEQ ID NO: 5), GSGGG (SEQ ID
NO: 6), GGGSG (SEQ ID NO: 7), and GSSSG (SEQ ID NO: 8).
[0357] In some embodiments, the multispecific activatable antibody
includes at least a first antibody or antigen-binding fragment
thereof (AB1) that specifically binds a first target or first
epitope and a second antibody or antigen-binding fragment thereof
(AB2) that specifically binds a second target or second epitope. In
some embodiments, each of the AB in the multispecific activatable
antibody is independently selected from the group consisting of 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, each of the AB in the multispecific activatable
antibody is a rodent (e.g., mouse or rat), chimeric, humanized or
fully human monoclonal antibody.
[0358] In some embodiments, each of the AB in the multispecific
activatable antibody has an equilibrium dissociation constant of
about 100 nM or less for binding to its corresponding target or
epitope.
[0359] In some embodiments, MM1 has an equilibrium dissociation
constant for binding to its corresponding AB that is greater than
the equilibrium dissociation constant of the AB to its
corresponding target or epitope.
[0360] In some embodiments, MM1 has an equilibrium dissociation
constant for binding to its corresponding AB that is no more than
the equilibrium dissociation constant of the AB to its
corresponding target or epitope.
[0361] In some embodiments, MM1 does not interfere or compete with
its corresponding AB for binding to the corresponding target or
epitope when the multispecific activatable antibody is in a cleaved
state.
[0362] In some embodiments, MM1 is a polypeptide of about 2 to 40
amino acids in length. In some embodiments, each of the MM in the
multispecific activatable antibody is a polypeptide of no more than
40 amino acids in length.
[0363] In some embodiments, MM1 has a polypeptide sequence that is
different from that of target of the corresponding AB.
[0364] In some embodiments, MM1 has a polypeptide sequence that is
no more than 50% identical to any natural binding partner of the
corresponding AB. In some embodiments, MM1 has a polypeptide
sequence that is no more than 25% identical to any natural binding
partner of the corresponding AB. In some embodiments, MM1 has a
polypeptide sequence that is no more than 10% identical to any
natural binding partner of the corresponding AB.
[0365] In some embodiments, the coupling of MM1 reduces the ability
of the corresponding AB to bind its target or epitope such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM1
towards its corresponding target or epitope is at least 20 times
greater than the K.sub.d of the AB when not coupled to the MM1
towards its corresponding target or epitope.
[0366] In some embodiments, the coupling of MM1 reduces the ability
of the corresponding AB to bind its target or epitope such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM1
towards its corresponding target or epitope is at least 40 times
greater than the K.sub.d of the AB when not coupled to the MM1
towards its corresponding target or epitope.
[0367] In some embodiments, the coupling of MM1 reduces the ability
of the corresponding AB to bind its target or epitope such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM1
towards its corresponding target or epitope is at least 100 times
greater than the K.sub.d of the AB when not coupled to the MM1
towards its corresponding target or epitope.
[0368] In some embodiments, the coupling of MM1 reduces the ability
of the corresponding AB to bind its target or epitope such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM1
towards its corresponding target or epitope is at least 1000 times
greater than the K.sub.d of the AB when not coupled to the MM1
towards its corresponding target or epitope.
[0369] In some embodiments, the coupling of MM1 reduces the ability
of the corresponding AB to bind its target or epitope such that the
dissociation constant (K.sub.d) of the AB when coupled to the MM1
towards its corresponding target or epitope is at least 10,000
times greater than the K.sub.d of the AB when not coupled to the
MM1 towards its corresponding target or epitope.
[0370] In some embodiments, MM1 is an amino acid sequence selected
from a MM disclosed herein.
[0371] In some embodiments, the multispecific activatable antibody
includes at least a second masking moiety (MM2) that inhibits the
binding of the AB2 to its target when the multispecific activatable
antibody is in an uncleaved state, and a second cleavable moiety
(CM2) coupled to the AB2, wherein the CM2 is a polypeptide that
functions as a substrate for a second protease. In some
embodiments, CM2 is a polypeptide of no more than 15 amino acids
long. In some embodiments, the second protease is co-localized with
the second target or epitope in a tissue, and wherein the second
protease cleaves the CM2 in the multispecific activatable antibody
when the multispecific activatable antibody is exposed to the
second protease. In some embodiments, the first protease and the
second protease are co-localized with the first target or epitope
and the second target or epitope in a tissue. In some embodiments,
the first protease and the second protease are the same protease.
In some embodiments, CM1 and CM2 are different substrates for the
same protease. In some embodiments, the protease is selected from
the group consisting of those shown in Table 7. In some
embodiments, the first protease and the second protease are
different proteases. In some embodiments, the first protease and
the second protease are different proteases selected from the group
consisting of those shown in Table 7.
[0372] In some embodiments, each of the MM in the multispecific
activatable antibody, e.g., MM1 and at least MM2, has an
equilibrium dissociation constant for binding to its corresponding
AB that is greater than the equilibrium dissociation constant of
the AB to its corresponding target or epitope.
[0373] In some embodiments, each of the MM in the multispecific
activatable antibody has an equilibrium dissociation constant for
binding to its corresponding AB that is no more than the
equilibrium dissociation constant of the AB to its corresponding
target or epitope.
[0374] In some embodiments, each of the MM in the multispecific
activatable antibody does not interfere or compete with its
corresponding AB for binding to the corresponding target or epitope
when the multispecific activatable antibody is in a cleaved
state.
[0375] In some embodiments, each of the MM in the multispecific
activatable antibody is a polypeptide of about 2 to 40 amino acids
in length. In some embodiments, each of the MM in the multispecific
activatable antibody is a polypeptide of no more than 40 amino
acids in length.
[0376] In some embodiments, each of the MM in the multispecific
activatable antibody has a polypeptide sequence that is different
from that of target of the corresponding AB.
[0377] In some embodiments, each of the MM in the multispecific
activatable antibody has a polypeptide sequence that is no more
than 50% identical to any natural binding partner of the
corresponding AB. In some embodiments, each of the MM in the
multispecific activatable antibody has a polypeptide sequence that
is no more than 25% identical to any natural binding partner of the
corresponding AB. In some embodiments, each of the MM in the
multispecific activatable antibody has a polypeptide sequence that
is no more than 10% identical to any natural binding partner of the
corresponding AB.
[0378] In some embodiments, the coupling of each of the MM reduces
the ability of the corresponding AB to bind its target or epitope
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards its corresponding target or epitope is at
least 20 times greater than the K.sub.d of the AB when not coupled
to the MM towards its corresponding target or epitope.
[0379] In some embodiments, the coupling of each of the MM reduces
the ability of the corresponding AB to bind its target or epitope
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards its corresponding target or epitope is at
least 40 times greater than the K.sub.d of the AB when not coupled
to the MM towards its corresponding target or epitope.
[0380] In some embodiments, the coupling of each of the MM reduces
the ability of the corresponding AB to bind its target or epitope
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards its corresponding target or epitope is at
least 100 times greater than the K.sub.d of the AB when not coupled
to the MM towards its corresponding target or epitope.
[0381] In some embodiments, the coupling of each of the MM reduces
the ability of the corresponding AB to bind its target or epitope
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards its corresponding target or epitope is at
least 1000 times greater than the K.sub.d of the AB when not
coupled to the MM towards its corresponding target or epitope.
[0382] In some embodiments, the coupling of each of the MM reduces
the ability of the corresponding AB to bind its target or epitope
such that the dissociation constant (K.sub.d) of the AB when
coupled to the MM towards its corresponding target or epitope is at
least 10,000 times greater than the K.sub.d of the AB when not
coupled to the MM towards its corresponding target or epitope.
[0383] In some embodiments, each of the MM is an amino acid
sequence selected from a MM disclosed herein.
[0384] In some embodiments, at least one of CM1 and/or CM2 is
cleaved by at least one MMP protease. In some embodiments, at least
one of CM1 and/or CM2 includes an amino acid sequence selected from
the group consisting of ISSGLLSS (SEQ ID NO: 14); QNQALRMA (SEQ ID
NO: 15); AQNLLGMV (SEQ ID NO: 16); STFPFGMF (SEQ ID NO: 17);
PVGYTSSL (SEQ ID NO: 18); DWLYWPGI (SEQ ID NO: 19); MIAPVAYR (SEQ
ID NO: 20); RPSPMWAY (SEQ ID NO: 21); WATPRPMR (SEQ ID NO: 22);
FRLLDWQW (SEQ ID NO: 23); LKAAPRWA (SEQ ID NO: 24); GPSHLVLT (SEQ
ID NO: 25); LPGGLSPW (SEQ ID NO: 26); MGLFSEAG (SEQ ID NO: 27);
SPLPLRVP (SEQ ID NO: 28); RMHLRSLG (SEQ ID NO: 29); LAAPLGLL (SEQ
ID NO: 30); AVGLLAPP (SEQ ID NO: 31); LLAPSHRA (SEQ ID NO: 32);
PAGLWLDP (SEQ ID NO: 33); and ISSGLSS (SEQ ID NO: 159).
[0385] In some embodiments, at least one of CM1 and/or CM2 includes
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 364-370, 379-393, 402-409, 420-424, 434, 435, 450-452, 457,
470-472, 474, and 483.
[0386] In some embodiments, at least one of CM1 and/or CM2 includes
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 328, 336-339, and 348-351.
[0387] In some embodiments, the protease that cleaves the first
cleavable moiety (CM1) sequence is co-localized with the target of
the AB1 in the multispecific activatable antibody in a tissue, and
the protease cleaves the CM1 in the multispecific activatable
antibody when the multispecific activatable antibody is exposed to
the protease.
[0388] In some embodiments, the multispecific activatable antibody
includes more than one cleavable moiety sequence, and the protease
that cleaves at least one cleavable moiety sequence is co-localized
with the target of at least one of the AB regions in the
multispecific activatable antibody in a tissue, and the protease
cleaves the CM in the multispecific activatable antibody when the
multispecific activatable antibody is exposed to the protease.
[0389] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least twofold
greater than the equilibrium dissociation constant of an unmodified
AB binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0390] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least
threefold greater than the equilibrium dissociation constant of an
unmodified AB binding to its target, and whereas in the cleaved
state, the AB binds its target.
[0391] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least fourfold
greater than the equilibrium dissociation constant of an unmodified
AB binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0392] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least fivefold
greater than the equilibrium dissociation constant of an unmodified
AB binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0393] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least tenfold
greater than the equilibrium dissociation constant of an unmodified
AB binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0394] In some embodiments, each CM, e.g., CM1 and at least CM2, is
positioned in the multispecific activatable antibody such that in
the uncleaved state, binding of the multispecific activatable
antibody to a target of one of the AB regions is reduced to occur
with an equilibrium dissociation constant that is at least 20-fold
greater than the equilibrium dissociation constant of an unmodified
AB binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0395] In some embodiments, each CM is positioned in the
multispecific activatable antibody such that in the uncleaved
state, binding of the multispecific activatable antibody to a
target of one of the AB regions is reduced to occur with an
equilibrium dissociation constant that is at least 40-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0396] In some embodiments, each CM is positioned in the
multispecific activatable antibody such that in the uncleaved
state, binding of the multispecific activatable antibody to a
target of one of the AB regions is reduced to occur with an
equilibrium dissociation constant that is at least 50-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0397] In some embodiments, each CM is positioned in the
multispecific activatable antibody such that in the uncleaved
state, binding of the multispecific activatable antibody to a
target of one of the AB regions is reduced to occur with an
equilibrium dissociation constant that is at least 100-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0398] In some embodiments, each CM is positioned in the
multispecific activatable antibody such that in the uncleaved
state, binding of the multispecific activatable antibody to a
target of one of the AB regions is reduced to occur with an
equilibrium dissociation constant that is at least 200-fold greater
than the equilibrium dissociation constant of an unmodified AB
binding to its target, and whereas in the cleaved state, the AB
binds its target.
[0399] In some embodiments, each CM in the multispecific
activatable antibody is a polypeptide of up to 15 amino acids in
length.
[0400] In some embodiments, at least one CM in the multispecific
activatable antibody includes an amino acid sequence selected from
the group consisting of SEQ ID NOs: 14-33 and 159 and the other CM
includes the amino acid sequence LSGRSDNH (SEQ ID NO: 26). In some
embodiments, at least one CM includes the amino acid sequence
LSGRSDNH (SEQ ID NO: 26). In some embodiments, at least one
cleavable moiety is selected for use with a specific protease, for
example a protease that is known to be co-localized with at least
one target of the multispecific activatable antibody. For example,
suitable cleavable moieties for use in the multispecific
activatable antibodies of the disclosure are cleaved by at least a
protease such as urokinase, legumain, and/or matriptase (also
referred to herein as MT-SP1 or MTSP1). In some embodiments, a
suitable cleavable moiety includes at least one of the following
sequences: TGRGPSWV (SEQ ID NO: 27); SARGPSRW (SEQ ID NO: 28);
TARGPSFK (SEQ ID NO: 29); LSGRSDNH (SEQ ID NO: 26); GGWHTGRN (SEQ
ID NO: 30); HTGRSGAL (SEQ ID NO: 31); PLTGRSGG (SEQ ID NO: 32);
AARGPAIH (SEQ ID NO: 33); RGPAFNPM (SEQ ID NO: 34); SSRGPAYL (SEQ
ID NO: 35); RGPATPIM (SEQ ID NO: 36); RGPA (SEQ ID NO: 37);
GGQPSGMWGW (SEQ ID NO: 38); FPRPLGITGL (SEQ ID NO: 39); VHMPLGFLGP
(SEQ ID NO: 40); SPLTGRSG (SEQ ID NO: 41); SAGFSLPA (SEQ ID NO:
42); LAPLGLQRR (SEQ ID NO: 43); SGGPLGVR (SEQ ID NO: 44); and/or
PLGL (SEQ ID NO: 45).
[0401] In some embodiments, one CM is a substrate for at least one
MMP protease and the other CM in the multispecific activatable
antibody is a substrate for a protease selected from the group
consisting of those shown in Table 7. In some embodiments, the
protease is selected from the group consisting of uPA, legumain,
matriptase, ADAM17, BMP-1, TMPRSS3, TMPRSS4, neutrophil elastase,
MMP-7, MMP-9, MMP-12, MMP-13, and MMP-14. In some embodiments, the
protease is a cathepsin, such as, but not limited to, cathepsin S.
In some embodiments, each CM in the multispecific activatable
antibody is a substrate for a protease selected from the group
consisting of uPA (urokinase plasminogen activator), legumain and
matriptase. In some embodiments, the protease comprises uPA. In
some embodiments, the protease comprises legumain. In some
embodiments, the protease comprises matriptase. In some
embodiments, the protease comprises a matrix metalloproteinase
(MMP).
[0402] In some embodiments, at least one CM in the multispecific
activatable antibody is a substrate for at least two proteases. In
some embodiments, each protease is selected from the group
consisting of those shown in Table 7. In some embodiments, at least
one CM in the multispecific activatable antibody is a substrate for
at least two proteases, wherein one of the proteases is selected
from the group consisting of uPA, legumain and matriptase and the
other protease is selected from the group consisting of those shown
in Table 7. In some embodiments, at least one CM in the
multispecific activatable antibody is a substrate for at least two
proteases selected from the group consisting of uPA, legumain and
matriptase.
[0403] In some embodiments, the multispecific activatable antibody
includes at least a first CM (CM1) and a second CM (CM2). In some
embodiments, CM1 and CM2 are part of a single cleavable linker that
joins an MM to an AB. In some embodiments, CM1 is part of a
cleavable linker that joins MM1 to AB1, and CM2 is part of a
separate cleavable linker that joins an MM2 to AB2. In some
embodiments, a multispecific activatable antibody comprises more
than two CMs. In some embodiments, such a multispecific activatable
antibody comprises more than two CMs and more than two MMs. In some
embodiments, CM1 and CM2 are each polypeptides of no more than 15
amino acids long. In some embodiments, at least one of the first CM
and the second CM is a polypeptide that functions as a substrate
for a protease selected from the group consisting of those listed
in Table 7. In some embodiments, at least one of the first CM and
the second CM is a polypeptide that functions as a substrate for a
protease selected from the group consisting of uPA, legumain, and
matriptase. In some embodiments, the first CM is cleaved by a first
cleaving agent selected from the group consisting of uPA, legumain,
and matriptase in a target tissue and the second CM is cleaved by a
second cleaving agent in a target tissue. In some embodiments, the
other protease is selected from the group consisting of those shown
in Table 7. In some embodiments, the first cleaving agent and the
second cleaving agent are the same protease selected from the group
consisting of those listed in Table 7, and the first CM and the
second CM are different substrates for the enzyme. In some
embodiments, the first cleaving agent and the second cleaving agent
are the same protease selected from the group consisting of uPA,
legumain, and matriptase, and the first CM and the second CM are
different substrates for the enzyme. In some embodiments, the first
cleaving agent and the second cleaving agent are the same protease
selected from the group listed in Table 7, and the first CM and the
second CM are the same substrate. In some embodiments, the first
cleaving agent and the second cleaving agent are different
proteases. In some embodiments, the first cleaving agent and the
second cleaving agent are different proteases selected from the
group consisting of those shown in Table 7. In some embodiments,
the first cleaving agent and the second cleaving agent are
co-localized in the target tissue. In some embodiments, the first
CM and the second CM are cleaved by at least one cleaving agent in
the target tissue.
[0404] In some embodiments, the multispecific activatable antibody
is exposed to and cleaved by a protease such that, in the activated
or cleaved state, the activated multispecific activatable 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.
[0405] The disclosure also provides compositions and methods that
include a multispecific activatable antibody that includes at least
a first antibody or antibody fragment (AB1) that specifically binds
a target and a second antibody or antibody fragment (AB2), where at
least the first AB in the multispecific activatable antibody is
coupled to a masking moiety (MM1) that decreases the ability of AB1
to bind its target. In some embodiments, each AB is coupled to a MM
that decreases the ability of its corresponding AB to each target.
For example, in bispecific activatable antibody embodiments, AB1 is
coupled to a first masking moiety (MM1) that decreases the ability
of AB1 to bind its target, and AB2 is coupled to a second masking
moiety (MM2) that decreases the ability of AB2 to bind its target.
In some embodiments, the multispecific activatable antibody
comprises more than two AB regions; in such embodiments, AB1 is
coupled to a first masking moiety (MM1) that decreases the ability
of AB1 to bind its target, AB2 is coupled to a second masking
moiety (MM2) that decreases the ability of AB2 to bind its target,
AB3 is coupled to a third masking moiety (MM3) that decreases the
ability of AB3 to bind its target, and so on for each AB in the
multispecific activatable antibody.
[0406] In some embodiments, the multispecific activatable antibody
further includes at least one cleavable moiety (CM) that is a
substrate for a protease, where the CM links a MM to an AB. For
example, in some embodiments, the multispecific activatable
antibody includes at least a first antibody or antibody fragment
(AB1) that specifically binds a target and a second antibody or
antibody fragment (AB2), where at least the first AB in the
multispecific activatable antibody is coupled via a first cleavable
moiety (CM1) to a masking moiety (MM1) that decreases the ability
of AB1 to bind its target. In some bispecific activatable antibody
embodiments, AB1 is coupled via CM1 to MM1, and AB2 is coupled via
a second cleavable moiety (CM2) to a second masking moiety (MM2)
that decreases the ability of AB2 to bind its target. In some
embodiments, the multispecific activatable antibody comprises more
than two AB regions; in some of these embodiments, AB1 is coupled
via CM1 to MM1, AB2 is coupled via CM2 to MM2, and AB3 is coupled
via a third cleavable moiety (CM3) to a third masking moiety (MM3)
that decreases the ability of AB3 to bind its target, and so on for
each AB in the multispecific activatable antibody.
[0407] Activatable Antibodies Having Non-Binding Steric Moieties or
Binding Partners for Non-Binding Steric Moieties
[0408] The disclosure also provides activatable antibodies that
include non-binding steric moieties (NB) or binding partners (BP)
for non-binding steric moieties, where the BP recruits or otherwise
attracts the NB to the activatable antibody. The activatable
antibodies provided herein include, for example, an activatable
antibody that includes a non-binding steric moiety (NB), a
cleavable linker (CL) and antibody or antibody fragment (AB) that
binds a target; an activatable antibody that includes a binding
partner for a non-binding steric moiety (BP), a CL and an AB; and
an activatable antibody that includes a BP to which an NB has been
recruited, a CL and an AB that binds the target. Activatable
antibodies in which the NB is covalently linked to the CL and AB of
the activatable antibody or is associated by interaction with a BP
that is covalently linked to the CL and AB of the activatable
antibody are referred to herein as "NB-containing activatable
antibodies." By activatable or switchable is meant that the
activatable antibody exhibits a first level of binding to a target
when the activatable antibody is in an inhibited, masked or
uncleaved state (i.e., a first conformation), and a second level of
binding to the target when the activatable antibody is in an
uninhibited, unmasked and/or cleaved state (i.e., a second
conformation, i.e., activated antibody), where the second level of
target binding is greater than the first level of target binding.
The activatable antibody compositions can exhibit increased
bioavailability and more favorable biodistribution compared to
conventional antibody therapeutics.
[0409] In some embodiments, activatable antibodies provide for
reduced toxicity and/or adverse side effects that could otherwise
result from binding of the at non-treatment sites and/or
non-diagnostic sites if the AB were not masked or otherwise
inhibited from binding to such a site.
[0410] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to the
target, wherein the NB is a polypeptide that does not bind
specifically to the AB; the CL is a polypeptide that includes a
substrate (S) for an enzyme; the CL is positioned such that in an
uncleaved state, the NB interferes with binding of the AB to the
target and in a cleaved state, the NB does not interfere with
binding of the AB to the target; and the NB does not inhibit
cleavage of the CL by the enzyme. As used herein and throughout,
the term polypeptide refers to any polypeptide that includes at
least two amino acid residues, including larger polypeptides,
full-length proteins and fragments thereof, and the term
polypeptide is not limited to single-chain polypeptides and can
include multi-unit, e.g., multi-chain, polypeptides. In cases where
the polypeptide is of a shorter length, for example, less than 50
amino acids total, the terms peptide and polypeptide are used
interchangeably herein, and in cases where the polypeptide is of a
longer length, e.g., 50 amino acids or greater, the terms
polypeptide and protein are used interchangeably herein.
[0411] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to the
target, wherein (i) the NB includes a polypeptide that does not
bind specifically to the AB; (ii) CL is a polypeptide of up to 50
amino acids in length that includes a substrate (S) for an enzyme;
(iii) the CL is positioned such that in an uncleaved state, the NB
interferes with binding of the AB to the target and in a cleaved
state, the NB does not interfere with binding of the AB to the
target; and (iv) the NB does not inhibit cleavage of the CL by the
enzyme. For example, the CL has a length of up to 15 amino acids, a
length of up to 20 amino acids, a length of up to 25 amino acids, a
length of up to 30 amino acids, a length of up to 35 amino acids, a
length of up to 40 amino acids, a length of up to 45 amino acids, a
length of up to 50 amino acids, a length in the range of 10-50
amino acids, a length in the range of 15-50 amino acids, a length
in the range of 20-50 amino acids, a length in the range of 25-50
amino acids, a length in the range of 30-50 amino acids, a length
in the range of 35-50 amino acids, a length in the range of 40-50
amino acids, a length in the range of 45-50 amino acids, a length
in the range of 10-40 amino acids, a length in the range of 15-40
amino acids, a length in the range of 20-40 amino acids, a length
in the range of 25-40 amino acids, a length in the range of 30-40
amino acids, a length in the range of 35-40 amino acids, a length
in the range of 10-30 amino acids, a length in the range of 15-30
amino acids, a length in the range of 20-30 amino acids, a length
in the range of 25-30 amino acids, a length in the range of 10-20
amino acids, or a length in the range of 10-15 amino acids.
[0412] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to the
target, wherein (i) the NB includes a polypeptide that does not
bind specifically to the AB; (ii) the CL is a polypeptide that
includes a substrate (S) for an enzyme; (iii) the CL is positioned
such that in an uncleaved state, the NB interferes with binding of
the AB to the target and in a cleaved state, the NB does not
interfere with binding of the AB to the target; (iv) the NB does
not inhibit cleavage of the CL by the enzyme; and (v) the
activatable antibody has the structural arrangement from N-terminus
to C-terminus as follows in the uncleaved state: NB-CL-AB or
AB-CL-NB.
[0413] In one embodiment, the activatable antibody includes a
non-binding steric moiety (NB); a cleavable linker (CL); and an
antibody or antibody fragment (AB) that binds specifically to the
target, wherein (i) the NB includes a polypeptide that does not
bind specifically to the AB; (ii) the CL is a polypeptide that
includes a substrate (S) for an enzyme; (iii) the CL is positioned
such that in an uncleaved state, the NB interferes with binding of
the AB to the target and in a cleaved state, the NB does not
interfere with binding of the AB to the target, and wherein the NB
in the uncleaved activatable antibody reduces the ability of the AB
to bind the target by at least 50%, for example, by at least 60%,
by at least 70%, by at least 75%, by at least 80%, by at least 85%,
by at least 90%, by at least 95%, by at least 96%, by at least 97%,
by at least 98%, by at least 99%, by at least 100% as compared to
the ability of the cleaved AB to bind the target; and (iv) the NB
does not inhibit cleavage of the CL by the enzyme. The reduction in
the ability of the AB to bind the target is determined, e.g., using
an assay as described herein or an in vitro target displacement
assay such as, for example, the assay described in PCT Publication
Nos. WO 2009/025846 and WO 2010/081173.
[0414] In one embodiment, the activatable antibody includes a
binding partner (BP) for a non-binding steric moiety (NB); a
cleavable linker (CL); and an antibody or antibody fragment (AB)
that binds specifically to the target, wherein the BP is a
polypeptide that binds to the NB when exposed thereto; the NB does
not bind specifically to the AB; the CL is a polypeptide that
includes a substrate (S) for an enzyme; the CL is positioned such
that in an uncleaved state in the presence of the NB, the NB
interferes with binding of the AB to the target and in a cleaved
state, the NB does not interfere with binding of the AB to the
target and the BP does not interfere with binding of the AB to the
target; and the NB and the BP do not inhibit cleavage of the CL by
the enzyme. In some examples of this embodiment, the BP of the
activatable antibody is optionally bound to the NB. In one
embodiment, the NB is recruited by the BP of the activatable
antibody in vivo.
[0415] In some examples of any of these activatable antibody
embodiments, the activatable antibody is formulated as a
composition. In some of these embodiments, the composition also
includes the NB, where the NB is co-formulated with the activatable
antibody that includes the BP, the CL, and the AB. In some examples
of this embodiment, 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.
[0416] In some examples of any of these activatable antibody
embodiments, the NB is a soluble, globular protein. In some
examples of any of these activatable antibody embodiments, the NB
is a protein that circulates in the bloodstream. In some examples
of any of these activatable antibody embodiments, the NB is
selected from the group consisting of albumin, fibrinogen,
fibronectin, hemoglobin, transferrin, an immunoglobulin domain, and
other serum proteins.
[0417] In some examples of any of these activatable antibody
embodiments, the CL is a polypeptide that includes a substrate (S)
for a protease. In some examples of any of these activatable
antibody embodiments, the protease is co-localized with the in a
tissue, and the protease cleaves the CL in the activatable antibody
when the activatable antibody is exposed to the protease. In some
examples of any of these activatable antibody embodiments, the CL
is a polypeptide of up to 50 amino acids in length. In some
examples of any of these activatable antibody embodiments, the CL
is a polypeptide that includes a substrate (S) having a length of
up to 15 amino acids, e.g., 3 amino acids long, 4 amino acids long,
5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino
acids long, 9 amino acids long, 10 amino acids long, 11 amino acids
long, 12 amino acids long, 13 amino acids long, 14 amino acids
long, or 15 amino acids long.
[0418] In some examples of any of these activatable antibody
embodiments, the activatable antibody has the structural
arrangement from N-terminus to C-terminus as follows in the
uncleaved state: NB-CL-AB, AB-CL-NB, BP-CL-AB or AB-CL-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.
[0419] In some examples of any of these activatable antibody
embodiments, the activatable antibody includes an antibody or
antigen-binding fragment thereof that specifically binds a given
target and 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, or a single domain light chain
antibody. In some embodiments, such an antibody or immunologically
active fragment thereof that binds the target a mouse, other
rodent, chimeric, humanized or fully human monoclonal antibody.
[0420] In some examples of any of these activatable antibody
embodiments, the activatable antibody includes a combination of a
variable heavy chain region comprising an amino acid sequence
presented herein and a variable light chain region comprising an
amino acid sequence presented herein. In some embodiments, the
activatable antibody includes a combination of a variable heavy
chain region comprising an amino acid sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to an amino acid sequence presented herein, and a variable light
chain region comprising an amino acid sequence that is at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical
to an amino acid sequence presented herein.
[0421] In some examples of any of these activatable antibody
embodiments, the activatable antibody also includes an agent
conjugated to the AB. In some embodiments, the agent is a
therapeutic agent. In some embodiments, the agent is an
antineoplastic agent. In some embodiments, the agent is a toxin or
fragment 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 conjugated to the AB via
a noncleavable linker. In some embodiments, the agent is an agent
selected from the group listed in Table 3. In some embodiments, the
agent is a microtubule inhibitor. In some embodiments, the agent is
a nucleic acid damaging agent, such as a DNA alkylator or DNA
intercalator, or other DNA damaging agent. 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 duocarmycin or derivative thereof. In some embodiments,
the agent is a calicheamicin or derivative thereof. In some
embodiments, the agent is a pyrrolobenzodiazepine.
[0422] In some examples of any of these activatable antibody
embodiments, the activatable antibody also includes a detectable
moiety. In some embodiments, the detectable moiety is a diagnostic
agent.
[0423] In some examples of any of these activatable antibody
embodiments, the activatable antibody also includes a spacer. In
some examples of any of these activatable antibody 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 examples of any of these activatable
antibody embodiments, the spacer is joined directly to the MM of
the activatable antibody.
[0424] 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 examples of any of these activatable antibody
embodiments, the serum half-life of the activatable antibody is at
least 5 days when administered to an organism. In some embodiments,
the serum half-life of the activatable antibody is at least 4 days
when administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 3 days when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 2 days when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 24 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 20 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 18 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 16 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 14 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 12 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 10 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 8 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 6 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 4 hours when
administered to an organism. In some embodiments, the serum
half-life of the activatable antibody is at least 3 hours when
administered to an organism.
[0425] The disclosure also provides an isolated nucleic acid
molecule encoding any of these activatable antibodies, as well as
vectors that include these isolated nucleic acid sequences. The
disclosure provides methods of producing an activatable antibody by
culturing a cell under conditions that lead to expression of the
activatable antibody, wherein the cell comprises such a nucleic
acid sequence. In some embodiments, the cell comprises such a
vector.
[0426] The dissociation constant (K.sub.d) of the NB-containing
activatable antibody toward the target is greater than the K.sub.d
of the AB towards the target when it is not associated with the NB
or NB:BP. The dissociation constant (K.sub.d) of the NB-containing
activatable antibody toward the target is greater than the K.sub.d
of the parental AB towards the target. For example, the K.sub.d of
the NB-containing activatable antibody toward the target is at
least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,
50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000,
50,000,000 or greater, or between 5-10, 10-100, 10-1,000,
10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000, 100-1,000,
100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times greater than the
K.sub.d of the AB when it is not associated with the NB or NB:BP or
the K.sub.d of the parental AB towards the target. Conversely, the
binding affinity of the NB-containing activatable antibody towards
the target is lower than the binding affinity of the AB when it is
not associated with the NB or NB:BP or lower than the binding
affinity of the parental AB towards the target. For example, the
binding affinity of the NB-containing activatable antibody toward
the target is at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500,
5,000, 10,000, 50,000, 100,000, 500,000, 1,000,000, 5,000,000,
10,000,000, 50,000,000 or greater, or between 5-10, 10-100,
10-1,000, 10-10,000, 10-100,000, 10-1,000,000, 10-10,000,000,
100-1,000, 100-10,000, 100-100,000, 100-1,000,000, 100-10,000,000,
1,000-10,000, 1,000-100,000, 1,000-1,000,000, 1000-10,000,000,
10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,
100,000-1,000,000, or 100,000-10,000,000 times lower than the
binding affinity of the AB when it is not associated with the NB or
NB:BP or lower than the binding affinity of the parental AB towards
the target.
[0427] When the NB-containing activatable antibody is in the
presence of the target, specific binding of the AB to the target is
reduced or inhibited, as compared to the specific binding of the AB
when it is not associated with the NB or NB:BP. When the
NB-containing activatable antibody is in the presence of the
target, specific binding of the AB to the target is reduced or
inhibited, as compared to the specific binding of the parental AB
to the target. When compared to the binding of the AB not
associated with an NB or NB:BP or the binding of the parental AB to
the target, the ability of the NB-containing activatable antibody
to bind the target is reduced, for example, by at least 50%, 60%,
70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100%
for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96
hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer when
measured in vitro and/or in vivo.
[0428] When the NB-containing activatable antibody is in the
presence of the target but not in the presence of a modifying agent
(for example a protease or other enzyme), specific binding of the
AB to the target is reduced or inhibited, as compared to the
specific binding of the AB when it is not associated with the NB or
NB:BP. When the NB-containing activatable antibody is in the
presence of the target but not in the presence of a modifying agent
(for example a protease, other enzyme, reduction agent, or light),
specific binding of the AB to the target is reduced or inhibited,
as compared to the specific binding of the parental AB to the
target. When compared to the binding of the AB not associated with
an NB or NB:BP or the binding of the parental AB to the target, the
ability of the NB-containing activatable antibody to bind the
target is reduced, for example, by at least 50%, 60%, 70%, 80%,
90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% for at
least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours,
or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 months or longer when measured in
vitro and/or in vivo.
[0429] In some examples of any of these activatable antibody
embodiments, the activatable antibody includes an agent conjugated
to the AB to produce an activatable antibody conjugate. In some
embodiments of the activatable antibody conjugate, the agent is a
therapeutic agent. In some embodiments, the agent is a diagnostic
agent. In some embodiments, the agent is a detectable marker. In
some embodiments of the activatable antibody conjugate, the agent
is an antineoplastic agent. In some embodiments of the activatable
antibody conjugate, the agent is a toxin or fragment thereof. In
some embodiments of the activatable antibody conjugate, the agent
is conjugated to the AB via a linker. In some embodiments of the
activatable antibody conjugate, the linker is a cleavable linker.
In some embodiments, the agent is conjugated to the AB via a
noncleavable linker. In some embodiments, the agent is a
microtubule inhibitor. In some embodiments, the agent is a nucleic
acid damaging agent, such as a DNA alkylator or DNA intercalator,
or other DNA damaging agent. In some embodiments, the agent is an
agent selected from the group listed in Table 3. 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 duocarmycin or derivative thereof. In
some embodiments, the agent is a calicheamicin or derivative
thereof. In some embodiments, the agent is a
pyrrolobenzodiazepine.
[0430] In some examples of any of these activatable antibody
embodiments, the activatable antibodies are dual-target binding
activatable antibodies. Such dual target binding activatable
antibodies contain two Abs that may bind the same or different
targets. In specific embodiments, dual-targeting activatable
antibodies contain bispecific antibodies or antibody fragments.
[0431] Dual target binding activatable antibodies are designed so
as to have a CL cleavable by a cleaving agent that is co-localized
in a target tissue with one or both of the targets capable of
binding to the ABs of the activatable antibodies. Dual target
binding activatable antibodies with more than one AB to the same or
different targets can be designed so as to have more than one CL,
wherein the first CL is cleavable by a cleaving agent in a first
target tissue and wherein the second CL is cleavable by a cleaving
agent in a second target tissue, with one or more of the targets
binding to the ABs of the activatable antibodies. In one
embodiment, the first and second target tissues are spatially
separated, for example, at different sites in the organism. In one
embodiment, the first and second target tissues are the same tissue
temporally separated, for example the same tissue at two different
points in time, for example the first time point is when the tissue
is an early stage tumor, and the second time point is when the
tissue is a late stage tumor.
[0432] The disclosure also provides nucleic acid molecules encoding
the activatable antibodies described herein. The disclosure also
provides vectors that include these nucleic acids. The activatable
antibodies described herein are produced by culturing a cell under
conditions that lead to expression of the activatable antibody,
wherein the cell includes these nucleic acid molecules or
vectors.
[0433] The disclosure also provides methods of manufacturing
activatable antibodies. In one embodiment, the method includes the
steps of (a) culturing a cell that includes a nucleic acid
construct that encodes the activatable antibody under conditions
that lead to expression of the activatable antibody, wherein the
activatable antibody includes (i) a non-binding steric moiety (NB);
(ii) a cleavable linker (CL); and (iii) an antibody or an antigen
binding fragment thereof (AB) that specifically binds a target,
wherein (1) the NB does not bind specifically to the AB; (2) the CL
is a polypeptide that includes a substrate (S) for an enzyme; (3)
the CL is positioned such that in an uncleaved state, the NB
interferes with binding of the AB to the target and in a cleaved
state, the NB does not interfere with binding of the AB to the
target; and (4) the NB does not inhibit cleavage of the CL by the
enzyme; and (b) recovering the activatable antibody.
[0434] In some embodiments, the method includes the steps of (a)
culturing a cell that includes a nucleic acid construct that
encodes the activatable antibody under conditions that lead to
expression of the activatable antibody, wherein the activatable
antibody includes (i) a binding partner (BP) for a non-binding
steric moiety (NB); (ii) a cleavable linker (CL); and (iii) an
antibody or an antigen binding fragment thereof (AB) that
specifically binds a target, wherein (1) the NB does not bind
specifically to the AB; (2) the CL is a polypeptide that includes a
substrate (S) for an enzyme; (3) the CL is positioned such that in
an uncleaved state in the presence of the NB, the NB interferes
with binding of the AB to the target and in a cleaved state, the NB
does not interfere with binding of the AB to the target and the BP
does not interfere with binding of the AB to the target; and (4)
the NB and the BP do not inhibit cleavage of the CL by the enzyme;
and (b) recovering the activatable antibody. In some examples of
this embodiment, the BP of the activatable antibody is bound to the
NB.
[0435] Use of Activatable Antibodies and Conjugated Activatable
Antibodies
[0436] It will be appreciated that administration of therapeutic
entities in accordance with the disclosure 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 disclosure, 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.
[0437] Therapeutic formulations of the disclosure, which include a
conjugated antibody, an activatable antibody and/or a conjugated
activatable antibody, are used to prevent, treat or otherwise
ameliorate a disease or disorder associated with aberrant target
expression and/or activity. For example, therapeutic formulations
of the disclosure, which include a conjugated antibody, an
activatable antibody and/or a conjugated activatable antibody, are
used to treat or otherwise ameliorate inflammation, an inflammatory
disorder, an autoimmune disease and/or 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.
[0438] 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, such as, for example, aberrant 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, e.g., aberrant target expression
and/or activity, in a subject indicates that the conjugated
antibody, activatable antibody and/or conjugated activatable
antibody confers a clinical benefit.
[0439] A conjugated antibody, an activatable antibody and/or a
conjugated activatable antibody 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, Langhorne, Pa., 1994; and Peptide And
Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4),
1991, M. Dekker, New York.
[0440] In some embodiments where antibody fragments are used, the
smallest fragment that specifically binds to the binding domain of
the target protein is selected. 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 compounds as necessary for
the particular indication being treated, for example, in some
embodiments, those with complementary activities that do not
adversely affect each other. In some embodiments, 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.
[0441] 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.
[0442] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0443] Sustained-release preparations can be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which 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.
[0444] In some embodiments, the conjugated antibody, activatable
antibody and/or conjugated 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 disclosure can be used to detect an analyte
mRNA, protein, or genomic DNA in a biological sample in vitro as
well as in vivo. For example, in vitro techniques for detection of
an analyte mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of an analyte
protein include enzyme linked immunosorbent assays (ELISAs),
Western blots, immunoprecipitations, immunochemical staining, and
immunofluorescence. In vitro techniques for detection of an analyte
genomic DNA include Southern hybridizations. 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.
[0445] The conjugated antibodies, activatable antibodies and/or
conjugated activatable antibodies of the disclosure are also useful
in a variety of diagnostic and prophylactic formulations. In one
embodiment, a conjugated antibody, an activatable antibody and/or a
conjugated activatable antibody is administered to patients that
are at risk of developing one or more of the aforementioned
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.
[0446] In some embodiments, a conjugated antibody, an activatable
antibody and/or 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 antibody, an activatable antibody and/or a
conjugated activatable antibody is administered to mitigate or
reverse the effects of the clinical indication.
[0447] A conjugated antibody, an activatable antibody and/or a
conjugated activatable antibody of the disclosure is also useful in
the detection of a target in patient samples and accordingly are
useful as diagnostics. For example, the antibodies and/or
activatable antibodies, and conjugated versions thereof, of the
disclosure are used in in vitro assays, e.g., ELISA, to detect
target levels in a patient sample.
[0448] In one embodiment, a conjugated antibody, an activatable
antibody and/or a conjugated activatable antibody of the disclosure
is immobilized on a solid support (e.g., the well(s) of a
microtiter plate). The immobilized conjugated antibody, activatable
antibody and/or conjugated activatable antibody serves as a capture
antibody for any target that may be present in a test sample. Prior
to contacting the immobilized antibody with a patient sample, the
solid support is rinsed and treated with a blocking agent such as
milk protein or albumin to prevent nonspecific adsorption of the
analyte.
[0449] Subsequently the wells are treated with a test sample
suspected of containing the antigen, or with a solution containing
a standard amount of the antigen. Such a sample is, e.g., a serum
sample from a subject suspected of having levels of circulating
antigen considered to be diagnostic of a pathology. After rinsing
away the test sample or standard, the solid support is treated with
a second antibody that is detectably labeled. The labeled second
antibody serves as a detecting antibody. The level of detectable
label is measured, and the concentration of target antigen in the
test sample is determined by comparison with a standard curve
developed from the standard samples.
[0450] It will be appreciated that based on the results obtained
using the antibodies of the disclosure, and conjugated versions
thereof, in an in vitro diagnostic assay, it is possible to stage a
disease in a subject based on expression levels of the target
antigen. For a given disease, samples of blood are taken from
subjects diagnosed as being at various stages in the progression of
the disease, and/or at various points in the therapeutic treatment
of the disease. Using a population of samples that provides
statistically significant results for each stage of progression or
therapy, a range of concentrations of the antigen that may be
considered characteristic of each stage is designated.
[0451] A conjugated antibody, an activatable antibody and/or a
conjugated activatable antibody 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,
activatable antibodies 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 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 antibodies (i.e., antibodies resulting
from cleavage of an activatable antibody) in a given cell or tissue
of a given host organism. Such accumulation of activated 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.
[0452] For example, the CM can be selected to be substrate for a
matrix metalloprotease (MMP) 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 antibody and/or 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 an MMP whose activity is elevated in the
disease tissue of interest, 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.
[0453] In another example, activatable antibodies can be used to
detect the presence or absence of a cleaving agent in a sample. For
example, where the activatable antibodies contain a CM susceptible
to cleavage by an enzyme, the activatable antibodies can be used to
detect (either qualitatively or quantitatively) the presence of an
enzyme in the sample. In another example, where the activatable
antibodies contain a CM susceptible to cleavage by reducing agent,
the 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 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.
[0454] 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 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.
[0455] 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).
[0456] 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.
[0457] In these techniques, an activatable antibody is labeled with
a detectable label. The detectable label may be a fluorescent dye,
(e.g. a fluorophore, Fluorescein Isothiocyanate (FITC), Rhodamine
Isothiocyanate (TRITC), an Alexa Fluor.RTM. label), 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).
[0458] 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 matrix metalloprotease (MMP)
that is specific for the CM of the activatable antibody. In some
embodiments, the presence of the MMP can be confirmed using broad
spectrum protease inhibitors such as those described herein, and/or
by 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
matriptase; see e.g., International Publication Number WO
2010/129609, published 11 Nov. 2010. The same approach of using
broad spectrum protease inhibitors such as those described herein,
and/or by using a more selective inhibitory agent can be used to
identify a MMP 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.
[0459] 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 MMP that is specific for the CM of the
activatable antibody.
[0460] 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.
[0461] The disclosure provides methods of using the antibodies
and/or activatable antibodies in a variety of diagnostic and/or
prophylactic indications. For example, the disclosure 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 an activatable antibody, wherein the
activatable antibody comprises a masking moiety (MM), a cleavable
moiety (CM) that is cleaved by the cleaving agent, and an antigen
binding domain or fragment thereof (AB) that specifically binds the
target of interest, wherein the activatable antibody in an
uncleaved, non-activated state comprises a structural arrangement
from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB and is not a
modified form of a natural binding partner of the AB; and (b)
wherein, in an uncleaved, non-activated state, the MM interferes
with specific binding of the AB to the target, and in a cleaved,
activated state the MM does not interfere or compete with specific
binding of the AB to the target; and (ii) measuring a level of
activated activatable antibody in the subject or sample, wherein a
detectable level of activated 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 activatable antibody in the subject or sample indicates
that the cleaving agent, the target or both the cleaving agent and
the target are absent and/or not sufficiently present in the
subject or sample. In some embodiments, the activatable antibody is
an activatable antibody to which a therapeutic agent is conjugated.
In some embodiments, the activatable antibody is not conjugated to
an agent. In some embodiments, the activatable antibody comprises a
detectable label. In some embodiments, the detectable label is
positioned on the AB. In some embodiments, measuring the level of
activatable antibody in the subject or sample is accomplished using
a secondary reagent that specifically binds to the activated
antibody, wherein the reagent comprises a detectable label. In some
embodiments, the secondary reagent is an antibody comprising a
detectable label.
[0462] The disclosure 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 an activatable antibody in the
presence of a target of interest, e.g., the target, wherein the
activatable antibody comprises a masking moiety (MM), a cleavable
moiety (CM) that is cleaved by the cleaving agent, and an antigen
binding domain or fragment thereof (AB) that specifically binds the
target of interest, wherein the activatable antibody in an
uncleaved, non-activated state comprises a structural arrangement
from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB and is not a
modified form of a natural binding partner of the AB; and (b)
wherein, in an uncleaved, non-activated state, the MM interferes
with specific binding of the AB to the target, and in a cleaved,
activated state the MM does not interfere or compete with specific
binding of the AB to the target; and (ii) measuring a level of
activated activatable antibody in the subject or sample, wherein a
detectable level of activated 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 activated
activatable antibody in the subject or sample indicates that the
cleaving agent is absent and/or not sufficiently present in the
subject or sample. In some embodiments, the activatable antibody is
an activatable antibody to which a therapeutic agent is conjugated.
In some embodiments, the activatable antibody is not conjugated to
an agent. In some embodiments, the activatable antibody comprises a
detectable label. In some embodiments, the detectable label is
positioned on the AB. In some embodiments, measuring the level of
activatable antibody in the subject or sample is accomplished using
a secondary reagent that specifically binds to the activated
antibody, wherein the reagent comprises a detectable label. In some
embodiments, the secondary reagent is an antibody comprising a
detectable label.
[0463] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent and the target in
a subject or a sample, where the kits include at least an
activatable antibody comprises a masking moiety (MM), a cleavable
moiety (CM) that is cleaved by the cleaving agent, and an antigen
binding domain or fragment thereof (AB) that specifically binds the
target of interest, wherein the activatable antibody in an
uncleaved, non-activated state comprises a structural arrangement
from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB and is not a
modified form of a natural binding partner of the AB; and (b)
wherein, in an uncleaved, non-activated state, the MM interferes
with specific binding of the AB to the target, and in a cleaved,
activated state the MM does not interfere or compete with specific
binding of the AB to the target; and (ii) measuring a level of
activated activatable antibody in the subject or sample, wherein a
detectable level of activated 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 activated
activatable antibody in the subject or sample indicates that the
cleaving agent is absent and/or not sufficiently present in the
subject or sample. In some embodiments, the activatable antibody is
an activatable antibody to which a therapeutic agent is conjugated.
In some embodiments, the activatable antibody is not conjugated to
an agent. In some embodiments, the activatable antibody comprises a
detectable label. In some embodiments, the detectable label is
positioned on the AB. In some embodiments, measuring the level of
activatable antibody in the subject or sample is accomplished using
a secondary reagent that specifically binds to the activated
antibody, wherein the reagent comprises a detectable label. In some
embodiments, the secondary reagent is an antibody comprising a
detectable label.
[0464] The disclosure 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 an activatable antibody,
wherein the activatable antibody comprises a masking moiety (MM), a
cleavable moiety (CM) that is cleaved by the cleaving agent, an
antigen binding domain (AB) that specifically binds the target, and
a detectable label, wherein the activatable antibody in an
uncleaved, non-activated state comprises a structural arrangement
from N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM;
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB and is not a
modified form of a natural binding partner of the AB; wherein, in
an uncleaved, non-activated state, the MM interferes with specific
binding of the AB to the target, and in a cleaved, activated state
the MM does not interfere or compete with specific binding of the
AB to the target; and wherein the detectable label is positioned on
a portion of the activatable antibody that is released following
cleavage of the CM; 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 and/or not sufficiently present 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, the
activatable antibody is an activatable antibody to which a
therapeutic agent is conjugated. In some embodiments, the
activatable antibody is not conjugated to an agent. In some
embodiments, the activatable antibody comprises a detectable label.
In some embodiments, the detectable label is positioned on the AB.
In some embodiments, measuring the level of activatable antibody in
the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0465] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent and the target in
a subject or a sample, where the kits include at least an
activatable antibody and/or conjugated activatable antibody (e.g.,
an activatable antibody to which a therapeutic agent is conjugated)
described herein for use in contacting a subject or biological
sample and means for detecting the level of activated activatable
antibody and/or conjugated activatable antibody in the subject or
biological sample, wherein a detectable level of activated
activatable antibody in the subject or biological sample indicates
that the cleaving agent and the target are present in the subject
or biological sample and wherein no detectable level of activated
activatable antibody in the subject or biological sample indicates
that the cleaving agent, the target or both the cleaving agent and
the target are absent and/or not sufficiently present in the
subject or biological sample, such that the target binding and/or
protease cleavage of the activatable antibody cannot be detected in
the subject or biological sample.
[0466] The disclosure also provides methods of detecting presence
or absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or biological sample with an activatable
antibody in the presence of the target, and (ii) measuring a level
of activated activatable antibody in the subject or biological
sample, wherein a detectable level of activated activatable
antibody in the subject or biological sample indicates that the
cleaving agent is present in the subject or biological sample and
wherein no detectable level of activated activatable antibody in
the subject or biological sample indicates that the cleaving agent
is absent and/or not sufficiently present in the subject or
biological sample at a detectable level, such that protease
cleavage of the activatable antibody cannot be detected in the
subject or biological sample. Such an activatable antibody includes
a masking moiety (MM), a cleavable moiety (CM) that is cleaved by
the cleaving agent, and an antigen binding domain or fragment
thereof (AB) that specifically binds the target, wherein the
activatable antibody in an uncleaved (i.e., non-activated) state
comprises a structural arrangement from N-terminus to C-terminus as
follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that
inhibits binding of the AB to the target, and wherein the MM does
not have an amino acid sequence of a naturally occurring binding
partner of the AB; and (b) wherein the MM of the activatable
antibody in an uncleaved state interferes with specific binding of
the AB to the target, and wherein the MM of an activatable antibody
in a cleaved (i.e., activated) state does not interfere or compete
with specific binding of the AB to the target. In some embodiments,
the activatable antibody is an activatable antibody to which a
therapeutic agent is conjugated. In some embodiments, the
activatable antibody is not conjugated to an agent. In some
embodiments, the detectable label is attached to the masking
moiety. In some embodiments, the detectable label is attached to
the cleavable moiety N-terminal to the protease cleavage site. In
some embodiments, a single antigen binding site of the AB is
masked. In some embodiments wherein an antibody of the disclosure
has at least two antigen binding sites, at least one antigen
binding site is masked and at least one antigen binding site is not
masked. In some embodiments, all antigen binding sites are masked.
In some embodiments, the measuring step includes use of a secondary
reagent comprising a detectable label.
[0467] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent and the target in
a subject or a sample, where the kits include at least an
activatable antibody and/or conjugated activatable antibody
described herein for use in contacting a subject or biological
sample with an activatable antibody in the presence of the target,
and measuring a level of activated activatable antibody in the
subject or biological sample, wherein a detectable level of
activated activatable antibody in the subject or biological sample
indicates that the cleaving agent is present in the subject or
biological sample and wherein no detectable level of activated
activatable antibody in the subject or biological sample indicates
that the cleaving agent is absent and/or not sufficiently present
in the subject or biological sample at a detectable level, such
that protease cleavage of the activatable antibody cannot be
detected in the subject or biological sample. Such an activatable
antibody includes a masking moiety (MM), a cleavable moiety (CM)
that is cleaved by the cleaving agent, and an antigen binding
domain or fragment thereof (AB) that specifically binds the target,
wherein the activatable antibody in an uncleaved (i.e.,
non-activated) state comprises a structural arrangement from
N-terminus to C-terminus as follows: MM-CM-AB or AB-CM-MM; (a)
wherein the MM is a peptide that inhibits binding of the AB to the
target, and wherein the MM does not have an amino acid sequence of
a naturally occurring binding partner of the AB; and (b) wherein
the MM of the activatable antibody in an uncleaved state interferes
with specific binding of the AB to the target, and wherein the MM
of an activatable antibody in a cleaved (i.e., activated) state
does not interfere or compete with specific binding of the AB to
the target. In some embodiments, the activatable antibody is an
activatable antibody to which a therapeutic agent is conjugated. In
some embodiments, the activatable antibody is not conjugated to an
agent. In some embodiments, the detectable label is attached to the
masking moiety. In some embodiments, the detectable label is
attached to the cleavable moiety N-terminal to the protease
cleavage site. In some embodiments, a single antigen binding site
of the AB is masked. In some embodiments wherein an antibody of the
disclosure has at least two antigen binding sites, at least one
antigen binding site is masked and at least one antigen binding
site is not masked. In some embodiments, all antigen binding sites
are masked. In some embodiments, the measuring step includes use of
a secondary reagent comprising a detectable label.
[0468] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent in a subject or a
sample, where the kits include at least an activatable antibody
and/or conjugated activatable antibody described herein for use in
contacting a subject or biological sample and means for detecting
the level of activated activatable antibody and/or conjugated
activatable antibody in the subject or biological sample, wherein
the activatable antibody includes a detectable label that is
positioned on a portion of the activatable antibody that is
released following cleavage of the CM, wherein a detectable level
of activated activatable antibody in the subject or biological
sample indicates that the cleaving agent is absent and/or not
sufficiently present in the subject or biological sample such that
the target binding and/or protease cleavage of the activatable
antibody cannot be detected in the subject or biological sample,
and wherein no detectable level of activated activatable antibody
in the subject or biological sample indicates that the cleaving
agent is present in the subject or biological sample at a
detectable level.
[0469] The disclosure provides methods of detecting presence or
absence of a cleaving agent and the target in a subject or a sample
by (i) contacting a subject or biological sample with an
activatable antibody, wherein the activatable antibody includes a
detectable label that is positioned on a portion of the activatable
antibody that is released following cleavage of the CM and (ii)
measuring a level of activated activatable antibody in the subject
or biological sample, wherein a detectable level of activated
activatable antibody in the subject or biological sample indicates
that the cleaving agent, the target or both the cleaving agent and
the target are absent and/or not sufficiently present in the
subject or biological sample, such that the target binding and/or
protease cleavage of the activatable antibody cannot be detected in
the subject or biological sample, and wherein a reduced detectable
level of activated activatable antibody in the subject or
biological sample indicates that the cleaving agent and the target
are present in the subject or biological sample. A reduced level of
detectable label is, for example, a reduction of about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or
about 100%. Such an activatable antibody includes a masking moiety
(MM), a cleavable moiety (CM) that is cleaved by the cleaving
agent, and an antigen binding domain or fragment thereof (AB) that
specifically binds the target, wherein the activatable antibody in
an uncleaved (i.e., non-activated) state comprises a structural
arrangement from N-terminus to C-terminus as follows: MM-CM-AB or
AB-CM-MM; (a) wherein the MM is a peptide that inhibits binding of
the AB to the target, and wherein the MM does not have an amino
acid sequence of a naturally occurring binding partner of the AB;
and (b) wherein the MM of the activatable antibody in an uncleaved
state interferes with specific binding of the AB to the target, and
wherein the MM of an activatable antibody in a cleaved (i.e.,
activated) state does not interfere or compete with specific
binding of the AB to the target. In some embodiments, the
activatable antibody is an activatable antibody to which a
therapeutic agent is conjugated. In some embodiments, the
activatable antibody is not conjugated to an agent. In some
embodiments, the activatable antibody comprises a detectable label.
In some embodiments, the detectable label is positioned on the AB.
In some embodiments, measuring the level of activatable antibody in
the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0470] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent and the target in
a subject or a sample, where the kits include at least an
activatable antibody and/or conjugated activatable antibody
described herein for use in contacting a subject or biological
sample and means for detecting the level of activated activatable
antibody and/or conjugated activatable antibody in the subject or
biological sample, wherein a detectable level of activated
activatable antibody in the subject or biological sample indicates
that the cleaving agent, the target or both the cleaving agent and
the target are absent and/or not sufficiently present in the
subject or biological sample, such that the target binding and/or
protease cleavage of the activatable antibody cannot be detected in
the subject or biological sample, and wherein a reduced detectable
level of activated activatable antibody in the subject or
biological sample indicates that the cleaving agent and the target
are present in the subject or biological sample. A reduced level of
detectable label is, for example, a reduction of about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or
about 100%.
[0471] The disclosure also provides methods of detecting presence
or absence of a cleaving agent in a subject or a sample by (i)
contacting a subject or biological sample with an activatable
antibody, wherein the activatable antibody includes a detectable
label that is positioned on a portion of the activatable antibody
that is released following cleavage of the CM; and (ii) measuring a
level of detectable label in the subject or biological sample,
wherein a detectable level of the detectable label in the subject
or biological sample indicates that the cleaving agent is absent
and/or not sufficiently present in the subject or biological sample
at a detectable level, such that protease cleavage of the
activatable antibody cannot be detected in the subject or
biological sample, and wherein a reduced detectable level of the
detectable label in the subject or biological sample indicates that
the cleaving agent is present in the subject or biological sample.
A reduced level of detectable label is, for example, a reduction of
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95% and/or about 100%. Such an activatable antibody includes
a masking moiety (MM), a cleavable moiety (CM) that is cleaved by
the cleaving agent, and an antigen binding domain or fragment
thereof (AB) that specifically binds the target, wherein the
activatable antibody in an uncleaved (i.e., non-activated) state
comprises a structural arrangement from N-terminus to C-terminus as
follows: MM-CM-AB or AB-CM-MM; (a) wherein the MM is a peptide that
inhibits binding of the AB to the target, and wherein the MM does
not have an amino acid sequence of a naturally occurring binding
partner of the AB; and (b) wherein the MM of the activatable
antibody in an uncleaved state interferes with specific binding of
the AB to the target, and wherein the MM of an activatable antibody
in a cleaved (i.e., activated) state does not interfere or compete
with specific binding of the AB to the target. In some embodiments,
the activatable antibody is an activatable antibody to which a
therapeutic agent is conjugated. In some embodiments, the
activatable antibody is not conjugated to an agent. In some
embodiments, the activatable antibody comprises a detectable label.
In some embodiments, the detectable label is positioned on the AB.
In some embodiments, measuring the level of activatable antibody in
the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0472] The disclosure also provides kits for use in methods of
detecting presence or absence of a cleaving agent of interest in a
subject or a sample, where the kits include at least an activatable
antibody and/or conjugated activatable antibody described herein
for use in contacting a subject or biological sample and means for
detecting the level of activated activatable antibody and/or
conjugated activatable antibody in the subject or biological
sample, wherein the activatable antibody includes a detectable
label that is positioned on a portion of the activatable antibody
that is released following cleavage of the CM, wherein a detectable
level of the detectable label in the subject or biological sample
indicates that the cleaving agent, the target, or both the cleaving
agent and the target are absent and/or not sufficiently present in
the subject or biological sample, such that the target binding
and/or protease cleavage of the activatable antibody cannot be
detected in the subject or biological sample, and wherein a reduced
detectable level of the detectable label in the subject or
biological sample indicates that the cleaving agent and the target
are present in the subject or biological sample. A reduced level of
detectable label is, for example, a reduction of about 5%, about
10%, about 15%, about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95% and/or
about 100%.
[0473] In some embodiments of these methods and kits, the
activatable antibody includes a detectable label. In some
embodiments of these methods and 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
kits, the imaging agent comprises a radioisotope. In some
embodiments of these methods and kits, the radioisotope is indium
or technetium. In some embodiments of these methods and kits, the
contrasting agent comprises iodine, gadolinium or iron oxide. In
some embodiments of these methods and kits, the enzyme comprises
horseradish peroxidase, alkaline phosphatase, or
.beta.-galactosidase. In some embodiments of these methods and
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 kits, 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 and kits, the ligand-based label comprises biotin,
avidin, streptavidin or one or more haptens.
[0474] In some embodiments of these methods and kits, the subject
is a mammal. In some embodiments of these methods and 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.
[0475] 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.
[0476] 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.
[0477] In some embodiments, in situ 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 (e.g., 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 (e.g., 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.
[0478] 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 (e.g., 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.
[0479] In some embodiments of the methods and kits, the method or
kit 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 (e.g., the target) and a protease that cleaves the substrate
in the cleavable moiety (CM) of the activatable antibody being
tested in these methods are identified as suitable candidates for
treatment with such an activatable antibody comprising such a CM.
Likewise, patients that test negative for both of the targets
(e.g., 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 can be tested with
other activatable antibodies 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, patients that test negative for
either of the target (e.g., the target) are identified as suitable
candidates for treatment with such an activatable antibody
comprising such a CM. In some embodiments, patients that test
negative for either of the target (e.g., the target) are identified
as not being suitable candidates for treatment with such an
activatable antibody comprising such a CM. In some embodiments,
such patients can be tested with other activatable antibodies 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
activatable antibody is an activatable antibody to which a
therapeutic agent is conjugated. In some embodiments, the
activatable antibody is not conjugated to an agent. In some
embodiments, the activatable antibody comprises a detectable label.
In some embodiments, the detectable label is positioned on the AB.
In some embodiments, measuring the level of activatable antibody in
the subject or sample is accomplished using a secondary reagent
that specifically binds to the activated antibody, wherein the
reagent comprises a detectable label. In some embodiments, the
secondary reagent is an antibody comprising a detectable label.
[0480] In some embodiments, a method or kit is used to identify or
otherwise refine a patient population suitable for treatment with
an anti-the target activatable antibody and/or conjugated
activatable antibody (e.g., activatable antibody to which a
therapeutic agent is conjugated) of the disclosure, followed by
treatment by administering that activatable antibody and/or
conjugated activatable antibody to a subject in need thereof. For
example, patients that test positive for both the targets (e.g.,
the target) and a protease that cleaves the substrate in the
cleavable moiety (CM) of the activatable antibody and/or conjugated
activatable antibody being tested in these methods are identified
as suitable candidates for treatment with such antibody and/or such
a conjugated activatable antibody comprising such a CM, and the
patient is then administered a therapeutically effective amount of
the activatable antibody and/or conjugated activatable antibody
that was tested. Likewise, patients that test negative for either
or both of the target (e.g., 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 can be tested with other antibody and/or conjugated
activatable antibody until a suitable antibody and/or conjugated
activatable antibody for treatment is identified (e.g., an
activatable antibody and/or 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 activatable antibody and/or
conjugated for which the patient tested positive.
[0481] In some embodiments of these methods and kits, the MM is a
peptide having a length from about 4 to 40 amino acids. In some
embodiments of these methods and kits, the activatable antibody
comprises a linker peptide, wherein the linker peptide is
positioned between the MM and the CM. In some embodiments of these
methods and kits, the activatable antibody comprises a linker
peptide, where the linker peptide is positioned between the AB and
the CM. In some embodiments of these methods and kits, the
activatable antibody comprises a first linker peptide (L1) and a
second linker peptide (L2), wherein the first linker peptide is
positioned between the MM and the CM and the second linker peptide
is positioned between the AB and the CM. In some embodiments of
these methods and kits, each of L1 and L2 is a peptide of about 1
to 20 amino acids in length, and wherein each of L1 and L2 need not
be the same linker. In some embodiments of these methods and kits,
one or both of L1 and L2 comprises a glycine-serine polymer. In
some embodiments of these methods and kits, at least one of L1 and
L2 comprises an amino acid sequence selected from the group
consisting of (GS)n, (GSGGS)n (SEQ ID NO: 1) and (GGGS)n (SEQ ID
NO: 2), where n is an integer of at least one. In some embodiments
of these methods and kits, at least one of L1 and L2 comprises an
amino acid sequence having the formula (GGS)n, where n is an
integer of at least one. In some embodiments of these methods and
kits, at least one of L1 and L2 comprises an amino acid sequence
selected from the group consisting of Gly-Gly-Ser-Gly (SEQ ID NO:
3), Gly-Gly-Ser-Gly-Gly (SEQ ID NO: 4), Gly-Ser-Gly-Ser-Gly (SEQ ID
NO: 5), Gly-Ser-Gly-Gly-Gly (SEQ ID NO: 6), Gly-Gly-Gly-Ser-Gly
(SEQ ID NO: 7), and Gly-Ser-Ser-Ser-Gly (SEQ ID NO: 8).
[0482] In some embodiments of these methods and kits, the AB
comprises an antibody or antibody fragment sequence selected from
the cross-reactive antibody sequences presented herein. In some
embodiments of these methods and kits, the AB comprises a Fab
fragment, a scFv or a single chain antibody (scAb).
[0483] In some embodiments of these methods and kits, the cleaving
agent is a protease that is co-localized in the subject or sample
with the target and the CM is a polypeptide that functions as a
substrate for the protease, wherein the protease cleaves the CM in
the activatable antibody when the activatable antibody is exposed
to the protease. In some embodiments of these methods and kits, the
CM is a polypeptide of up to 15 amino acids in length. In some
embodiments of these methods and kits, the CM is coupled to the
N-terminus of the AB. In some embodiments of these methods and
kits, the CM is coupled to the C-terminus of the AB. In some
embodiments of these methods and kits, the CM is coupled to the
N-terminus of a VL chain of the AB.
[0484] The activatable antibodies and/or conjugated activatable
antibodies of the disclosure are used in diagnostic and
prophylactic formulations. In one embodiment, an activatable
antibody is administered to patients that are at risk of developing
one or more of the aforementioned inflammation, inflammatory
disorders, cancer or other disorders.
[0485] A patient's or organ's predisposition to one or more of the
aforementioned disorders can be determined using genotypic,
serological or biochemical markers.
[0486] In some embodiments, an activatable antibody and/or
conjugated activatable antibodies is administered to human
individuals diagnosed with a clinical indication associated with
one or more of the aforementioned disorders. Upon diagnosis, an
activatable antibody and/or conjugated activatable antibodies is
administered to mitigate or reverse the effects of the clinical
indication.
[0487] Activatable antibodies and/or conjugated activatable
antibodies of the disclosure are also useful in the detection of
the target in patient samples and accordingly are useful as
diagnostics. For example, the activatable antibodies and/or
conjugated activatable antibodies of the disclosure are used in in
vitro assays, e.g., ELISA, to detect target levels in a patient
sample.
[0488] In one embodiment, an activatable antibody of the disclosure
is immobilized on a solid support (e.g., the well(s) of a
microtiter plate). The immobilized activatable antibody serves as a
capture antibody for any target that may be present in a test
sample. Prior to contacting the immobilized antibody with a patient
sample, the solid support is rinsed and treated with a blocking
agent such as milk protein or albumin to prevent nonspecific
adsorption of the analyte.
[0489] Subsequently the wells are treated with a test sample
suspected of containing the antigen, or with a solution containing
a standard amount of the antigen. Such a sample is, e.g., a serum
sample from a subject suspected of having levels of circulating
antigen considered to be diagnostic of a pathology. After rinsing
away the test sample or standard, the solid support is treated with
a second antibody that is detectably labeled. The labeled second
antibody serves as a detecting antibody. The level of detectable
label is measured, and the concentration of target antigen in the
test sample is determined by comparison with a standard curve
developed from the standard samples.
[0490] It will be appreciated that based on the results obtained
using the antibodies of the disclosure in an in vitro diagnostic
assay, it is possible to stage a disease in a subject based on
expression levels of the Target antigen. For a given disease,
samples of blood are taken from subjects diagnosed as being at
various stages in the progression of the disease, and/or at various
points in the therapeutic treatment of the disease. Using a
population of samples that provides statistically significant
results for each stage of progression or therapy, a range of
concentrations of the antigen that may be considered characteristic
of each stage is designated.
[0491] Activatable antibodies and/or 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, activatable antibodies 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
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 antibodies (i.e., antibodies resulting from cleavage of
an activatable antibody) in a given cell or tissue of a given host
organism. Such accumulation of activated 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.
[0492] 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
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, 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.
[0493] In another example, activatable antibodies can be used to
detect the presence or absence of a cleaving agent in a sample. For
example, where the activatable antibodies contain a CM susceptible
to cleavage by an enzyme, the activatable antibodies can be used to
detect (either qualitatively or quantitatively) the presence of an
enzyme in the sample. In another example, where the activatable
antibodies contain a CM susceptible to cleavage by reducing agent,
the 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 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.
[0494] 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 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.
[0495] 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).
[0496] 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.
[0497] In these techniques, an activatable antibody is labeled with
a detectable label. The detectable label may be a fluorescent dye,
(e.g. Fluorescein Isothiocyanate (FITC), Rhodamine Isothiocyanate
(TRITC), 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).
[0498] 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 such as those described herein, and/or by 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 matriptase; see
e.g., International Publication Number WO 2010/129609, published 11
Nov. 2010. The same approach of using broad spectrum protease
inhibitors such as those described herein, 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.
[0499] 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.
[0500] 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.
[0501] 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.
[0502] In some embodiments, in situ 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 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 are
identified as suitable candidates for another form of therapy
(i.e., not suitable for treatment with the activatable antibody
being tested). 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).
[0503] 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 are identified as suitable candidates
for another form of therapy (i.e., not suitable for treatment with
the activatable antibody being tested). 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).
[0504] Pharmaceutical Compositions
[0505] The conjugated antibodies, activatable antibodies and/or
conjugated activatable antibodies of the disclosure (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 antibody,
activatable antibody and/or 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. Suitable 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.
[0506] A pharmaceutical composition of the disclosure 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.
[0507] 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 some embodiments, it
will be desirable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, 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.
[0508] 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.
[0509] 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.
[0510] 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.
[0511] 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.
[0512] 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.
[0513] 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.
[0514] 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 disclosure 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.
[0515] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0516] 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
Ability of Synovial Fluid to Activate Quenched Probes Comprising
Substrates of the Disclosure
[0517] This Example demonstrates the ability of synovial fluid
samples to cleave MMP substrate sequences of the disclosure. In
particular, the MMP cleavable sequences were tested in the context
of an activatable antibody construct comprising a masking moiety
linked to an anti-IL-6R antibody sequence via a linker region that
includes the MMP cleavable sequence being evaluated.
[0518] The following MMP-cleavable activatable antibodies were
incubated with synovial fluid:
TABLE-US-00032 4792.sup.10419AV1 amino acid (SEQ ID NO: 115)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSGISSGLSSGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.10419AV1 nucleotide (SEQ ID NO: 116)
Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcaggtattagtagtggtcttagcagtggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt 4792.sup.559AV1 amino acid (SEQ ID NO: 117)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSQNQALRMAGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.559AV1 nucleotide (SEQ ID NO: 118)
caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcacagaatcaggcattacgtatggcaggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt 4792.sup.601AV1 amino acid (SEQ ID NO: 119)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSAQNLLGMVGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.601AV1 nucleotide (SEQ ID NO: 120)
Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcagcacagaatctgttaggtatggtaggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt 4792.sup.3457AV1 amino acid (SEQ ID NO: 121)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSSTFPFGMFGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.3457AV1 nucleotide (SEQ ID NO: 122)
Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcaagtacatttccattcggtatgttcggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt 4792.sup.3458AV1 amino acid (SEQ ID NO: 123)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSPVGYTSSLGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.3458AV1 nucleotide (SEQ ID NO: 124)
Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcacctgttggatatacgagtagtctgggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt 4792.sup.3463AV1 amino acid (SEQ ID NO: 125)
QGQSGQYGSCSWNYVHIFMDCGSSGGSGGSGGSDWLYWPGIGGSDIQMTQSPSSLSASVGDRVT
ITCRASQDISSYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTFTISSLQPEDIA
TYYCQQGNTLPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC
4792.sup.3463AV1 nucleotide (SEQ ID NO: 66)
Caaggccagtctggccagtatgggtcctgcagttggaactatgtacacatattcatggattgcg
gctcgagcggtggcagcggtggctctggtggctcagactggttatactggcctggtattggcgg
ttctgacatccagatgactcagtctcctagctccctgtccgcctctgtgggggaccgagtcacc
atcacatgcagagccagccaggatatttctagttacctgaactggtatcagcagaagcccggaa
aagcacctaagctgctgatctactatacctccaggctgcactctggcgtgcccagtcggttcag
tggctcagggagcggaaccgacttcacttttaccatctcaagcctgcagccagaggatattgcc
acatactattgtcagcagggcaatacactgccctacacttttggccaggggaccaaggtggaaa
tcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatc
tggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtgg
aaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagg
acagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagt
ctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga
gagtgt
[0519] The extent of activatable antibody activation was determined
by an ELISA format that measured the ability of the activatable
antibody, following incubation in synovial fluid, to bind to human
IL6R as compared to the binding of anti-IL6R parental antibody to
IL6R. Briefly, Nunc Maxisorp plates were coated overnight at
4.degree. C. with 100 .mu.l/well (microliters/well) of a 500-ng/mL
solution of human IL6R (R and D Systems, Cat No. 227-SR/CF) in PBS,
pH 7.4. Plates were washed 3 times with PBST (PBS, pH 7.4, 0.05%
Tween-20). Wells were then blocked with 200 .mu.l/well, 2% NFDM
(non-fat dry milk) in PBST for 2 hours at room temperature. The
IL6R-coated plates were washed 3 times with PBST (PBS, pH 7.4,
0.05% Tween-20). A dilution series of each activatable
antibody--synovial fluid reaction mixture, as well as a dilution
series of the parental anti-IL6R antibody, was added to appropriate
wells of the IL6R-coated ELISA plate. The plates were incubated 1
hour at room temperature, and then washed 3 times with PBST (PBS,
pH 7.4, 0.05% Tween-20). One hundred .mu.l/well 1:3000 dilution
goat-anti-human IgG (Fab specific, Sigma Cat No. A0293) in 2%
NFDM-PBST was added, and the plate incubated for 1 hour at room
temperature. The plates were washed 6 times with PBST (PBS, pH 7.4,
0.05% Tween-20) and then developed with TMB and 1N HCl.
[0520] Table 6 provides the results of this experiment. The data
indicate that anti-IL6R activatable antibodies comprising the
substrates in Table 6 are cleaved by at least some synovial fluid
samples (SyF) obtained from RA patients.
TABLE-US-00033 TABLE 6 Activatable Antibody Activation Substrate/
Activation Activation Incidence Sequence in vivo in SyF in SyF
10419 <5% >30% 3/3 ISSGLSS (SEQ ID NO: 159) 559 <5% 20%
3/3 QNQALRMA (SEQ ID NO: 15) 601 <5% >30% 3/3 AQNLLGMV (SEQ
ID NO: 16) 3457 10% >50% 3/3 STFPFGMF (SEQ ID NO: 17) 3458 10%
20% 3/3 PVGYTSSL (SEQ ID NO: 18) 3463 <5% >30% 3/3 DWLYWPGI
(SEQ ID NO: 19)
Example 2
Activatable Anti-EGFR Antibody with MMP-Cleavable Substrate to
Inhibit Tumor Growth
[0521] This Example demonstrates the ability of an activatable
anti-EGFR antibody that contains a masking moiety comprising the
amino acid sequence CISPRGCPDGPYVMY (SEQ ID NO: 160), a cleavage
moiety comprising the MMP14 substrate 520 (also referred to herein
as MN520) ISSGLLSS (SEQ ID NO: 14), and the heavy chain (SEQ ID NO:
56) and light chain (SEQ ID NO: 59) of the anti-EGFR antibody
C225v5, where the entire activatable antibody construct is referred
to herein as Pb-MN520, to inhibit tumor growth in the H292
xenograft lung cancer model. The configuration of the light chain
of the activatable antibody was masking moiety--MMP
substrate--light chain of C225v5.
[0522] FIG. 1A is a graph depicting the effects seen in H292
xenograft tumor-bearing mice that were treated using Pb-520 (12.5
mg/kg, solid blue line) and IVIG (12.5 mg/kg, green dashed line)
dosed at different times. Data are presented as mean tumor
volume.+-.SEM. FIG. 1B is a graph depicting systemic stability of
the Pb-520 activatable antibody in H292 tumor bearing mice. Blood
samples were taken through retro-orbital bleeds at Day 7 and the
circulating stability of substrate 520 was determined by analysis
of IgG pull-downs with capillary electrophoresis (GXII; Caliper
LifeSciences). Concentrations of cleaved and uncleaved light chain
were determined using LabChip GX software (Caliper
LifeSciences).
Example 3
Materials and Methods
[0523] Reagents and Strains:
[0524] Streptavidin-conjugated phycoerythrin (SA-PE) (Invitrogen,
Life Technologies) was used without modifications. Human MMP9
(Research & Diagnostics Systems, Inc.) was activated following
the supplied protocol and used without modifications. Human MMP14
(Research & Diagnostics Systems, Inc.) was activated following
the supplied protocol and used without modifications. Human Plasmin
(Haematologic Technologies Inc.) was used without modifications.
Human tPA (Molecular Innovations) was used without modifications.
YPet fused to the SH3 domain of Mona (monocytic adaptor protein)
was produced at CytomX Therapeutics and used without modifications.
MMP14 Buffer HCM (50 mM HEPES (pH 6.8), 10 mM CaCl.sub.2, 0.5 mM
MgCl.sub.2), was used. MMP9 Buffer TCNB (50 mM Tris-HCl, 10 mM
CaCl.sub.2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5) was used.
Plasmin Buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 0.01% Tween20
and 1 mM EDTA was used. TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05%
Tween20, pH 7.4) was used. E. coli MC1061 (Casadaban et al., JMB
138(2):179-207 (1980) was used. All bacterial growth was performed
at 37.degree. C. with vigorous shaking in Luria-Bertani broth (LB)
supplemented with 34 .mu.g/mL chloramphenicol, unless another
antibiotic is specified.
[0525] Substrate Cleavage and Scaffold Stability Analysis:
[0526] For screening and clone analysis, overnight cultures were
subcultured by dilution into fresh medium (1:50) and grown for
1.5-2 hours. The subculture was then induced with 0.04% arabinose
and incubated with shaking at 37.degree. C. for 45 minutes to 1
hour. To stop further growth cells were incubated on ice for 15
minutes to 1 hour. Cell aliquots were harvested and washed with PBS
(pH 7.4). Cells were pelleted by centrifugation, the supernatant
removed and the cells resuspended in reaction buffer containing the
enzyme; the reaction mixture was incubated at 37.degree. C. static.
To stop the reaction, cells were removed and diluted 10-fold in
PBS, pelleted by centrifugation, and resuspended in PBS containing
either (CLiPS) SA-PE (20 .mu.g/mL) or YPet-MONA (50 nM). After
incubation on ice (30 min), cells were washed with PBS and analyzed
using a FACSAria.TM. cell sorter.
[0527] For MMP9 protease cleavage assays, cultures were induced for
45 minutes to 1 hour. The reaction buffer for MMP9 was TCNB. Assays
for MMP9 hydrolysis were performed after fresh cells were incubated
with 5 nM-25 nM MMP9 for 1 hr. Background hydrolysis of the regions
flanking the substrate site (using platform eCLiPS3.0-NSUB_SP
described in PCT patent application PCT/US13/54378, filed Aug. 9,
2013, which was published as International Publication No. WO
2014/026136 on 13 Feb. 2014, the contents of which are hereby
incorporated by reference in their entirety) was measured under
each reaction condition to ensure that hydrolysis occurred in the
designated substrate region.
[0528] For MMP14 protease cleavage assays, cultures were induced
for 45 minutes to 1 hour. The reaction buffer for MMP14 was HCM.
Assays for MMP14 hydrolysis were performed after reactions with 3
nM-250 nM MMP14 or 1 hr. Background hydrolysis of the regions
flanking the substrate site (using platform eCLiPS3.0-NSUB_SP
described herein) was measured under each reaction condition to
ensure that hydrolysis occurred in the designated substrate
region.
[0529] For human plasmin stability assays, platform
eCLiPS3.0-NSUB_SP is used; cultures are induced for 45 minutes to 1
hr. The reaction buffer for plasmin is 50 mM Tris-HCl pH 7.5
supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA. Assays
for plasmin hydrolysis are performed after reactions with plasmin
for 1 hr.
[0530] For human tPA stability assays, platform eCLiPS3.0-NSUB_SP
is used; cultures are induced for 45 minutes to 1 hr. The reaction
buffer for tPA is TBST. Assays for tPA hydrolysis are performed
after reactions with tPA for 1 hr.
[0531] Amino and Carboxy Terminus Labeling Conditions:
[0532] Streptavidin conjugated phycoerythrin (SAPE) was used for
labeling streptavidin binding affinity ligand on the N-termini of
CPX. Fluorescent protein YPet fused to the SH3 domain of Mona was
used for labeling the MONA binding affinity ligand on the C-termini
of CPX. For optimum labeling of cells without protease reaction,
the cells were incubated for 30 min at 4.degree. C. with SAPE (20
.mu.g/mL) or YPet-MONA (50 nM). For the described example below 30
min incubation was used.
[0533] Kinetic Data Analysis:
[0534] The extent of conversion of cell surface displayed peptide
substrates was measured directly, using flow cytometry to measure
changes in mean fluorescence of clonal cell populations upon
protease treatment. Specifically, for each sample, conversion was
determined by flow cytometry analyses using the relationship
Conversion = F L - - F L + F L - - F L 0 [ 1 ] ##EQU00001##
where (FL.sub.-) is the fluorescence after incubating without
enzyme, (FL.sub.+) is fluorescence after incubation with enzyme,
and (FL.sub.0) is fluorescence of unlabeled cells. Given that the
expected substrate concentrations that were used are significantly
below the expected K.sub.M of the substrate for the target
protease, the Michaelis-Menten model simplifies to:
[ S ] t .apprxeq. k cat k M [ S ] [ E ] [ 2 ] ##EQU00002##
allowing substrate conversion to be expressed as
Conversion = 1 - exp ( - k cat k M [ E ] t ) [ 3 ] ##EQU00003##
where [S] is the substrate concentration, [E] is enzyme
concentration and t is time. To determine the second order rate
constant (k.sub.cat/K.sub.M), equation [3] was simplified to:
k cat K m = - ln ( 1 - C ) / ( t * p ) ##EQU00004##
where C is product conversion, t is time and p is protease
concentration.
[0535] Sequence Data Analysis--Directed Families:
[0536] Substrates were submitted to Ion Torrent.TM. sequencing
(see, e.g., Rothenberg, J M, Nature 475, 348-352). Raw Ion Torrent
reads were cropped by invariant vector sequences to obtain just the
variable peptide insert. Insert sequences were translated, and
sequences with stop codons were excluded from further analysis. The
frequency of each sequence was obtained by number of times observed
out of all viable peptide reads observed. Enrichment of sequences
was obtained by comparison of observed frequency of each sequence
post selection to the frequency of each sequence pre-selection.
Individual sequences were identified and isolated from these data,
and sequences were aligned in CLC main lab (CLC Main Workbench
6.6.2, available online). The alignment file was imported to
Jalview (see, e.g., Waterhouse, A. M., et al., 2009, Bioinformatics
9, 1189-1191), and an average distance tree was assembled using the
BLOSUM62 algorithm (S Henikoff S et al., 1992, Proc Natl Acad Sci
USA. 89, 10915-10919). The restricted group of sequences includes
members of the cluster closest to the sequence of interest. The
extended group of sequences includes the restricted group of
sequences plus members of the branch that shares the closest common
ancestor (where applicable).
Example 4
Selection and Characterization of Substrate Pools in a Platform
Scaffold
[0537] The use of multi-copy substrate display on whole cells
enabled selection of populations of substrates cleaved by MMP9.
Selections were performed as described in U.S. Pat. No. 7,666,817
B2, issued Feb. 23, 2010, using recombinant human MMP9. Background
hydrolysis of the regions flanking the substrate site (using
platform eCLiPS3.0-NSUB_SP) was measured under each reaction
condition to ensure that hydrolysis occurred in the designated
substrate region. Selected pools were tested with MMP9 and MMP14.
FIGS. 2A and 2B show cleavage of pool SMP87 by MMP9 at 5 nM in TCNB
buffer.
Example 5
Characterization of Substrate Cleavage Kinetics in the Platform
Scaffold
[0538] The use of multi-copy substrate display on whole cells
enabled simple and direct quantitative characterization of cleavage
kinetics. Consequently, flow cytometry was used to rank individual
isolated clones on the basis of substrate conversion, and clones
were identified by DNA sequencing. In this way, the extent of
conversion for each clone could be determined at several different
protease concentrations and fit to a Michaelis-Menten model
(Kinetic Data Analysis Section). The observed second order rate
constant (k.sub.cat/K.sub.M) was determined for each substrate
versus MMP9. Background hydrolysis of the regions flanking the
substrate site (using platform eCLiPS3.0-NSUB_SP), was measured
under each reaction condition to ensure that hydrolysis occurred in
the designated substrate region. For example, FIGS. 3A and 3B show
cleavage of a substrate comprising amino acid sequence VAGRSMRP
(SEQ ID NO: 484) by 5 nM MMP9 in TBST.
Example 6
Correlation of Next Generation Sequencing Frequency and Substrate
Cleavage Kinetics in the Platform Scaffold
[0539] Final pools of enriched substrates were sequenced using Ion
Torrent Next-Generation Sequencing. Raw Ion Torrent reads were
cropped by invariant vector sequences to obtain just the variable
peptide insert. Insert sequences were translated, and sequences
with stop codons were excluded from further analysis. A selection
of clones (displaying a range of frequencies) was selected for
functional analysis. Selected clones were cleaved with human MMP9,
and a k.sub.cat/K.sub.M was determined for each. The log of the
clone copy number in the pool was then plotted versus the log of
the k.sub.cat/K.sub.M. FIG. 4 shows the correlation between
frequency of particular cleavage moieties (Copy Number) and their
abilities to be cleaved by MMP9 (MMP9 k.sub.cat/K.sub.M M.sup.-1
s.sup.-1).
Example 7
Selection and Characterization of Substrate Pools in a Platform
Scaffold
[0540] The use of multi-copy substrate display on whole cells
enabled selection of populations of substrates cleaved by MMP14.
Selections were performed as described in U.S. Pat. No. 7,666,817
B2, using recombinant human MMP14. Background hydrolysis of the
regions flanking the substrate site (using platform
eCLiPS3.0-NSUB_SP) was measured under each reaction condition to
ensure that hydrolysis occurred in the designated substrate region.
Selected pools were tested with MMP9 and MMP14. FIGS. 5A and 5B
show cleavage of pool SMP39 by MMP14 at 60 nM in HCM buffer.
Example 8
Characterization of Substrate Cleavage Kinetics in the Platform
Scaffold
[0541] The use of multi-copy substrate display on whole cells
enabled simple and direct quantitative characterization of cleavage
kinetics. Consequently, flow cytometry was used to rank individual
isolated clones on the basis of substrate conversion, and clones
were identified by DNA sequencing. In this way, the extent of
conversion for each clone could be determined at several different
protease concentrations and fit to a Michaelis-Menten model
(Kinetic Data Analysis Section). The observed second order rate
constant (k.sub.cat/K.sub.M) was determined for each substrate
versus MMP14. Background hydrolysis of the regions flanking the
substrate site (using platform eCLiPS3.0-NSUB_SP), was measured
under each reaction condition to ensure that hydrolysis occurred in
the designated substrate region. For example, FIGS. 6A and 6B show
cleavage of a substrate comprising amino acid sequence QNQALRMA
(SEQ ID NO: 15) by 30 nM MMP14 in HCM buffer.
Example 9
In Vitro Substrate Activity in Activatable Antibodies
[0542] This Example demonstrates the in vitro activity of
substrates of the disclosure when they are incorporated into
activatable antibodies.
[0543] Several substrates identified in these studies were inserted
into activatable antibodies having the 3954 mask and C225v5 variant
of cetuximab, which is described in PCT Publication No. WO
2013/163631, and which is incorporated herein by reference in its
entirety.
[0544] The ability of substrates in the resultant activatable
antibodies to be cleaved by MMP9 or MMP14 was determined as
follows. MMP9 protease digests were performed in TCNB, 50 mM
Tris-HCl, 10 mM CaCl.sub.2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH
7.5. MMP14 digests were performed in 50 mM HEPES (pH 6.8), 10 mM
CaCl.sub.2, 0.5 mM MgCl.sub.2. Varying concentrations of active
site titrated MMP9 or MMP14 were combined with a fixed activatable
antibody concentration to maintain a substrate to protease ratio of
at least 50. Samples comprising MMP9 substrates were incubated at
37.degree. C. for up to 24 hr. Samples comprising MMP14 substrates
were incubated at 37.degree. C. for 4 hr. To stop the reaction, 5
.mu.l of the digest was added to 7 .mu.l of HT Protein Express
Sample Buffer (Caliper LifeSciences) containing 20 mM
2-Mercaptoethanol for 10 minutes at 95.degree. C. After heat
denaturation, 32 .mu.l of ddH.sub.2O was added and samples analyzed
on a LabChip GXII per manufacturer's instructions. The LabChip GXII
software was used to quantify light chain peak area. Product
conversion was calculated by plugging the light chain peak areas
into the following equation: cleaved LC/(cleaved LC+uncleaved LC),
LC=light chain. k.sub.cat/K.sub.M values were determined with the
following equation
k cat K m = - ln ( 1 - C ) / ( t * p ) ##EQU00005##
where C is product conversion, t is time (s), and p is protease
concentration (M), which assumes that the substrate concentration
is below the K.sub.M and in excess of the protease
concentration.
[0545] Resultant activatable antibodies comprising substrates
selected for cleavage by MMP14 tested for cleavage by MMP14 had
k.sub.cat/K.sub.M values ranging from about 400 to 60,000 M.sup.-1
s.sup.-1 for MMP14. Resultant activatable antibodies comprising
substrates selected for cleavage by MMP9 tested for cleavage by
MMP9 were cleaved by MMP9.
Example 10
Substrate Stability of Activatable Antibodies In Vivo
[0546] This Example demonstrates the in vivo stability of
substrates of the disclosure when they are incorporated into
activatable antibodies and injected into mice.
[0547] Three nude mice (Crl:NU-Foxn1nu) received a single IP dose
of each activatable antibody at 12.5 mg/kg on Day 0. Mice were
euthanized on day 4 (.about.96 h post-dose) by CO.sub.2
asphyxiation, and blood was collected immediately as plasma-EDTA
and stored at -80.degree. C.
[0548] Activatable antibodies were purified from plasma by
anti-human IgG immunoprecipitation using magnetic beads. Eluted
activatable antibodies were prepared for analysis by capillary
electrophoresis as described in the k.sub.cat/K.sub.M section.
Briefly, 5 .mu.l of eluted IgG was added to 7 .mu.l Protein Express
Sample Buffer with 2-mercaptoethanol. Quantification of circulating
stability was identical to quantification of product
conversion.
[0549] Of ten activatable antibodies comprising substrates of the
disclosure selected for cleavage by MMP14, seven demonstrated less
than 20% cleavage in the collected plasma samples. Of seven
activatable antibodies comprising substrates of the disclosure
selected for cleavage by MMP9, four demonstrated no more than 20%
cleavage in the collected plasma samples.
Example 11
Materials and Methods
[0550] Reagents and Strains:
[0551] Human MMP9 (catalog no. 911-MP, Research & Diagnostics
Systems, Inc.) was activated following the supplied protocol and
used without modifications. Human MMP14 (catalog no. 918-M),
Research & Diagnostics Systems, Inc.) was activated following
the supplied protocol and used without modifications. Human Plasmin
(catalog no. HCPM-0140, Haematologic Technologies Inc.) was used
without modifications. Anti-EE monoclonal antibody (Covance,
Princeton, N.J.) was labeled with Alexa 647 (Life Sciences) and
used with no other modifications (named EE647). E. coli MC1061 or
MC1061 derived strains (DH10.beta.) were used for all experiments
(Casadaban et al., JMB 138(2):179-207 (1980)). All bacterial growth
was performed at 37.degree. C. with vigorous shaking in
Luria-Bertani broth (LB) supplemented with 34 .mu.g/mL
chloramphenicol (cm), unless another antibiotic is specified.
[0552] Display Platforms:
[0553] Display platforms, each engineered to contain an 8-amino
acid substrate of the embodiments, were produced and used as
described in International Publication No. WO 2014/026136,
published 13 Feb. 2014, the contents of which are hereby
incorporated by reference in their entirety. The amino acid
sequence of the mature (i.e., without a signal peptide)
CYTX-DP-XXXXXXXX display platform (SEQ ID NO: 512) is shown in FIG.
7A. XXXXXXXX indicates the location into which each substrate is
inserted. The amino acid sequence of CYTX-DP-XXXXXXXX display
platform also including its signal peptide, i.e.,
SP-CYTX-DP-XXXXXXXX display platform (SEQ ID NO: 513) is shown in
FIG. 7B.
CYTX-DP-XXXXXXXX Display Platform:
TABLE-US-00034 [0554] (SEQ ID NO: 512)
GQSGQEYMPMEGGSGQXXXXXXXXSGGQGGSGGSGGSGGSGGSAYYGITA
GPAYRINDWASIYGVVGVGYGSGPGGSYGFSYGAGLQFNPMENVALDFSY
EQSRIRSVDVGTWILSVGYRFGSKSRRATSTVTGGYAQSDAQGQMNKMGG
FNLKYRYEEDNSPLGVIGSFTYTGGSGGSSGQAAAGHHHHHHHH
SP-CYTX-DP-XXXXXXXX Display Platform:
TABLE-US-00035 [0555] (SEQ ID NO: 513)
MKKIACLSALAAVLAFTAGTSVAGQSGQEYMPMEGGSGQXXXXXXXXSGG
QGGSGGSGGSGGSGGSAYYGITAGPAYRINDWASIYGVVGVGYGSGPGGS
YGFSYGAGLQFNPMENVALDFSYEQSRIRSVDVGTWILSVGYRFGSKSRR
ATSTVTGGYAQSDAQGQMNKMGGFNLKYRYEEDNSPLGVIGSFTYTGGSG
GSSGQAAAGHHHHHHHH
[0556] Substrate Cleavage and Cleavage Kinetics Analysis:
[0557] For clone analysis, overnight cultures were subcultured by
dilution into fresh medium (1:40) and grown for 1.5-2 hours. The
subculture was then induced with 0.04% arabinose and incubated with
shaking at 37.degree. C. for 40 minutes to 1 hour. To stop further
growth, cells were then incubated on ice for 15 minutes to 1 hour.
Cell aliquots were harvested and washed with PBS (pH 7.4). Cells
were pelleted by centrifugation, the supernatant removed and the
cells resuspended in reaction buffer containing the enzyme; the
reaction mixture was incubated at 37.degree. C. with shaking To
stop the reaction cells were removed and diluted 10-fold in PBS,
pelleted by centrifugation, and resuspended in PBS containing EE647
(20 micrograms per ml (also referred to herein as ug/ml or
.mu.g/ml)). After incubation on ice (1 hour), cells were washed
with PBS and analyzed using an Accuri C6 cell sorter.
[0558] For MMP9 protease cleavage assays, cultures were induced for
45 minutes. The reaction buffer for MMP9 was 50 mM Tris-HCl, pH
7.4, supplemented with 150 mM NaCl, 10 mM CaCl.sub.2 and 0.05%
(w/v) Brij-35. Assays for MMP9 hydrolysis, were performed after
cleavage with 5 nM-150 nM MMP9 for 1 hour. Background hydrolysis of
the regions flanking the substrate site (using, e.g., CYTX-DP-NSUB,
a display platform in which the "Substrate" is non-cleavable linker
GGGSGGGS) was measured under each reaction condition to ensure that
hydrolysis occurred in the designated substrate region.
[0559] For MMP14 protease cleavage assays, cultures were induced
for 45 minutes. The reaction buffer for MMP14 was 50 mM HEPES, pH
6.8, supplemented with 10 mM CaCl.sub.2 and 0.5 mM MgCl.sub.2.
Assays for MMP14 hydrolysis, were performed after cleavage with 5
nM-150 nM MMP14 for 1 hr. Background hydrolysis of the regions
flanking the substrate site (using, e.g., CYTX-DP-NSUB) was
measured under each reaction condition to ensure that hydrolysis
occurred in the designated substrate region.
[0560] For human plasmin stability assays, cultures were induced
for 45 minutes. The reaction buffer for plasmin was 50 mM Tris-HCl
pH 7.4, supplemented with 100 mM NaCl, 0.01% Tween20 and 1 mM EDTA.
Assays for plasmin hydrolysis were performed after cleavage with
500 pM plasmin for 1 hr. Background hydrolysis of the regions
flanking the substrate site (using, e.g., CYTX-DP-NSUB) was
measured under each reaction condition to ensure that hydrolysis
occurred in the designated substrate region.
[0561] Amino and Carboxyl Terminus Labeling Conditions:
[0562] Alexa-647 conjugated anti-EE antibody (EE647) was used for
labeling EE binding affinity ligand on the N-termini of the CYTX-DP
display platform. Alexa-647 conjugated anti-His antibody (His647)
was used for labeling the 8His binding affinity ligand on the
C-termini of the CYTX-DP display platform. For optimum labeling of
cells without protease reaction, the cells were incubated for 1
hour at 4.degree. C. with EE647 (20 .mu.g/mL) or His647 (2
.mu.g/mL). For the example described below, a 1-hour incubation was
used.
[0563] Kinetic Data Analysis:
[0564] The extent of conversion of cell surface displayed peptide
substrates was measured directly, using flow cytometry to measure
changes in mean fluorescence of clonal cell populations upon
protease treatment. Specifically, for each sample, conversion was
determined by flow cytometry analyses using the relationship
Conversion CLiPS = F L - - F L + F L - - F L 0 [ 1 ]
##EQU00006##
where (FL.sub.-) is the fluorescence after incubating without
enzyme, (FL.sub.+) is fluorescence after incubation with enzyme,
and (FL.sub.0) is fluorescence of unlabeled cells. Given that the
expected substrate concentrations that were used are significantly
below the expected K.sub.M of the substrate for the target
protease, the Michaelis-Menten model simplifies to
[ S ] t .apprxeq. k cat k M [ S ] [ E ] [ 2 ] ##EQU00007##
allowing substrate conversion to be expressed as
Conversion MM = 1 - exp ( - k cat k M [ E ] t ) [ 3 ]
##EQU00008##
where [S] is the substrate concentration, [E] is enzyme
concentration and t is time. To determine the second order rate
constant (k.sub.cat/K.sub.M), the time dependent conversion for
each substrate was fit to equation [3].
Example 12
Characterization of Substrate Cleavability in CYTX-DP Display
Platform
[0565] This Example demonstrates the ability of substrates of the
embodiments to be cleaved by MMP but not by plasmin.
[0566] The use of multi-copy substrate display on whole cells
enabled simple and direct quantitative characterization of cleavage
kinetics. Clones encoding substrates were identified by DNA
sequencing and subcloned into the CYTX-DP display platform such
that the expressed display platform contained the 8-amino acid
substrate in place of XXXXXXXX. Individual substrate displaying
clones (127 independent substrate-containing display platforms in
total) were assessed for cleavage by either MMP9 or MMP14 (target
proteases, i.e., the proteases used to select the substrate) and
plasmin (off-target protease); turnover was determined by flow
cytometry. Thirty-one of the MMP9-selected substrates were selected
from the same pool that was the source of substrates comprising
amino acid sequences SEQ ID NOs: 17, 18, 19, 20, 21, 22, or 23
(MMP9 substrates from pool). Nine of the MMP9-selected substrates
comprise consensus amino acid sequences SEQ ID NOs: 328, 336, 337,
338, 339, 348, 349, 350 or 351 (MMP9 consensus sequences).
Thirty-eight of the MMP14-selected substrates were selected from
the same pool that was the source of substrates comprising amino
acid sequences SEQ ID NOs: 14, 15, 16, 24, 25, 26, 27, 28, 29, 30,
or 33 (MMP14 substrates from 1.sup.st pool). Ten of the
MMP14-selected substrates were selected from the same pool that was
the source of substrates comprising amino acid sequences SEQ ID
NOs: 31 or 32 (MMP14 substrates from 2.sup.nd pool). Thirty-nine of
the MMP14-selected substrates were chosen from consensus amino acid
sequences SEQ ID NOs: 364-370, 379-393, 402-409, 420-424, 434-435,
450-452, 457, 470-472, 474, or 483 (MMP14 consensus sequences).
[0567] In this way, the extent of cleavage for each clone could be
determined and the data aggregated to determine a percent of clones
that are cleaved by the target protease and not the off-target
protease. Background hydrolysis of the regions flanking the
substrate site (using, e.g., CYTX-DP-NSUB) was measured under each
reaction condition to ensure that hydrolysis occurred in the
designated substrate region. Results are presented in Table 9.
TABLE-US-00036 TABLE 9 Summary statistics of substrate cleavability
>20% Cleavage with <20% Cleavage Discovery 50 nM MMP9 or with
500 pM effort Substrate Group MMP14 Plasmin MMP9- All MMP9 35% (14
of 40) 85% (34 of 40) selected substrates tested Substrates MMP9
substrates 39% (12 of 31) 84% (26 of 31) from pool MMP9 consensus
22% (2 of 9) 89% (8 of 9) substrates MMP14- All MMP14 85% (74 of
87) 94% (82 of 87) selected substrates tested Substrates MMP14 79%
(38 of 48) 94% (45 of 48) substrates from 1.sup.st and 2.sup.nd
pools MMP14 79% (30 of 38) 95% (36 of 38) substrates from 1.sup.st
pool MMP14 80% (8 of 10) 100% (9 of 10) substrates from 2.sup.nd
pool MMP14 92% (36 of 39) 95% (37 of 39) consensus substrates
Combined Total 69% (88 of 127) 91% (116 of 127) MMP9 and MMP14
[0568] Table 9 depicts (a) the percentage of MMP9-selected
substrates tested in the CYTX-DP display platform that exhibited at
least 20% cleavage when incubated with 50 nM human MMP9 (catalog
no. 911-MP, Research & Diagnostics Systems, Inc., activated
following the supplied protocol and used without modifications) for
1 hour at 37.degree. C. in 50 mM Tris-HCl, pH 7.4, supplemented
with 150 mM NaCl, 10 mM CaCl.sub.2, and 0.05% (w/v) Brij-35
(>20% Cleavage with 50 nM MMP9); (b) the percentage of
MMP14-selected substrates tested in the CYTX-DP display platform
that exhibited at least 20% cleavage when incubated with 50 nM
human MMP14 (catalog no. 918-MP, Research & Diagnostics
Systems, Inc., activated following the supplied protocol and used
without modifications) for 1 hour at 37.degree. C. in 50 mM HEPES,
pH 6.8, supplemented with 10 mM CaCl.sub.2, and 0.5 mM MgCl.sub.2
(>20% Cleavage with 50 nM MMP14); and (c) the percentage of
MMP9-selected or MMP-14-selected substrates tested in the CYTX-DP
display platform that exhibited less than 20% cleavage when
incubated with 500 pM human plasmin (catalog number HCPM-0140,
Haematologic Technologies, Inc., used without modifications) for 1
hour at 37.degree. C. in 50 mM Tris-HCl, pH 7.4, supplemented with
100 mM NaCl, 0.01% Tween20 and 1 mM EDTA (<20% cleavage with 500
pM plasmin).
Example 13
Characterization of Substrate Cleavage Kinetics in CYTX-DP Display
Platforms
[0569] This Example demonstrates the cleavage kinetics of various
substrates.
[0570] The use of multi-copy substrate display on whole cells
enabled simple and direct quantitative characterization of cleavage
kinetics. Clones were identified by DNA sequencing and subcloned
into the CYTX-DP-XXXXXXXX display platform as described in the
preceding example. Seventy-two individual substrate-displaying
clones were assessed for cleavage and a subset were chosen to
assess cleavage kinetics by their target protease. The extent of
conversion for each clone could be determined at several different
protease concentrations and fit to the Michaelis-Menten model
described herein. Observed k.sub.cat/K.sub.M values were then
plotted versus frequency of the clone within the substrate pool and
a correlation between frequency and k.sub.cat/K.sub.M was seen.
Background hydrolysis of the regions flanking the substrate site
(using, e.g., CYTX-DP-NSUB) was measured under each reaction
condition to ensure that hydrolysis occurred in the designated
substrate region. Results are presented in Table 10.
TABLE-US-00037 TABLE 10 Summary statistics of substrate kinetics
Target Target Protease Protease Substrate Target Protease
k.sub.cat/K.sub.M > k.sub.cal/K.sub.M > Group
k.sub.cat/K.sub.M > 1 .times. 10E2 1 .times. 10E3 1 .times. 10E4
MMP9 All MMP9 100% (16 of 16) 100% (16 of 16) 63% (10 of 16)
Substrates substrates tested MMP9 100% (15 of 15) 100% (15 of 15)
67% (10 of 15) substrates from pool MMP9 100% (1 of 1) 100% (1 of
1) 0% (0 of 1) consensus substrates MMP14 All MMP-14 100% (55 of
55) 98% (54 of 55) 36% (20 of 55) Substrates MMP14 100% (47 of 47)
98% (46 of 47) 36% (17 of 47) substrates from 1.sup.st and 2.sup.nd
pools MMP14 100% (38 of 38) 100% (38 of 38) 39% (15 of 38)
substrates from 1.sup.st pool MMP14 100% (9 of 9) 89% (8 of 9) 22%
(2 of 9) substrates from 2.sup.nd pool MMP14 100% (8 of 8) 100% (8
of 8) 38% (3 of 8) consensus substrates Combined Total 100% (71 of
71) 99% (70 of 71) 42% (30 of 71) MMP9 and MMP14
Example 14
In Vivo Efficacy and In Situ Activation of Activatable Antibodies
Comprising a MMP Substrate
[0571] This Example demonstrates that activatable antibodies
comprising MMP substrates of the embodiments are efficacious in
vivo. This Example also demonstrates that such activatable
antibodies are activatable in an in situ imaging assay, such as
that described in International Publication No. WO 2014/107559,
published 10 Jul. 2014, the contents of which are hereby
incorporated by reference in their entirety. Six activatable
antibodies comprising different MMP substrates (one MMP9-selected
and five MMP14-selected) of the embodiments were administered at 10
mg/kg or 12.5 mg/kg to H292 xenograft tumor-bearing (lung cancer)
mice. All six activatable antibodies also comprised the masking
moiety comprising the amino acid sequence CISPRGCPDGPYVMY (SEQ ID
NO: 160) and anti-EGFR antibody C225v5 antibody comprising a light
chain (SEQ ID NO: 59) and a heavy chain (SEQ ID NO: 56). The
configuration of the light chain of the activatable antibody was
masking moiety--MMP substrate--light chain of C225v5. All six
activatable antibodies demonstrated tumor growth inhibition ranging
from 22% to 81% as measured by mean % .DELTA. inhibition. Mean %
.DELTA. inhibition is calculated as
(mean(C)-mean(C0))-(mean(T)-mean(T0))/(mean(C)-mean(C0))*100%,
wherein T is the current test group value, T0 is the current test
group initial value, C is the control group value, and C0 is the
control group initial value. The EGFR antibody cetuximab
demonstrated 96-98% inhibition in this study.
[0572] The same six activatable antibodies were submitted to in
situ imaging assays of H292 tumor tissue, using the conditions
described in the examples of WO 2014/107559. All six activatable
antibodies were activated, demonstrating that all six MMP
substrates were cleaved and the released antibodies bound to EGFR
on the tumor tissue. The staining signals ranged from 15% to 65% of
the IHC signal intensity of cetuximab. In general, the percentage
of activation of each activatable antibody demonstrated a positive
correlation with the efficacy that activatable antibody
demonstrated in the H292 mouse model.
[0573] Tissue from ten triple negative breast cancer patients was
submitted to in situ imaging using an anti-Jagged activatable
antibody (e.g., an anti-Jagged activatable antibody cited in
International Publication No. WO 2013/192550, published 27 Dec.
2013, the contents of which are hereby incorporated by reference in
their entirety) comprising an MMP 14-selected substrate under the
conditions described in the examples of WO 2014/107559. Nine of the
ten tissue samples demonstrated activatable antibody activation
staining scores ranging from 15% to 100% as compared to the IHC
signal intensity of cetuximab: Eight of the ten tissue samples
demonstrated activatable antibody activation staining scores
ranging from 30% to 100% as compared to the IHC signal intensity of
cetuximab.
OTHER EMBODIMENTS
[0574] 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
1
51515PRTArtificial Sequencelinking peptide 1Gly Ser Gly Gly Ser 1 5
24PRTArtificial Sequencelinking peptide 2Gly Gly Gly Ser 1
34PRTArtificial Sequencelinking peptide 3Gly Gly Ser Gly 1
45PRTArtificial Sequencelinking peptide 4Gly Gly Ser Gly Gly 1 5
55PRTArtificial Sequencelinking peptide 5Gly Ser Gly Ser Gly 1 5
65PRTArtificial Sequencelinking peptide 6Gly Ser Gly Gly Gly 1 5
75PRTArtificial Sequencelinking peptide 7Gly Gly Gly Ser Gly 1 5
85PRTArtificial Sequencelinking peptide 8Gly Ser Ser Ser Gly 1 5
913PRTArtificial Sequencelinking peptide 9Gly Ser Ser Gly Gly Ser
Gly Gly Ser Gly Gly Ser Gly 1 5 10 1011PRTArtificial
Sequencelinking peptide 10Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly
Gly 1 5 10 1112PRTArtificial Sequencelinking peptide 11Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Gly Ser 1 5 10 125PRTArtificial
Sequencelinking peptide 12Gly Ser Ser Gly Thr 1 5 134PRTArtificial
Sequencelinking peptide 13Gly Ser Ser Gly 1 148PRTArtificial
Sequencecleavable moiety 14Ile Ser Ser Gly Leu Leu Ser Ser 1 5
158PRTArtificial Sequencecleavable moiety 15Gln Asn Gln Ala Leu Arg
Met Ala 1 5 168PRTArtificial Sequencecleavable moiety 16Ala Gln Asn
Leu Leu Gly Met Val 1 5 178PRTArtificial Sequencecleavable moiety
17Ser Thr Phe Pro Phe Gly Met Phe 1 5 188PRTArtificial
Sequencecleavable moiety 18Pro Val Gly Tyr Thr Ser Ser Leu 1 5
198PRTArtificial Sequencecleavable moiety 19Asp Trp Leu Tyr Trp Pro
Gly Ile 1 5 208PRTArtificial Sequencecleavable moiety 20Met Ile Ala
Pro Val Ala Tyr Arg 1 5 218PRTArtificial Sequencecleavable moiety
21Arg Pro Ser Pro Met Trp Ala Tyr 1 5 228PRTArtificial
Sequencecleavable moiety 22Trp Ala Thr Pro Arg Pro Met Arg 1 5
238PRTArtificial Sequencecleavable moiety 23Phe Arg Leu Leu Asp Trp
Gln Trp 1 5 248PRTArtificial Sequencecleavable moiety 24Leu Lys Ala
Ala Pro Arg Trp Ala 1 5 258PRTArtificial Sequencecleavable moiety
25Gly Pro Ser His Leu Val Leu Thr 1 5 268PRTArtificial
Sequencecleavable moiety 26Leu Pro Gly Gly Leu Ser Pro Trp 1 5
278PRTArtificial Sequencecleavable moiety 27Met Gly Leu Phe Ser Glu
Ala Gly 1 5 288PRTArtificial Sequencecleavable moiety 28Ser Pro Leu
Pro Leu Arg Val Pro 1 5 298PRTArtificial Sequencecleavable moiety
29Arg Met His Leu Arg Ser Leu Gly 1 5 308PRTArtificial
Sequencecleavable moiety 30Leu Ala Ala Pro Leu Gly Leu Leu 1 5
318PRTArtificial Sequencecleavable moiety 31Ala Val Gly Leu Leu Ala
Pro Pro 1 5 328PRTArtificial Sequencecleavable moiety 32Leu Leu Ala
Pro Ser His Arg Ala 1 5 338PRTArtificial Sequencecleavable moiety
33Pro Ala Gly Leu Trp Leu Asp Pro 1 5 348PRTArtificial
Sequencecleavable moiety 34Thr Gly Arg Gly Pro Ser Trp Val 1 5
358PRTArtificial Sequencecleavable moiety 35Ser Ala Arg Gly Pro Ser
Arg Trp 1 5 368PRTArtificial Sequencecleavable moiety 36Thr Ala Arg
Gly Pro Ser Phe Lys 1 5 378PRTArtificial Sequencecleavable moiety
37Leu Ser Gly Arg Ser Asp Asn His 1 5 388PRTArtificial
Sequencecleavable moiety 38Gly Gly Trp His Thr Gly Arg Asn 1 5
398PRTArtificial Sequencecleavable moiety 39His Thr Gly Arg Ser Gly
Ala Leu 1 5 408PRTArtificial Sequencecleavable moiety 40Pro Leu Thr
Gly Arg Ser Gly Gly 1 5 418PRTArtificial Sequencecleavable moiety
41Ala Ala Arg Gly Pro Ala Ile His 1 5 428PRTArtificial
Sequencecleavable moiety 42Arg Gly Pro Ala Phe Asn Pro Met 1 5
438PRTArtificial Sequencecleavable moiety 43Ser Ser Arg Gly Pro Ala
Tyr Leu 1 5 448PRTArtificial Sequencecleavable moiety 44Arg Gly Pro
Ala Thr Pro Ile Met 1 5 454PRTArtificial Sequencecleavable moiety
45Arg Gly Pro Ala 1 4610PRTArtificial Sequencecleavable moiety
46Gly Gly Gln Pro Ser Gly Met Trp Gly Trp 1 5 10 4710PRTArtificial
Sequencecleavable moiety 47Phe Pro Arg Pro Leu Gly Ile Thr Gly Leu
1 5 10 4810PRTArtificial Sequencecleavable moiety 48Val His Met Pro
Leu Gly Phe Leu Gly Pro 1 5 10 498PRTArtificial Sequencecleavable
moiety 49Ser Pro Leu Thr Gly Arg Ser Gly 1 5 509PRTArtificial
Sequencecleavable moiety 50Leu Ala Pro Leu Gly Leu Gln Arg Arg 1 5
518PRTArtificial Sequencecleavable moiety 51Ser Gly Gly Pro Leu Gly
Val Arg 1 5 524PRTArtificial Sequencecleavable moiety 52Pro Leu Gly
Leu 1 536PRTArtificial Sequencespacer peptide 53Gln Gly Gln Ser Gly
Gln 1 5 54449PRTArtificial SequenceAv1 Antibody Heavy Chain 54Gln
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 55214PRTArtificial SequenceAv1
Antibody Light Chain 55Asp 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 56449PRTArtificial SequenceC225v5 Antibody
Heavy Chain 56Gln 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
57449PRTArtificial SequenceC225v4 Antibody Heavy Chain 57Gln 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 58449PRTArtificial SequenceC225v6
Antibody
Heavy Chain 58Gln 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
59214PRTArtificial SequenceC225 Antibody Light Chain 59Gln 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
60108PRTArtificial SequenceVariable Light Chain Lc4 60Asp 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 61119PRTArtificial SequenceVariable Heavy
Chain Hc4 61Glu 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 62108PRTArtificial SequenceVariable
Light Chain Lc5 62Asp 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
63119PRTArtificial SequenceVariable Heavy Chain Hc5 63Glu 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 64108PRTArtificial SequenceVariable Light Chain Lc7 64Asp
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 65119PRTArtificial SequenceVariable
Heavy Chain Hc7 65Glu 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 66774DNAArtificial
Sequence4792 3463 AV1 66caaggccagt ctggccagta tgggtcctgc agttggaact
atgtacacat attcatggat 60tgcggctcga gcggtggcag cggtggctct ggtggctcag
actggttata ctggcctggt 120attggcggtt ctgacatcca gatgactcag
tctcctagct ccctgtccgc ctctgtgggg 180gaccgagtca ccatcacatg
cagagccagc caggatattt ctagttacct gaactggtat 240cagcagaagc
ccggaaaagc acctaagctg ctgatctact atacctccag gctgcactct
300ggcgtgccca gtcggttcag tggctcaggg agcggaaccg acttcacttt
taccatctca 360agcctgcagc cagaggatat tgccacatac tattgtcagc
agggcaatac actgccctac 420acttttggcc aggggaccaa ggtggaaatc
aaacgtacgg tggctgcacc atctgtcttc 480atcttcccgc catctgatga
gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 540aataacttct
atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
600ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 660agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 720acccatcagg gcctgagctc gcccgtcaca
aagagcttca acaggggaga gtgt 77467108PRTArtificial SequenceVariable
Light Chain Lc8 67Asp 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
68119PRTArtificial SequenceVariable Heavy Chain Hc8 68Glu 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 69108PRTArtificial SequenceVariable Light Chain Lc13 69Asp
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 70116PRTArtificial SequenceVariable
Heavy Chain Hc13 70Glu 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 71108PRTArtificial SequenceVariable
Light Chain Lc16 71Asp 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
72119PRTArtificial SequenceVariable Heavy Chain Hc16 72Glu 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 73108PRTArtificial SequenceVariable Light Chain Lc19 73Asp
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 74119PRTArtificial SequenceVariable Heavy Chain
Hc19 74Glu 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 75108PRTArtificial SequenceVariable
Light Chain Lc21 75Asp 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
76119PRTArtificial SequenceVariable Heavy Chain Hc21 76Glu 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 77108PRTArtificial SequenceVariable Light Chain Lc24 77Asp
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 78116PRTArtificial SequenceVariable
Heavy Chain Hc24 78Glu 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 79108PRTArtificial SequenceVariable
Light Chain Lc26 79Asp 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
80119PRTArtificial SequenceVariable Heavy Chain Hc26 80Glu 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 81108PRTArtificial SequenceVariable Light Chain Lc27 81Asp
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 82119PRTArtificial SequenceVariable
Heavy Chain Hc27 82Glu 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 83108PRTArtificial
SequenceVariable Light Chain Lc28 83Asp 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 84119PRTArtificial SequenceVariable Heavy Chain Hc28 84Glu
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 85108PRTArtificial SequenceVariable Light Chain
Lc30 85Asp 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 86115PRTArtificial
SequenceVariable Heavy Chain Hc30 86Glu 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 87108PRTArtificial
SequenceVariable Light Chain Lc31 87Asp 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 88119PRTArtificial SequenceVariable Heavy Chain Hc31 88Glu
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 89108PRTArtificial SequenceVariable Light Chain
Lc32 89Asp 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 90116PRTArtificial
SequenceVariable Heavy Chain Hc32 90Glu 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 91108PRTArtificial
SequenceVariable Light Chain Lc37 91Asp 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 92119PRTArtificial SequenceVariable Heavy Chain Hc37 92Glu
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
93108PRTArtificial SequenceVariable Light Chain Lc39 93Asp 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 94116PRTArtificial SequenceVariable Heavy
Chain Hc39 94Glu 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 95108PRTArtificial SequenceVariable Light
Chain Lc40 95Asp 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
96119PRTArtificial SequenceHeavy Chain Hc40 96Glu 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
97108PRTArtificial SequenceVariable Light Chain Lc47 97Asp 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 98116PRTArtificial SequenceVariable Heavy
Chain Hc47 98Glu 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 99108PRTArtificial SequenceVariable 4B2
Light Chain 99Asp 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
100119PRTArtificial SequenceVariable 4B2 Heavy Chain 100Glu 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 101108PRTArtificial SequenceVariable 4D11 Light Chain
101Asp 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 102119PRTArtificial
SequenceVariable 4D11 Heavy Chain 102Glu 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
103108PRTArtificial SequenceVariable 4E7 Light Chain 103Asp 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 104116PRTArtificial SequenceVariable 4E7
Heavy Chain 104Glu 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 105108PRTArtificial SequenceVariable 4E11
Light Chain 105Asp 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
106119PRTArtificial SequenceVariable 4E11 Heavy Chain 106Glu 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 107108PRTArtificial SequenceVariable 6B7 Light Chain
107Asp 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 108116PRTArtificial
SequenceVariable 6B7 Heavy Chain 108Glu 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 109108PRTArtificial
SequenceVariable 6F8 Light Chain 109Asp 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 110116PRTArtificial SequenceVariable 6F8 Heavy Chain 110Glu
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 111214PRTArtificial Sequence4D11 Light Chain 111Asp 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 112449PRTArtificial Sequence4D11 Heavy Chain 112Glu 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 113449PRTArtificial Sequence4D11v2
Heavy Chain 113Glu 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
114214PRTArtificial Sequence4D11v2 Light Chain 114Asp 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
115258PRTArtificial Sequence4792 10419 AV1 115Gln Gly Gln Ser Gly
Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met
Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Gly Ile Ser Ser Gly Leu Ser Ser 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 Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr 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
116774DNAArtificial Sequence4792 10419 AV1 116caaggccagt ctggccagta
tgggtcctgc agttggaact atgtacacat attcatggat 60tgcggctcga gcggtggcag
cggtggctct ggtggctcag gtattagtag tggtcttagc 120agtggcggtt
ctgacatcca gatgactcag tctcctagct ccctgtccgc ctctgtgggg
180gaccgagtca ccatcacatg cagagccagc caggatattt ctagttacct
gaactggtat 240cagcagaagc ccggaaaagc acctaagctg ctgatctact
atacctccag gctgcactct 300ggcgtgccca gtcggttcag tggctcaggg
agcggaaccg acttcacttt taccatctca 360agcctgcagc cagaggatat
tgccacatac tattgtcagc agggcaatac actgccctac 420acttttggcc
aggggaccaa ggtggaaatc aaacgtacgg tggctgcacc atctgtcttc
480atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 540aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 600ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 660agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 720acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt
774117258PRTArtificial Sequence4792 559 AV1 117Gln Gly Gln Ser Gly
Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met
Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Gln Asn Gln Ala Leu Arg Met Ala 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 Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr 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
118774DNAArtificial Sequence4792 559 AV1 118caaggccagt ctggccagta
tgggtcctgc agttggaact atgtacacat attcatggat 60tgcggctcga gcggtggcag
cggtggctct ggtggctcac agaatcaggc attacgtatg 120gcaggcggtt
ctgacatcca gatgactcag tctcctagct ccctgtccgc ctctgtgggg
180gaccgagtca ccatcacatg cagagccagc caggatattt ctagttacct
gaactggtat 240cagcagaagc ccggaaaagc acctaagctg ctgatctact
atacctccag gctgcactct 300ggcgtgccca gtcggttcag tggctcaggg
agcggaaccg acttcacttt taccatctca 360agcctgcagc cagaggatat
tgccacatac tattgtcagc agggcaatac actgccctac 420acttttggcc
aggggaccaa ggtggaaatc aaacgtacgg tggctgcacc atctgtcttc
480atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt
gtgcctgctg 540aataacttct atcccagaga ggccaaagta cagtggaagg
tggataacgc cctccaatcg 600ggtaactccc aggagagtgt cacagagcag
gacagcaagg acagcaccta cagcctcagc 660agcaccctga cgctgagcaa
agcagactac gagaaacaca aagtctacgc ctgcgaagtc 720acccatcagg
gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt
774119258PRTArtificial Sequence4792 601 AV1 119Gln Gly Gln Ser Gly
Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met
Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Ala Gln Asn Leu Leu Gly Met Val 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 Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr 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 120774DNAArtificial
Sequence4792 601 AV1 120caaggccagt ctggccagta tgggtcctgc agttggaact
atgtacacat attcatggat 60tgcggctcga gcggtggcag cggtggctct ggtggctcag
cacagaatct gttaggtatg 120gtaggcggtt ctgacatcca gatgactcag
tctcctagct ccctgtccgc ctctgtgggg 180gaccgagtca ccatcacatg
cagagccagc caggatattt ctagttacct gaactggtat 240cagcagaagc
ccggaaaagc acctaagctg ctgatctact atacctccag gctgcactct
300ggcgtgccca gtcggttcag tggctcaggg agcggaaccg acttcacttt
taccatctca 360agcctgcagc cagaggatat tgccacatac tattgtcagc
agggcaatac actgccctac 420acttttggcc aggggaccaa ggtggaaatc
aaacgtacgg tggctgcacc atctgtcttc 480atcttcccgc catctgatga
gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 540aataacttct
atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg
600ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta
cagcctcagc 660agcaccctga cgctgagcaa agcagactac gagaaacaca
aagtctacgc ctgcgaagtc 720acccatcagg gcctgagctc gcccgtcaca
aagagcttca acaggggaga gtgt 774121258PRTArtificial Sequence4792 3457
AV1 121Gln Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val
His 1 5 10 15 Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly
Ser Gly Gly 20 25 30 Ser Ser Thr Phe Pro Phe Gly Met Phe 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
Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110 Thr
Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120
125 Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 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 122774DNAArtificial Sequence4792 3457 AV1
122caaggccagt ctggccagta tgggtcctgc agttggaact atgtacacat
attcatggat 60tgcggctcga gcggtggcag cggtggctct ggtggctcaa gtacatttcc
attcggtatg 120ttcggcggtt ctgacatcca gatgactcag tctcctagct
ccctgtccgc ctctgtgggg 180gaccgagtca ccatcacatg cagagccagc
caggatattt ctagttacct gaactggtat 240cagcagaagc ccggaaaagc
acctaagctg ctgatctact atacctccag gctgcactct 300ggcgtgccca
gtcggttcag tggctcaggg agcggaaccg acttcacttt taccatctca
360agcctgcagc cagaggatat tgccacatac tattgtcagc agggcaatac
actgccctac 420acttttggcc aggggaccaa ggtggaaatc aaacgtacgg
tggctgcacc atctgtcttc 480atcttcccgc catctgatga gcagttgaaa
tctggaactg cctctgttgt gtgcctgctg 540aataacttct atcccagaga
ggccaaagta cagtggaagg tggataacgc cctccaatcg 600ggtaactccc
aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc
660agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc
ctgcgaagtc 720acccatcagg gcctgagctc gcccgtcaca aagagcttca
acaggggaga gtgt 774123258PRTArtificial Sequence4792 3458 AV1 123Gln
Gly Gln Ser Gly Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10
15 Ile Phe Met Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly
20 25 30 Ser Pro Val Gly Tyr Thr Ser Ser Leu 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 Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His Ser Gly Val
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr
Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125 Thr Tyr
Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 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 124774DNAArtificial Sequence4792 3458 AV1 124caaggccagt
ctggccagta tgggtcctgc agttggaact atgtacacat attcatggat 60tgcggctcga
gcggtggcag cggtggctct ggtggctcac ctgttggata tacgagtagt
120ctgggcggtt ctgacatcca gatgactcag tctcctagct ccctgtccgc
ctctgtgggg 180gaccgagtca ccatcacatg cagagccagc caggatattt
ctagttacct gaactggtat 240cagcagaagc ccggaaaagc acctaagctg
ctgatctact atacctccag gctgcactct 300ggcgtgccca gtcggttcag
tggctcaggg agcggaaccg acttcacttt taccatctca 360agcctgcagc
cagaggatat tgccacatac tattgtcagc agggcaatac actgccctac
420acttttggcc aggggaccaa ggtggaaatc aaacgtacgg tggctgcacc
atctgtcttc 480atcttcccgc catctgatga gcagttgaaa tctggaactg
cctctgttgt gtgcctgctg 540aataacttct atcccagaga ggccaaagta
cagtggaagg tggataacgc cctccaatcg 600ggtaactccc aggagagtgt
cacagagcag gacagcaagg acagcaccta cagcctcagc 660agcaccctga
cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc
720acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt
774125258PRTArtificial Sequence4792 3463 AV1 125Gln Gly Gln Ser Gly
Gln Tyr Gly Ser Cys Ser Trp Asn Tyr Val His 1 5 10 15 Ile Phe Met
Asp Cys Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly 20 25 30 Ser
Asp Trp Leu Tyr Trp Pro Gly Ile 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 Asp 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 Tyr Thr Ser 85 90 95 Arg Leu His Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly 100 105 110 Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro Glu Asp Ile Ala 115 120 125 Thr Tyr Tyr Cys Gln Gln
Gly Asn Thr Leu Pro Tyr Thr 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
1268PRTArtificial Sequencecleavable moiety 126Ser Ala Gly Phe Ser
Leu Pro Ala 1 5 1278PRTArtificial Sequencecleavable linker sequence
127Pro Arg Phe Lys Ile Ile Gly Gly 1 5 1288PRTArtificial
Sequencecleavable linker sequence 128Pro Arg Phe Arg Ile Ile Gly
Gly 1 5 1299PRTArtificial Sequencecleavable linker sequence 129Ser
Ser Arg His Arg Arg Ala Leu Asp 1 5 13014PRTArtificial
Sequencecleavable linker sequence 130Arg Lys Ser Ser Ile Ile Ile
Arg Met Arg Asp Val Val Leu 1 5 10 13115PRTArtificial
Sequencecleavable linker sequence 131Ser Ser Ser Phe Asp Lys Gly
Lys Tyr Lys Lys Gly Asp Asp Ala 1 5 10 15 13215PRTArtificial
Sequencecleavable linker sequence 132Ser Ser Ser Phe Asp Lys Gly
Lys Tyr Lys Arg Gly Asp Asp Ala 1 5 10 15 1334PRTArtificial
Sequencecleavable linker sequence 133Ile Glu Gly Arg 1
1344PRTArtificial Sequencecleavable linker sequence 134Ile Asp Gly
Arg 1 1357PRTArtificial Sequencecleavable linker sequence 135Gly
Gly Ser Ile Asp Gly Arg 1 5 1366PRTArtificial Sequencecleavable
linker sequence 136Pro Leu Gly Leu Trp Ala 1 5 1378PRTArtificial
Sequencecleavable linker sequence 137Gly Pro Gln Gly Ile Ala Gly
Gln 1 5 1388PRTArtificial Sequencecleavable linker sequence 138Gly
Pro Gln Gly Leu Leu Gly Ala 1 5 1395PRTArtificial Sequencecleavable
linker sequence 139Gly Ile Ala Gly Gln 1 5 1408PRTArtificial
Sequencecleavable linker sequence 140Gly Pro Leu Gly Ile Ala Gly
Ile 1 5 1418PRTArtificial Sequencecleavable linker sequence 141Gly
Pro Glu Gly Leu Arg Val Gly 1 5 1428PRTArtificial Sequencecleavable
linker sequence 142Tyr Gly Ala Gly Leu Gly Val Val 1 5
1438PRTArtificial Sequencecleavable linker sequence 143Ala Gly Leu
Gly Val Val Glu Arg 1 5 1448PRTArtificial Sequencecleavable linker
sequence 144Ala Gly Leu Gly Ile Ser Ser Thr 1 5 1458PRTArtificial
Sequencecleavable linker sequence 145Glu Pro Gln Ala Leu Ala Met
Ser 1 5 1468PRTArtificial Sequencecleavable linker sequence 146Gln
Ala Leu Ala Met Ser Ala Ile 1 5 1478PRTArtificial Sequencecleavable
linker sequence 147Ala Ala Tyr His Leu Val Ser Gln 1 5
1488PRTArtificial Sequencecleavable linker sequence 148Met Asp Ala
Phe Leu Glu Ser Ser 1 5 1498PRTArtificial Sequencecleavable linker
sequence 149Glu Ser Leu Pro Val Val Ala Val 1 5 1508PRTArtificial
Sequencecleavable linker sequence 150Ser Ala Pro Ala Val Glu Ser
Glu 1 5 1518PRTArtificial Sequencecleavable linker sequence 151Asp
Val Ala Gln Phe Val Leu Thr 1 5 1528PRTArtificial Sequencecleavable
linker sequence 152Val Ala Gln Phe Val Leu Thr Glu 1 5
1538PRTArtificial Sequencecleavable linker sequence 153Ala Gln Phe
Val Leu Thr Glu Gly 1 5 1548PRTArtificial Sequencecleavable linker
sequence 154Pro Val Gln Pro Ile Gly Pro Gln 1 5 15516PRTArtificial
Sequencelinking peptide 155Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly
Gly Ser Gly Gly Gly Ser 1 5 10 15 15610PRTArtificial
Sequencelinking peptide 156Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly
1 5 10 15711PRTArtificial Sequencelinking peptide 157Gly Ser Ser
Gly Gly Ser Gly Gly Ser Gly Ser 1 5 10 1584PRTArtificial
Sequencelinking peptide 158Gly Gly Gly Ser 1 1597PRTArtificial
Sequencecleavable moiety 159Ile Ser Ser Gly Leu Ser Ser 1 5
16015PRTArtificial Sequencemasking moiety 160Cys Ile Ser Pro Arg
Gly Cys Pro Asp Gly Pro Tyr Val Met Tyr 1 5 10 15
16115PRTArtificial SequenceN-terminal AV1 light chain sequence
161Tyr Gly Ser Cys Ser Trp Asn Tyr Val His Ile Phe Met Asp Cys 1 5
10 15 16216PRTArtificial SequenceN-terminal AV1 light chain
sequence 162Gln Gly Asp Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro
Ile Thr 1 5 10 15 16318PRTArtificial SequenceN-terminal AV1 light
chain sequence 163Met Gly Val Pro Ala Gly Cys Val Trp Asn Tyr Ala
His Ile Phe Met 1 5 10 15 Asp Cys 16421PRTArtificial
SequenceN-terminal AV1 light chain sequence 164Gln 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 16521PRTArtificial SequenceN-terminal AV1 light chain
sequence 165Gln 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 16624PRTArtificial
SequenceN-terminal AV1 light chain sequence 166Gln 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 1676PRTArtificial Sequencemasking moiety
167Cys Ile Ser Pro Arg Gly 1 5 1688PRTArtificial Sequencemasking
moiety 168Cys Ile Ser Pro Arg Gly Cys Gly 1 5 16914PRTArtificial
Sequencemasking moiety 169Cys Ile Ser Pro Arg Gly Cys Pro Asp Gly
Pro Tyr Val Met 1 5 10 17015PRTArtificial Sequencemasking moiety
170Cys Ile Ser Pro Arg Gly Cys Glu Pro Gly Thr Tyr Val Pro Thr 1 5
10 15 17115PRTArtificial Sequencemasking moiety 171Cys Ile Ser Pro
Arg Gly Cys Pro Gly Gln Ile Trp His Pro Pro 1 5 10 15
17215PRTArtificial Sequencemasking moiety 172Gly Ser His Cys Leu
Ile Pro Ile Asn Met Gly Ala Pro Ser Cys 1 5 10 15
17332PRTArtificial Sequencemasking moiety 173Cys 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
17419PRTArtificial Sequencemasking moiety 174Cys Asn His His Tyr
Phe Tyr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
17519PRTArtificial Sequencemasking moiety 175Ala Asp His Val Phe
Trp Gly Ser Tyr Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
17619PRTArtificial Sequencemasking moiety 176Cys His His Val Tyr
Trp Gly His Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
17719PRTArtificial Sequencemasking moiety 177Cys Pro His Phe Thr
Thr Thr Ser Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
17819PRTArtificial Sequencemasking moiety 178Cys Asn His His Tyr
His Tyr Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
17919PRTArtificial Sequencemasking moiety 179Cys Pro His Val Ser
Phe Gly Ser Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
18019PRTArtificial Sequencemasking moiety 180Cys Pro Tyr Tyr Thr
Leu Ser Tyr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
18119PRTArtificial Sequencemasking moiety 181Cys Asn His Val Tyr
Phe Gly Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
18219PRTArtificial Sequencemasking moiety 182Cys Asn His Phe Thr
Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
18319PRTArtificial Sequencemasking moiety 183Cys His His Phe Thr
Leu Thr Thr Cys Gly Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Pro Gly
18418PRTArtificial Sequencemasking moiety 184Tyr Asn Pro Cys Ala
Thr Pro Met Cys Cys Ile Ser
Pro Arg Gly Cys 1 5 10 15 Pro Gly 18518PRTArtificial
Sequencemasking moiety 185Cys Asn His His Tyr Phe Tyr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 18618PRTArtificial
Sequencemasking moiety 186Cys Asn His His Tyr His Tyr Tyr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 18718PRTArtificial
Sequencemasking moiety 187Cys Asn His Val Tyr Phe Gly Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 18818PRTArtificial
Sequencemasking moiety 188Cys His His Val Tyr Trp Gly His Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 18918PRTArtificial
Sequencemasking moiety 189Cys Pro His Phe Thr Thr Thr Ser Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19018PRTArtificial
Sequencemasking moiety 190Cys Asn His Phe Thr Leu Thr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19118PRTArtificial
Sequencemasking moiety 191Cys His His Phe Thr Leu Thr Thr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19218PRTArtificial
Sequencemasking moiety 192Cys Pro Tyr Tyr Thr Leu Ser Tyr Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19318PRTArtificial
Sequencemasking moiety 193Cys Pro His Val Ser Phe Gly Ser Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19418PRTArtificial
Sequencemasking moiety 194Ala Asp His Val Phe Trp Gly Ser Tyr Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19517PRTArtificial
Sequencemasking moiety 195Tyr Asn Pro Cys Ala Thr Pro Met Cys Cys
Ile Ser Pro Arg Gly Cys 1 5 10 15 Gly 19618PRTArtificial
Sequencemasking moiety 196Cys His His Val Tyr Trp Gly His Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19718PRTArtificial
Sequencemasking moiety 197Cys Xaa His Xaa Xaa Xaa Xaa Xaa Cys Gly
Cys Ile Ser Pro Arg Gly 1 5 10 15 Cys Gly 19815PRTArtificial
Sequencemasking moiety 198Cys Ile Ser Pro Arg Gly Cys Gly Gln Pro
Ile Pro Ser Val Lys 1 5 10 15 19915PRTArtificial Sequencemasking
moiety 199Cys Ile Ser Pro Arg Gly Cys Thr Gln Pro Tyr His Val Ser
Arg 1 5 10 15 20015PRTArtificial Sequencemasking moiety 200Cys Ile
Ser Pro Arg Gly Cys Asn Ala Val Ser Gly Leu Gly Ser 1 5 10 15
20126PRTArtificial Sequencemasking moiety 201Gln 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 20215PRTArtificial
Sequencemasking moiety 202Pro Trp Cys Met Gln Arg Gln Asp Phe Leu
Arg Cys Pro Gln Pro 1 5 10 15 20315PRTArtificial Sequencemasking
moiety 203Gln Leu Gly Leu Pro Ala Tyr Met Cys Thr Phe Glu Cys Leu
Arg 1 5 10 15 20415PRTArtificial Sequencemasking moiety 204Cys Asn
Leu Trp Val Ser Gly Gly Asp Cys Gly Gly Leu Gln Gly 1 5 10 15
20515PRTArtificial Sequencemasking moiety 205Ser Cys Ser Leu Trp
Thr Ser Gly Ser Cys Leu Pro His Ser Pro 1 5 10 15
20615PRTArtificial Sequencemasking moiety 206Tyr Cys Leu Gln Leu
Pro His Tyr Met Gln Ala Met Cys Gly Arg 1 5 10 15
20715PRTArtificial Sequencemasking moiety 207Cys Phe Leu Tyr Ser
Cys Thr Asp Val Ser Tyr Trp Asn Asn Thr 1 5 10 15
20815PRTArtificial Sequencemasking moiety 208Pro Trp Cys Met Gln
Arg Gln Asp Tyr Leu Arg Cys Pro Gln Pro 1 5 10 15
20915PRTArtificial Sequencemasking moiety 209Cys Asn Leu Trp Ile
Ser Gly Gly Asp Cys Arg Gly Leu Ala Gly 1 5 10 15
21015PRTArtificial Sequencemasking moiety 210Cys Asn Leu Trp Val
Ser Gly Gly Asp Cys Arg Gly Val Gln Gly 1 5 10 15
21115PRTArtificial Sequencemasking moiety 211Cys Asn Leu Trp Val
Ser Gly Gly Asp Cys Arg Gly Leu Arg Gly 1 5 10 15
21215PRTArtificial Sequencemasking moiety 212Cys Asn Leu Trp Ile
Ser Gly Gly Asp Cys Arg Gly Leu Pro Gly 1 5 10 15
21315PRTArtificial Sequencemasking moiety 213Cys Asn Leu Trp Val
Ser Gly Gly Asp Cys Arg Asp Ala Pro Trp 1 5 10 15
21415PRTArtificial Sequencemasking moiety 214Cys Asn Leu Trp Val
Ser Gly Gly Asp Cys Arg Asp Leu Leu Gly 1 5 10 15
21515PRTArtificial Sequencemasking moiety 215Cys Asn Leu Trp Val
Ser Gly Gly Asp Cys Arg Gly Leu Gln Gly 1 5 10 15
21615PRTArtificial Sequencemasking moiety 216Cys Asn Leu Trp Leu
His Gly Gly Asp Cys Arg Gly Trp Gln Gly 1 5 10 15
21715PRTArtificial Sequencemasking moiety 217Cys Asn Ile Trp Leu
Val Gly Gly Asp Cys Arg Gly Trp Gln Gly 1 5 10 15
21815PRTArtificial Sequencemasking moiety 218Cys Thr Thr Trp Phe
Cys Gly Gly Asp Cys Gly Val Met Arg Gly 1 5 10 15
21915PRTArtificial Sequencemasking moiety 219Cys Asn Ile Trp Gly
Pro Ser Val Asp Cys Gly Ala Leu Leu Gly 1 5 10 15
22015PRTArtificial Sequencemasking moiety 220Cys Asn Ile Trp Val
Asn Gly Gly Asp Cys Arg Ser Phe Glu Gly 1 5 10 15
22115PRTArtificial Sequencemasking moiety 221Tyr Cys Leu Asn Leu
Pro Arg Tyr Met Gln Asp Met Cys Trp Ala 1 5 10 15
22215PRTArtificial Sequencemasking moiety 222Tyr Cys Leu Ala Leu
Pro His Tyr Met Gln Ala Asp Cys Ala Arg 1 5 10 15
22315PRTArtificial Sequencemasking moiety 223Cys Phe Leu Tyr Ser
Cys Gly Asp Val Ser Tyr Trp Gly Ser Ala 1 5 10 15
22415PRTArtificial Sequencemasking moiety 224Cys Tyr Leu Tyr Ser
Cys Thr Asp Ser Ala Phe Trp Asn Asn Arg 1 5 10 15
22515PRTArtificial Sequencemasking moiety 225Cys Tyr Leu Tyr Ser
Cys Asn Asp Val Ser Tyr Trp Ser Asn Thr 1 5 10 15
22612PRTArtificial Sequencemasking moiety 226Cys Phe Leu Tyr Ser
Cys Thr Asp Val Ser Tyr Trp 1 5 10 22715PRTArtificial
Sequencemasking moiety 227Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala
Tyr Trp Asn Ser Ala 1 5 10 15 22815PRTArtificial Sequencemasking
moiety 228Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Asp
Thr 1 5 10 15 22915PRTArtificial Sequencemasking moiety 229Cys Phe
Leu Tyr Ser Cys Thr Asp Val Ser Tyr Trp Gly Asn Ser 1 5 10 15
23015PRTArtificial Sequencemasking moiety 230Cys Phe Leu Tyr Ser
Cys Thr Asp Val Ala Tyr Trp Asn Asn Thr 1 5 10 15
23118PRTArtificial Sequencemasking moiety 231Cys Phe Leu Tyr Ser
Cys Gly Asp Val Ser Tyr Trp Gly Asn Pro Gly 1 5 10 15 Leu Ser
23215PRTArtificial Sequencemasking moiety 232Cys Phe Leu Tyr Ser
Cys Thr Asp Val Ala Tyr Trp Ser Gly Leu 1 5 10 15
23315PRTArtificial Sequencemasking moiety 233Cys Tyr Leu Tyr Ser
Cys Thr Asp Gly Ser Tyr Trp Asn Ser Thr 1 5 10 15
23415PRTArtificial Sequencemasking moiety 234Cys Phe Leu Tyr Ser
Cys Ser Asp Val Ser Tyr Trp Gly Asn Ile 1 5 10 15
23512PRTArtificial Sequencemasking moiety 235Cys Phe Leu Tyr Ser
Cys Thr Asp Val Ala Tyr Trp 1 5 10 23615PRTArtificial
Sequencemasking moiety 236Cys Phe Leu Tyr Ser Cys Thr Asp Val Ser
Tyr Trp Gly Ser Thr 1 5 10 15 23715PRTArtificial Sequencemasking
moiety 237Cys Phe Leu Tyr Ser Cys Thr Asp Val Ala Tyr Trp Gly Asp
Thr 1 5 10 15 23820PRTArtificial Sequencemasking moiety 238Gly Cys
Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg Gly Trp Val Asp 1 5 10 15
Pro Leu Gln Gly 20 23920PRTArtificial Sequencemasking moiety 239Gly
Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Ile Gly 1 5 10
15 Asp Thr Asn Gly 20 24020PRTArtificial Sequencemasking moiety
240Gly Cys Asn Ile Trp Leu Val Gly Gly Asp Cys Arg Gly Trp Ile Glu
1 5 10 15 Asp Ser Asn Gly 20 24120PRTArtificial Sequencemasking
moiety 241Gly Cys Asn Ile Trp Ala Asn Gly Gly Asp Cys Arg Gly Trp
Ile Asp 1 5 10 15 Asn Ile Asp Gly 20 24220PRTArtificial
Sequencemasking moiety 242Gly Cys Asn Ile Trp Leu Val Gly Gly Asp
Cys Arg Gly Trp Leu Gly 1 5 10 15 Glu Ala Val Gly 20
24320PRTArtificial Sequencemasking moiety 243Gly Cys Asn Ile Trp
Leu Val Gly Gly Asp Cys Arg Gly Trp Leu Glu 1 5 10 15 Glu Ala Val
Gly 20 24420PRTArtificial Sequencemasking moiety 244Gly Gly Pro Ala
Leu Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly Trp
Ser Gly 20 24520PRTArtificial Sequencemasking moiety 245Gly Ala Pro
Val Phe Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg 1 5 10 15 Gly
Trp Met Gly 20 24620PRTArtificial Sequencemasking moiety 246Gly Gln
Gln Gln Trp Cys Asn Ile Trp Ile Asn Gly Gly Asp Cys Arg 1 5 10 15
Gly Trp Asn Gly 20 24720PRTArtificial Sequencemasking moiety 247Gly
Lys Ser Glu Phe Cys Asn Ile Trp Leu Asn Gly Gly Asp Cys Arg 1 5 10
15 Gly Trp Ile Gly 20 24820PRTArtificial Sequencemasking moiety
248Gly Thr Pro Gly Gly Cys Asn Ile Trp Ala Asn Gly Gly Asp Cys Arg
1 5 10 15 Gly Trp Glu Gly 20 24920PRTArtificial Sequencemasking
moiety 249Gly Ala Ser Gln Tyr Cys Asn Leu Trp Ile Asn Gly Gly Asp
Cys Arg 1 5 10 15 Gly Trp Arg Gly 20 25018PRTArtificial
Sequencemasking moiety 250Gly Cys Asn Ile Trp Leu Val Gly Gly Asp
Cys Arg Pro Trp Val Glu 1 5 10 15 Gly Gly 25118PRTArtificial
Sequencemasking moiety 251Gly Cys Asn Ile Trp Ala Val Gly Gly Asp
Cys Arg Pro Phe Val Asp 1 5 10 15 Gly Gly 25218PRTArtificial
Sequencemasking moiety 252Gly Cys Asn Ile Trp Leu Asn Gly Gly Asp
Cys Arg Ala Trp Val Asp 1 5 10 15 Thr Gly 25318PRTArtificial
Sequencemasking moiety 253Gly Cys Asn Ile Trp Ile Val Gly Gly Asp
Cys Arg Pro Phe Ile Asn 1 5 10 15 Asp Gly 25418PRTArtificial
Sequencemasking moiety 254Gly Cys Asn Ile Trp Leu Asn Gly Gly Asp
Cys Arg Pro Val Val Phe 1 5 10 15 Gly Gly 25518PRTArtificial
Sequencemasking moiety 255Gly Cys Asn Ile Trp Leu Ser Gly Gly Asp
Cys Arg Met Phe Met Asn 1 5 10 15 Glu Gly 25618PRTArtificial
Sequencemasking moiety 256Gly Cys Asn Ile Trp Val Asn Gly Gly Asp
Cys Arg Ser Phe Val Tyr 1 5 10 15 Ser Gly 25718PRTArtificial
Sequencemasking moiety 257Gly Cys Asn Ile Trp Leu Asn Gly Gly Asp
Cys Arg Gly Trp Glu Ala 1 5 10 15 Ser Gly 25818PRTArtificial
Sequencemasking moiety 258Gly Cys Asn Ile Trp Ala His Gly Gly Asp
Cys Arg Gly Phe Ile Glu 1 5 10 15 Pro Gly 25918PRTArtificial
Sequencemasking moiety 259Gly Cys Asn Ile Trp Leu Asn Gly Gly Asp
Cys Arg Thr Phe Val Ala 1 5 10 15 Ser Gly 26018PRTArtificial
Sequencemasking moiety 260Gly Cys Asn Ile Trp Ala His Gly Gly Asp
Cys Arg Gly Phe Ile Glu 1 5 10 15 Pro Gly 26118PRTArtificial
Sequencemasking moiety 261Gly Phe Leu Glu Asn Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Thr Gly 26218PRTArtificial
Sequencemasking moiety 262Gly Ile Tyr Glu Asn Cys Asn Ile Trp Leu
Asn Gly Gly Asp Cys Arg 1 5 10 15 Met Gly 26318PRTArtificial
Sequencemasking moiety 263Gly Ile Pro Asp Asn Cys Asn Ile Trp Ile
Asn Gly Gly Asp Cys Arg 1 5 10 15 Tyr Gly 26421PRTArtificial
Sequencemasking moiety 264Gln 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
26521PRTArtificial Sequencemasking moiety 265Gln 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 26624PRTArtificial Sequencemasking moiety 266Gln 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 26715PRTArtificial Sequencemasking
moiety 267Tyr Arg Ser Cys Asn Trp Asn Tyr Val Ser Ile Phe Leu Asp
Cys 1 5 10 15 26816PRTArtificial Sequencemasking moiety 268Pro Gly
Ala Phe Asp Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr 1 5 10 15
26915PRTArtificial Sequencemasking moiety 269Glu Ser Ser Cys Val
Trp Asn Tyr Val His Ile Tyr Met Asp Cys 1 5 10 15
27015PRTArtificial Sequencemasking moiety 270Tyr Pro Gly Cys Lys
Trp Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15
27115PRTArtificial Sequencemasking moiety 271Tyr Arg Thr Cys Ser
Trp Asn Tyr Val Gly Ile Phe Leu Asp Cys 1 5 10 15
27215PRTArtificial Sequencemasking moiety 272Tyr Gly Ser Cys Ser
Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
27315PRTArtificial Sequencemasking moiety 273Tyr Gly Ser Cys Asn
Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
27415PRTArtificial Sequencemasking moiety 274Tyr Thr Ser Cys Asn
Trp Asn Tyr Val His Ile Phe Met Asp Cys 1 5 10 15
27515PRTArtificial Sequencemasking moiety 275Tyr Pro Gly Cys Lys
Trp Asn Tyr Asp Arg Ile Phe Leu Asp Cys 1 5 10 15
27615PRTArtificial Sequencemasking moiety 276Trp Arg Ser Cys Asn
Trp Asn Tyr Ala His Ile Phe Leu Asp Cys 1 5 10 15
27715PRTArtificial Sequencemasking moiety 277Trp Ser Asn Cys His
Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
27815PRTArtificial Sequencemasking moiety 278Asp Arg Ser Cys Thr
Trp Asn Tyr Val Arg Ile Ser Tyr Asp Cys 1 5 10 15
27915PRTArtificial Sequencemasking moiety 279Ser Gly Ser Cys Lys
Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
28015PRTArtificial Sequencemasking moiety 280Ser Arg Ser Cys Ile
Trp Asn Tyr Ala His Ile His Leu Asp Cys 1 5 10 15
28115PRTArtificial Sequencemasking moiety 281Ser Met Ser Cys Tyr
Trp Gln Tyr Glu Arg Ile Phe Leu Asp Cys 1 5 10 15
28215PRTArtificial Sequencemasking moiety 282Tyr Arg Ser Cys Asn
Trp Asn Tyr Val Ser Ile Phe Leu Asp Cys 1 5 10 15
28315PRTArtificial Sequencemasking moiety 283Ser Gly Ser Cys Lys
Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
28415PRTArtificial Sequencemasking moiety 284Tyr Lys Ser Cys His
Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
28515PRTArtificial Sequencemasking moiety 285Tyr Gly Ser Cys Thr
Trp Asn Tyr Val His Ile Phe Met Glu Cys 1 5
10 15 28615PRTArtificial Sequencemasking moiety 286Phe Ser Ser Cys
Asn Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
28715PRTArtificial Sequencemasking moiety 287Trp Arg Ser Cys Asn
Trp Asn Tyr Ala His Ile Phe Leu Asp Cys 1 5 10 15
28815PRTArtificial Sequencemasking moiety 288Tyr Gly Ser Cys Gln
Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
28915PRTArtificial Sequencemasking moiety 289Tyr Arg Ser Cys Asn
Trp Asn Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
29015PRTArtificial Sequencemasking moiety 290Asn Met Ser Cys His
Trp Asp Tyr Val His Ile Phe Leu Asp Cys 1 5 10 15
29115PRTArtificial Sequencemasking moiety 291Phe Gly Pro Cys Thr
Trp Asn Tyr Ala Arg Ile Ser Trp Asp Cys 1 5 10 15
29215PRTArtificial Sequencemasking moiety 292Xaa Xaa Ser Cys Xaa
Trp Xaa Tyr Val His Ile Phe Xaa Asp Cys 1 5 10 15
29318PRTArtificial Sequencemasking moiety 293Arg Asp Thr Gly Gly
Gln Cys Arg Trp Asp Tyr Val His Ile Phe Met 1 5 10 15 Asp Cys
29418PRTArtificial Sequencemasking moiety 294Ala Gly Val Pro Ala
Gly Cys Thr Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
29518PRTArtificial Sequencemasking moiety 295Val Gly Val Pro Asn
Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu Cys
29618PRTArtificial Sequencemasking moiety 296Asp Gly Gly Pro Ala
Gly Cys Ser Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
29718PRTArtificial Sequencemasking moiety 297Ala Val Gly Pro Ala
Gly Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
29818PRTArtificial Sequencemasking moiety 298Cys Thr Trp Asn Tyr
Val His Ile Phe Met Asp Cys Gly Glu Gly Glu 1 5 10 15 Gly Pro
29918PRTArtificial Sequencemasking moiety 299Gly Gly Val Pro Glu
Gly Cys Thr Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu Cys
30018PRTArtificial Sequencemasking moiety 300Ala Glu Val Pro Ala
Gly Cys Trp Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
30118PRTArtificial Sequencemasking moiety 301Ala Gly Val Pro Ala
Gly Cys Thr Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Glu Cys
30218PRTArtificial Sequencemasking moiety 302Ser Gly Ala Ser Gly
Gly Cys Lys Trp Asn Tyr Val His Ile Phe Met 1 5 10 15 Asp Cys
30318PRTArtificial Sequencemasking moiety 303Thr Pro Gly Cys Arg
Trp Asn Tyr Val His Ile Phe Met Glu Cys Glu 1 5 10 15 Ala Leu
30418PRTArtificial Sequencemasking moiety 304Val Gly Val Pro Asn
Gly Cys Val Trp Asn Tyr Ala His Ile Phe Met 1 5 10 15 Glu Cys
30516PRTArtificial Sequencemasking moiety 305Pro Gly Ala Phe Asp
Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr 1 5 10 15
30616PRTArtificial Sequencemasking moiety 306Arg Gly Ala Cys Asp
Ile Pro Phe Pro Ala His Trp Ile Pro Asn Thr 1 5 10 15
30716PRTArtificial Sequencemasking moiety 307Xaa Gly Ala Phe Asp
Ile Pro Phe Pro Ala His Trp Val Pro Asn Thr 1 5 10 15
30819PRTArtificial Sequencemasking moiety 308Arg Gly Asp Gly Asn
Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr
30919PRTArtificial Sequencemasking moiety 309Ser Gly Val Gly Arg
Asp Arg Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr
31019PRTArtificial Sequencemasking moiety 310Trp Ala Gly Gly Asn
Asp Cys Asp Ile Pro Phe Pro Ala His Trp Ile 1 5 10 15 Pro Asn Thr
31119PRTArtificial Sequencemasking moiety 311Trp Gly Asp Gly Met
Asp Val Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Val Thr
31219PRTArtificial Sequencemasking moiety 312Ala Gly Ser Gly Asn
Asp Ser Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr
31319PRTArtificial Sequencemasking moiety 313Glu Ser Arg Ser Gly
Tyr Ala Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr
31419PRTArtificial Sequencemasking moiety 314Arg Glu Cys Gly Arg
Cys Gly Asp Ile Pro Phe Pro Ala His Trp Val 1 5 10 15 Pro Arg Thr
3157PRTArtificial Sequencecleavable moiety 315Gly Pro Arg Ser Phe
Gly Leu 1 5 3166PRTArtificial Sequencecleavable moiety 316Gly Pro
Arg Ser Phe Gly 1 5 3178PRTArtificial SequenceMMP9 Cleavable Core
CM Consensus Sequence 1 317Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3188PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.1 318Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3198PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.2 319Xaa Pro Xaa Xaa Xaa Trp Xaa Xaa 1 5
3208PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.3 320Xaa Pro Xaa Xaa Xaa Trp Xaa Xaa 1 5
3218PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.4 321Xaa Pro Ser Xaa Xaa Trp Xaa Xaa 1 5
3228PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.5 322Xaa Pro Ser Xaa Xaa Trp Xaa Tyr 1 5
3238PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
1 subgenus 1.6 323Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3248PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
2 324Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3258PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 2 subgenus 2.1
325Trp Xaa Xaa Xaa Xaa Ser Xaa Xaa 1 5 3268PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 2 subgenus 2.2
326Trp Asp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3278PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 2 subgenus 2.3
327Trp Asp Xaa Pro Xaa Ser Xaa Leu 1 5 3288PRTArtificial
Sequencecleavable moiety 328Trp Asp His Pro Ile Ser Leu Leu 1 5
3298PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
3 329Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3308PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.1
330Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3318PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.2
331Xaa Xaa Xaa Pro Xaa Xaa Xaa Phe 1 5 3328PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.3
332Xaa Xaa Phe Pro Xaa Xaa Xaa Phe 1 5 3338PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.4
333Xaa Xaa Phe Pro Xaa Xaa Xaa Phe 1 5 3348PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.5
334Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3358PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 3 subgenus 3.6
335Ser Thr Xaa Xaa Xaa Gly Xaa Phe 1 5 3368PRTArtificial
Sequencecleavable moiety 336Leu Thr Phe Pro Thr Tyr Ile Phe 1 5
3378PRTArtificial Sequencecleavable moiety 337Met Thr Phe Pro Thr
Tyr Ile Phe 1 5 3388PRTArtificial Sequencecleavable moiety 338Leu
Thr Phe Pro Thr Tyr Trp Phe 1 5 3398PRTArtificial Sequencecleavable
moiety 339Met Thr Phe Pro Thr Tyr Trp Phe 1 5 3408PRTArtificial
SequenceMMP9 Cleavable Core CM Consensus Sequence 4 340Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 1 5 3418PRTArtificial SequenceMMP9 Cleavable
Core CM Consensus Sequence 4 subgenus 4.1 341Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 1 5 3428PRTArtificial SequenceMMP9 Cleavable Core CM
Consensus Sequence 4 subgenus 4.2 342Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 3438PRTArtificial SequenceMMP9 Cleavable Core CM Consensus
Sequence 4 subgenus 4.3 343His Trp Xaa Xaa Xaa Pro Xaa Xaa 1 5
3448PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
4 subgenus 4.4 344His Trp Xaa Xaa Gly Pro Xaa Xaa 1 5
3458PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
4 subgenus 4.5 345His Trp Xaa Xaa Gly Pro Pro Thr 1 5
3468PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
4 subgenus 4.6 346Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3478PRTArtificial SequenceMMP9 Cleavable Core CM Consensus Sequence
4 subgenus 4.7 347Xaa Trp Leu Tyr Xaa Xaa Xaa Xaa 1 5
3488PRTArtificial Sequencecleavable moiety 348Asp Trp Leu Tyr Trp
Met Gly Ile 1 5 3498PRTArtificial Sequencecleavable moiety 349Asp
Trp Leu Tyr Trp Met Ser Ile 1 5 3508PRTArtificial Sequencecleavable
moiety 350Asp Trp Leu Tyr Trp Pro Ser Ile 1 5 3518PRTArtificial
Sequencecleavable moiety 351His Trp His Leu Gly Pro Pro Thr 1 5
3528PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 5 352Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3538PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.1
353Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3548PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.2
354Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa 1 5 3558PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.3
355Xaa Xaa Xaa Gly Leu Xaa Xaa Xaa 1 5 3568PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.4
356Xaa Xaa Xaa Gly Leu Xaa Xaa Xaa 1 5 3578PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.5
357Xaa Xaa Xaa Gly Leu Xaa Ser Xaa 1 5 3588PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.6
358Thr Xaa Ser Gly Leu Arg Ser Pro 1 5 3598PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.7
359Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 3608PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.8
360Xaa Xaa Xaa Gly Leu Xaa Ser Xaa 1 5 3618PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.9
361Xaa Xaa Xaa Gly Leu Xaa Ser Xaa 1 5 3628PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.10
362Xaa Xaa Xaa Gly Leu Xaa Ser Xaa 1 5 3638PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 5 subgenus 5.11
363Xaa Xaa Ser Gly Leu Xaa Ser Xaa 1 5 3648PRTArtificial
Sequencecleavable moiety 364Ser Val Ser Gly Leu Leu Ser His 1 5
3658PRTArtificial Sequencecleavable moiety 365Ser Val Ser Gly Leu
Leu Ser Ser 1 5 3668PRTArtificial Sequencecleavable moiety 366Ser
Val Ser Gly Leu Arg Ser His 1 5 3678PRTArtificial Sequencecleavable
moiety 367Ser Val Ser Gly Leu Arg Ser Ser 1 5 3688PRTArtificial
Sequencecleavable moiety 368Thr Leu Ser Gly Leu Arg Ser Pro 1 5
3698PRTArtificial Sequencecleavable moiety 369Thr Ser Ser Gly Leu
Arg Ser Pro 1 5 3708PRTArtificial Sequencecleavable moiety 370Thr
Val Ser Gly Leu Arg Ser Pro 1 5 3718PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 6 371Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 3728PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 6 Subgenus 6.1 372Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 3738PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6 Subgenus 6.2 373Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3748PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6 Subgenus 6.3 374Leu Xaa Xaa Xaa Xaa Arg Xaa Xaa 1 5
3758PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6 Subgenus 6.4 375Leu Xaa Xaa Xaa Xaa Arg Ala Xaa 1 5
3767PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6A Subgenus 6A.1 376Xaa Xaa Xaa Ala Xaa Xaa Xaa 1 5
3777PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6A Subgenus 6A.2 377Xaa Xaa Gln Ala Xaa Xaa Xaa 1 5
3787PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 6A Subgenus 6A.3 378Ala Xaa Gln Ala Leu Arg Met 1 5
3797PRTArtificial Sequencecleavable moiety 379Ala Phe Gln Ala Leu
Arg Met 1 5 3807PRTArtificial Sequencecleavable moiety 380Ala His
Gln Ala Leu Arg Met 1 5 3817PRTArtificial Sequencecleavable moiety
381Ala Asn Gln Ala Leu Arg Met 1 5 3828PRTArtificial
Sequencecleavable moiety 382Ala Asn Gln Ala Leu Arg Met Ala 1 5
3838PRTArtificial Sequencecleavable moiety 383Leu Leu Glu Ala Leu
Arg Ala Leu 1 5 3848PRTArtificial Sequencecleavable moiety 384Leu
Leu Asn Ala Leu Arg Ala Leu 1 5 3858PRTArtificial Sequencecleavable
moiety 385Leu Leu Gln Ala Leu Arg Ala Leu 1 5 3868PRTArtificial
Sequencecleavable moiety 386Leu Leu Ser Ala Leu Arg Ala Leu 1 5
3878PRTArtificial Sequencecleavable moiety 387Leu Leu Glu Ser Leu
Arg Ala Leu 1 5 3888PRTArtificial Sequencecleavable moiety 388Leu
Leu Asn Ser Leu Arg Ala Leu 1 5 3898PRTArtificial Sequencecleavable
moiety 389Leu Leu Gln Ser Leu Arg Ala Leu 1 5 3908PRTArtificial
Sequencecleavable moiety 390Leu Leu Ser Ser Leu Arg Ala Leu 1 5
3917PRTArtificial Sequencecleavable moiety 391Gln Phe Gln Ala Leu
Arg Met 1 5 3927PRTArtificial Sequencecleavable moiety 392Gln His
Gln Ala Leu Arg Met 1 5 3937PRTArtificial Sequencecleavable moiety
393Gln Asn Gln Ala Leu Arg Met 1 5 3948PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 7 394Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 3958PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 7 Subgenus 7.1 395Leu Xaa Xaa Xaa Pro Xaa Xaa
Xaa 1 5 3968PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.2 396Leu Xaa Xaa Xaa Pro Xaa Xaa Xaa 1 5
3978PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.3 397Leu Xaa Ala Xaa Pro Xaa Trp Xaa 1 5
3988PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.4 398Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
3998PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.5 399Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa 1 5
4008PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.6 400Leu Xaa Xaa Xaa Pro Xaa Trp Xaa 1 5
4018PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 7 Subgenus 7.7 401Leu Xaa Ala Xaa Pro Xaa Trp Xaa 1 5
4027PRTArtificial Sequencecleavable moiety 402Leu Pro Ala Gly Leu
Leu Leu 1 5 4038PRTArtificial Sequencecleavable moiety 403Leu Lys
Ala Ala Pro Val Trp Ala 1 5 4048PRTArtificial Sequencecleavable
moiety 404Leu Lys Ala Ala Pro Arg Trp Phe 1 5 4058PRTArtificial
Sequencecleavable moiety 405Leu Lys Ala Ala Pro Val Trp Phe 1 5
4068PRTArtificial Sequencecleavable moiety 406Leu Tyr Ala Ala Pro
Arg Trp Ala 1 5 4078PRTArtificial Sequencecleavable moiety 407Leu
Tyr Ala Ala Pro Val Trp Ala 1 5 4088PRTArtificial Sequencecleavable
moiety 408Leu Tyr Ala Ala Pro Arg Trp Phe 1 5 4098PRTArtificial
Sequencecleavable moiety 409Leu Tyr Ala Ala Pro Val Trp Phe 1 5
4107PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8
410Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4117PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 8 Subgenus 8.1 411Xaa Pro Xaa
Xaa Xaa Xaa Xaa 1 5 4127PRTArtificial SequenceMMP14 Cleavable Core
CM Consensus Sequence 8 Subgenus 8.2 412Xaa Pro Xaa Xaa Xaa Xaa Xaa
1 5 4137PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.3 413Xaa Pro Xaa Xaa Xaa Xaa Xaa 1 5
4147PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.4 414Xaa Pro Xaa Xaa Xaa Xaa Xaa 1 5
4157PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.5 415Xaa Pro Xaa Gly Xaa Xaa Leu 1 5
4167PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.6 416Xaa Pro Xaa Gly Xaa Xaa Leu 1 5
4177PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.7 417Xaa Pro Xaa Xaa Xaa Xaa Xaa 1 5
4187PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.8 418Xaa Pro Xaa Xaa Xaa Xaa Xaa 1 5
4197PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 8 Subgenus 8.9 419Leu Pro Xaa His Xaa Val Leu 1 5
4208PRTArtificial Sequencecleavable moiety 420Leu Pro Ala Gly Leu
Leu Leu Arg 1 5 4218PRTArtificial Sequencecleavable moiety 421Leu
Pro Ala His Leu Val Leu Leu 1 5 4228PRTArtificial Sequencecleavable
moiety 422Leu Pro Ser His Leu Val Leu Leu 1 5 4238PRTArtificial
Sequencecleavable moiety 423Leu Pro Ala His Leu Val Leu Val 1 5
4248PRTArtificial Sequencecleavable moiety 424Leu Pro Ser His Leu
Val Leu Val 1 5 4258PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 9 425Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4268PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.1 426Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4278PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.2 427Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4288PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.3 428Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4298PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.4 429Arg Arg Xaa Xaa Gly Xaa Arg Xaa 1 5
4308PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.5 430Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4318PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.6 431Xaa Xaa His Xaa Xaa Xaa Xaa Xaa 1 5
4328PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.7 432Xaa Xaa His Xaa Xaa Xaa Xaa Xaa 1 5
4338PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 9 Subgenus 9.8 433Xaa Arg His Asp Xaa Xaa Xaa Xaa 1 5
4348PRTArtificial Sequencecleavable moiety 434Arg Arg His Asp Gly
Leu Arg Ala 1 5 4358PRTArtificial Sequencecleavable moiety 435Arg
Arg His Asp Gly Leu Arg Ser 1 5 4368PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 10 436Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 4378PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 10 Subgenus 10.1 437Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4388PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.2 438Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4398PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.3 439Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4408PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.4 440Xaa Tyr Xaa Xaa Leu Xaa Xaa Xaa 1 5
4418PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.5 441Xaa Tyr Xaa Xaa Leu Xaa Xaa Xaa 1 5
4428PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.6 442Xaa Tyr Xaa Xaa Leu Ser Xaa Xaa 1 5
4438PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.7 443Ala Tyr Xaa Xaa Leu Ser Arg Xaa 1 5
4448PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.8 444Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4458PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.9 445Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 1 5
4468PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.10 446Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 1 5
4478PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.11 447Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 1 5
4488PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.12 448Ile Xaa Asn Xaa Leu Xaa Xaa Xaa 1 5
4498PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 10 Subgenus 10.13 449Ile Xaa Asn Xaa Leu Ser Met Xaa 1 5
4508PRTArtificial Sequencecleavable moiety 450Ile Ala Asn Leu Leu
Ser Met Val 1 5 4518PRTArtificial Sequencecleavable moiety 451Ile
Leu Asn Leu Leu Ser Met Val 1 5 4528PRTArtificial Sequencecleavable
moiety 452Ile Gln Asn Leu Leu Ser Met Val 1 5 4538PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 11 453Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4548PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 11 Subgenus 11.1 454Xaa Xaa
Xaa Xaa Trp Xaa Xaa Xaa 1 5 4558PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 11 Subgenus 11.2 455Xaa Xaa
Xaa Xaa Trp Xaa Xaa Gln 1 5 4568PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 11 Subgenus 11.3 456Pro Ala
Xaa Xaa Trp Tyr Thr Gln 1 5 4578PRTArtificial Sequencecleavable
moiety 457Pro Ala Ser Leu Trp Tyr Thr Gln 1 5 4588PRTArtificial
SequenceMMP14 Cleavable Core CM Consensus Sequence 12 458Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4598PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.1 459Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4608PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.2 460Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa 1 5 4618PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.3 461Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa 1 5 4628PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.4 462Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa 1 5 4638PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.5 463Xaa Leu
Xaa Xaa Leu Xaa Leu Pro 1 5 4648PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.6 464Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 1 5 4658PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.7 465Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa 1 5 4668PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.8 466Xaa Xaa
Xaa Xaa Leu Xaa Xaa Xaa 1 5 4678PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.9 467Xaa Xaa
Xaa Leu Leu Xaa Xaa Pro 1 5 4688PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.10 468Xaa Xaa
Xaa Leu Leu Xaa Xaa Pro 1 5 4698PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 12 Subgenus 12.11 469Xaa Ser
Xaa Leu Leu Arg Phe Pro 1 5 4708PRTArtificial Sequencecleavable
moiety 470Ala Leu Gly Leu Leu Arg Leu Pro 1 5 4718PRTArtificial
Sequencecleavable moiety 471Ala Leu Gly Leu Leu Ser Leu Pro 1 5
4728PRTArtificial Sequencecleavable moiety 472Ala Ser Gly Leu Leu
Arg Phe Pro 1 5 4738PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 13 473Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4748PRTArtificial Sequencecleavable moiety 474Leu Leu Leu Pro Ala
His Gly Gly 1 5 4758PRTArtificial SequenceMMP14 Cleavable Core CM
Consensus Sequence 13 Subgenus 13.1 475Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4768PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.2 476Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa 1 5
4778PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.3 477Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa 1 5
4788PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.4 478Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa 1 5
4798PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.5 479Xaa Xaa Xaa Pro Leu Xaa Gly Ser 1 5
4808PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.6 480Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4818PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13. 481Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4828PRTArtificial SequenceMMP14 Cleavable Core CM Consensus
Sequence 13 Subgenus 13.7 482Xaa Xaa Leu Xaa Xaa His Gly Xaa 1 5
4838PRTArtificial Sequencecleavable moiety 483Leu Leu Leu Pro Leu
Leu Gly Ser 1 5 4848PRTArtificial Sequencecleavable moiety 484Val
Ala Gly Arg Ser Met Arg Pro 1 5 4857PRTArtificial SequenceMMP14
Cleavable Core CM Consensus Sequence 6A 485Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4868PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A 486Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
4878PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.1 487Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4888PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.2 488Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4898PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.3 489Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4908PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.4 490Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4918PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.5 491Xaa Pro Xaa Gly Xaa Xaa Leu
Arg 1 5 4928PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.6 492Xaa Pro Xaa Gly Xaa Xaa Leu
Arg 1 5 4938PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.7 493Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4948PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.8 494Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 4958PRTArtificial SequenceMMP14 Cleavable Extended Core CM
Consensus Sequence 8A Subgenus 8A.9 495Leu Pro Xaa His Xaa Val Leu
Xaa 1 5 49621PRTArtificial Sequencemasking moiety 496Gln 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 4977PRTArtificial Sequencemasking moiety 497Cys
Ile Ser Pro Arg Gly Cys 1 5 4985PRTArtificial Sequenceanti-Jagged
antigen binding fragment VH CDR1 sequence 498Ser Tyr Ala Met Ser 1
5 49917PRTArtificial Sequenceanti-Jagged antigen binding fragment
VH CDR2 sequence 499Ser Ile Asp Pro Glu Gly Arg Gln Thr Tyr Tyr Ala
Asp Ser Val Lys 1 5 10 15 Gly 50010PRTArtificial
Sequenceanti-Jagged antigen binding fragment VH CDR3 sequence
500Asp Ile Gly Gly Arg Ser Ala Phe Asp Tyr 1 5 10 5019PRTArtificial
Sequenceanti-Jagged antigen binding fragment VL CDR1 sequence
501Arg Ala Ser Gln Ser Ile Ser Ser Tyr 1 5 5027PRTArtificial
Sequenceanti-Jagged antigen binding fragment VL CDR2 sequence
502Ala Ala Ser Ser Leu Gln Ser 1 5 5039PRTArtificial
Sequenceanti-Jagged antigen binding fragment VL CDR3 sequence
503Gln Gln Thr Val Val Ala Pro Pro Leu 1 5 5045PRTArtificial
Sequenceanti-EGFR antigen binding fragment VH CDR1 sequence 504Asn
Tyr Gly Val His 1 5 50516PRTArtificial Sequenceanti-EGFR antigen
binding fragment VH CDR2 sequence 505Val Ile Trp Ser Gly Gly Asn
Thr Asp Tyr Asn Thr Pro Phe Thr Ser 1 5 10 15 50611PRTArtificial
Sequenceanti-EGFR antigen binding fragment VH CDR3 sequence 506Ala
Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr 1 5 10 50711PRTArtificial
Sequenceanti-EGFR antigen binding fragment VL CDR1 sequence 507Arg
Ala Ser Gln Ser Ile Gly Thr Asn Ile His 1 5 10 5088PRTArtificial
Sequenceanti-EGFR antigen binding fragment VL CDR2 sequence 508Lys
Tyr Ala Ser Glu Ser Ile Ser 1 5 5099PRTArtificial Sequenceanti-EGFR
antigen binding fragment VL CDR3 sequence 509Gln Gln Asn Asn Asn
Trp Pro Thr Thr 1 5 510273PRTArtificial Sequenceanti-CTLA-4 scFv
510Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Ser Gly
1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Glu Ile Val Leu Thr Gln Ser
Pro Gly 20 25 30 Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
Ser Cys Arg Ala 35 40 45 Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro 50 55 60 Gly Gln Ala Pro Arg Leu Leu Ile
Tyr Gly Ala Ser Ser Arg Ala Thr 65 70 75 80 Gly Ile Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Thr Ile Ser
Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys 100 105 110 Gln Gln
Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 115 120 125
Glu Ile Lys Arg Ser Gly Gly Ser Thr Ile Thr Ser Tyr Asn Val Tyr 130
135 140 Tyr Thr Lys Leu Ser Ser Ser Gly Thr Gln Val Gln Leu Val Gln
Thr 145 150 155 160 Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg
Leu Ser Cys Ala 165 170 175 Ala Ser Gly Ser Thr Phe Ser Ser Tyr Ala
Met Ser Trp Val Arg Gln 180 185 190 Ala Pro Gly Lys Gly Leu Glu Trp
Val Ser Ala Ile Ser Gly Ser Gly 195 200 205 Gly Ser Thr Tyr Tyr Ala
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 210 215 220 Arg Asp Asn Ser
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 225 230 235 240 Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Asn Ser Leu Tyr Trp 245 250
255 Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala
260 265 270 Ser 511264PRTArtificial Sequenceanti-CD3 epsilon scFv
511Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Gly Gly Gly Ser Gly
1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gln Val Gln Leu Gln Gln Ser
Gly Ala 20 25 30 Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser
Cys Lys Ala Ser 35 40 45 Gly Tyr Thr Phe Thr Arg Tyr Thr Met His
Trp Val Lys Gln Arg Pro 50 55 60 Gly Gln Gly Leu Glu Trp Ile Gly
Tyr Ile Asn Pro Ser Arg Gly Tyr 65 70 75 80 Thr Asn Tyr Asn Gln Lys
Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp 85 90 95 Lys Ser Ser Ser
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu 100 105 110 Asp Ser
Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys 115 120
125 Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly
130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
Ile Val 145 150 155 160 Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Pro Gly Glu Lys Val 165 170 175 Thr Met Thr Cys Ser Ala Ser Ser Ser
Val Ser Tyr Met Asn Trp Tyr 180 185 190 Gln Gln Lys Ser Gly Thr Ser
Pro Lys Arg Trp Ile Tyr Asp Thr Ser 195 200 205 Lys Leu Ala Ser Gly
Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly 210 215 220 Thr Ser Tyr
Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala 225 230 235 240
Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser 245
250 255 Gly Thr Lys Leu Glu Ile Asn Arg 260 512194PRTArtificial
SequenceDisplay Platform CYTX-DP-XXXXXXXX peptide 512Gly Gln Ser
Gly Gln Glu Tyr Met Pro Met Glu Gly Gly Ser Gly Gln 1 5 10 15 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Gly Gly Gln Gly Gly Ser Gly 20 25
30 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ala Tyr Tyr Gly Ile
35 40 45 Thr Ala Gly Pro Ala Tyr Arg Ile Asn Asp Trp Ala Ser Ile
Tyr Gly 50 55 60 Val Val Gly Val Gly Tyr Gly Ser Gly Pro Gly Gly
Ser Tyr Gly Phe 65 70 75 80 Ser Tyr Gly Ala Gly Leu Gln Phe Asn Pro
Met Glu Asn Val Ala Leu 85 90 95 Asp Phe Ser Tyr Glu Gln Ser Arg
Ile Arg Ser Val Asp Val Gly Thr 100 105 110 Trp Ile Leu Ser Val Gly
Tyr Arg Phe Gly Ser Lys Ser Arg Arg Ala 115 120 125 Thr Ser Thr Val
Thr Gly Gly Tyr Ala Gln Ser Asp Ala Gln Gly Gln 130 135 140 Met Asn
Lys Met Gly Gly Phe Asn Leu Lys Tyr Arg Tyr Glu Glu Asp 145 150 155
160 Asn Ser Pro Leu Gly Val Ile Gly Ser Phe Thr Tyr Thr Gly Gly Ser
165 170 175 Gly Gly Ser Ser Gly Gln Ala Ala Ala Gly His His His His
His His 180 185 190 His His 513217PRTArtificial
SequenceSP-CYTX-DP-XXXXXXXX peptide 513Met Lys Lys Ile Ala Cys Leu
Ser Ala Leu Ala Ala Val Leu Ala Phe 1 5 10 15 Thr Ala Gly Thr Ser
Val Ala Gly Gln Ser Gly Gln Glu Tyr Met Pro 20 25 30 Met Glu Gly
Gly Ser Gly Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 35 40 45 Gly
Gly Gln Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 50 55
60 Gly Ser Ala Tyr Tyr Gly Ile Thr Ala Gly Pro Ala Tyr Arg Ile Asn
65 70 75 80 Asp Trp Ala Ser Ile Tyr Gly Val Val Gly Val Gly Tyr Gly
Ser Gly 85 90 95 Pro Gly Gly Ser Tyr Gly Phe Ser Tyr Gly Ala Gly
Leu Gln Phe Asn 100 105 110 Pro Met Glu Asn Val Ala Leu Asp Phe Ser
Tyr Glu Gln Ser Arg Ile 115 120 125 Arg Ser Val Asp Val Gly Thr Trp
Ile Leu Ser Val Gly Tyr Arg Phe 130 135 140 Gly Ser Lys Ser Arg Arg
Ala Thr Ser Thr Val Thr Gly Gly Tyr Ala 145 150 155 160 Gln Ser Asp
Ala Gln Gly Gln Met Asn Lys Met Gly Gly Phe Asn Leu 165 170 175 Lys
Tyr Arg Tyr Glu Glu Asp Asn Ser Pro Leu Gly Val Ile Gly Ser 180 185
190 Phe Thr Tyr Thr Gly Gly Ser Gly Gly Ser Ser Gly Gln Ala Ala Ala
195 200 205 Gly His His His His His His His His 210 215
5148PRTArtificial Sequenceamino acid substrate 514Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 1 5 5158PRTArtificial Sequencelinker sequence
515Gly Gly Gly Ser Gly Gly Gly Ser 1 5
* * * * *