U.S. patent application number 17/541021 was filed with the patent office on 2022-09-01 for protease-activated t cell bispecific molecules.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Peter BRUENKER, Rebecca CROASDALE-WOOD, Martina GEIGER, Christian KLEIN, Jigar PATEL, Juergen Michael SCHANZER, Kay-Gunnar STUBENRAUCH, Eric SULLIVAN, Pablo UMANA.
Application Number | 20220275087 17/541021 |
Document ID | / |
Family ID | 1000006330309 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220275087 |
Kind Code |
A1 |
BRUENKER; Peter ; et
al. |
September 1, 2022 |
PROTEASE-ACTIVATED T CELL BISPECIFIC MOLECULES
Abstract
The present invention generally relates to novel
protease-activatable T cell activating bispecific molecules and
idiotype-specific polypeptides. The present invention also relates
to polynucleotides encoding such protease-activatable T cell
activating bispecific molecules and idiotype-specific polypeptides,
and vectors and host cells comprising such polynucleotides. The
invention further relates to methods for producing the
protease-activatable T cell activating bispecific molecules and
idiotype-specific polypeptides of the invention, and to methods of
using these protease-activatable T cell activating bispecific
molecules and idiotype-specific polypeptides in the treatment of
disease.
Inventors: |
BRUENKER; Peter; (Hittnau,
CH) ; CROASDALE-WOOD; Rebecca; (Preston, GB) ;
KLEIN; Christian; (Bonstetten, CH) ; SCHANZER;
Juergen Michael; (Munchen, DE) ; STUBENRAUCH;
Kay-Gunnar; (Penzberg, DE) ; UMANA; Pablo;
(Wollerau, CH) ; GEIGER; Martina; (Obfelden,
CH) ; SULLIVAN; Eric; (Pleasanton, CA) ;
PATEL; Jigar; (Pleasanton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc. |
Little Falls |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Little Falls
NJ
|
Family ID: |
1000006330309 |
Appl. No.: |
17/541021 |
Filed: |
December 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16138417 |
Sep 21, 2018 |
11242390 |
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17541021 |
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PCT/EP2017/056556 |
Mar 20, 2017 |
|
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16138417 |
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62433327 |
Dec 13, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/4208 20130101; C07K 16/2809 20130101; A61P 35/00 20180101;
C07K 2319/50 20130101; C07K 16/28 20130101; C07K 2317/55 20130101;
C07K 16/2863 20130101; C07K 2317/73 20130101; C07K 2317/526
20130101; C07K 2317/31 20130101; C07K 2317/64 20130101; C07K
2317/71 20130101; C07K 2317/622 20130101; C07K 2317/524
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00; C07K 16/42 20060101
C07K016/42 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 22, 2016 |
EP |
16161740.2 |
Claims
1. A protease-activatable T cell activating bispecific molecule
comprising (a) a first antigen binding moiety capable of specific
binding to CD3; (b) a second antigen binding moiety capable of
specific binding to a target cell antigen; and (c) a masking moiety
covalently attached to the T cell bispecific binding molecule
through a protease-cleavable linker, wherein the masking moiety is
capable of specific binding to the idiotype of the first or the
second antigen binding moiety thereby reversibly concealing the
first or the second antigen binding moiety.
2. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein the masking moiety is: (a) covalently attached
to the first antigen binding moiety and reversibly conceals the
first antigen binding moiety; and/or (b) an anti-idiotypic
scFv.
3. The protease-activatable T cell activating bispecific molecule
of claim 2, wherein the masking moiety is covalently attached to
the heavy chain variable region of the first antigen binding
moiety.
4. (canceled)
5. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein: (a) the second antigen binding moiety is a
crossover Fab molecule wherein either the variable or the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged; and/or (b) the first antigen binding moiety is a
conventional Fab molecule.
6. (canceled)
7. The protease-activatable T cell activating bispecific molecule
of claim 1, comprising not more than one antigen binding moiety
capable of specific binding to CD3.
8. The protease-activatable T cell activating bispecific molecule
of claim 1, comprising a third antigen binding moiety, wherein: (a)
the third antigen binding moiety is a Fab molecule capable of
specific binding to a target cell antigen; and/or (b) the third
antigen binding moiety is identical to the second antigen binding
moiety.
9. (canceled)
10. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein the second antigen binding moiety is capable of
specific binding to a target cell antigen selected from the group
consisting of FolR1, HER1, HER2 and Mesothelin.
11. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein the first and the second antigen binding moiety
are fused to each other via a peptide linker.
12. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein: (a) the second antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
Fab heavy chain of the first antigen binding moiety; (b) the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety; and/or (c) the T cell activating bispecific
molecule additionally comprises an Fc domain composed of a first
and a second subunit capable of stable association.
13-14. (canceled)
15. The protease-activatable T cell activating bispecific molecule
of claim 12, wherein the Fc domain is an IgG Fc domain.
16. The protease-activatable T cell activating bispecific molecule
of claim 15, wherein the Fc domain exhibits reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a native IgG.sub.1 Fc domain.
17. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein the masking moiety comprises a heavy chain
variable region comprising: (a) a heavy chain complementarity
determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID
NO:26); (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ
ID NO:27); (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ
ID NO:28); and a light chain variable region comprising: (d) a
light chain (CDR L)1 amino acid sequence of RASENIDSYLA (SEQ ID
NO:29); (e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);
and (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID
NO:31).
18. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein the protease cleavable linker comprises at
least one protease recognition sequence.
19. The protease-activatable T cell activating bispecific molecule
of claim 18, wherein the protease cleavable linker comprises the
protease recognition sequence RQARVVNG (SEQ ID NO:36).
20. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein: (i) the first antigen binding moiety is
capable of specific binding to CD3 and comprises a heavy chain
variable region comprising: a) a CDR H1 amino acid sequence of
TYAMN (SEQ ID NO:44); b) a CDR H2 amino acid sequence of
RIRSKYNNYATYYADSVKG (SEQ ID NO:45); and c) a CDR H3 amino acid
sequence of HGNFGNSYVSWFAY (SEQ ID NO:46); and a light chain
variable region comprising: d) a CDR L1 amino acid sequence of
GSSTGAVTTSNYAN (SEQ ID NO:17); e) a CDR L2 amino acid sequence of
GTNKRAP (SEQ ID NO:18); and f) a CDR L3 amino acid sequence of
ALWYSNLWV (SEQ ID NO:19); and/or (ii) the first antigen binding
moiety is capable of specific binding to CD3 and comprises a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 43 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 55.
21. (canceled)
22. The protease-activatable T cell activating bispecific molecule
of claim 1, wherein: (i) the second antigen binding moiety is
capable of specific binding to FolR1 and comprises a heavy chain
variable region comprising: a) a CDR H1 amino acid sequence of
NAWMS (SEQ ID NO:14); b) a CDR H2 amino acid sequence of
RIKSKTDGGTTDYAAPVKG (SEQ ID NO:15); and c) a CDR H3 amino acid
sequence of PWEWSWYDY (SEQ ID NO:16); and a light chain variable
region comprising: d) a CDR L1 amino acid sequence of
GSSTGAVTTSNYAN (SEQ ID NO:17); e) a CDR L2 amino acid sequence of
GTNKRAP (SEQ ID NO:18); and f) a CDR L3 amino acid sequence of
ALWYSNLWV (SEQ ID NO:19); or (ii) the second antigen binding moiety
is capable of specific binding to Mesothelin and comprises a heavy
chain variable region comprising: a) a CDR H1 amino acid sequence
of GYTMN (SEQ ID NO:107); b) a CDR H2 amino acid sequence of
LITPYNGASSYNQKFRG (SEQ ID NO:108); and c) a CDR H3 amino acid
sequence of GGYDGRGFDY (SEQ ID NO:109); and a light chain variable
region comprising: d) a CDR L1 amino acid sequence of SASSSVSYMH
(SEQ ID NO:110); e) a CDR L2 amino acid sequence of DTSKLAS (SEQ ID
NO:111); and f) a CDR L3 amino acid sequence of QQWSKHPLT (SEQ ID
NO:112).
23. (canceled)
24. An idiotype-specific polypeptide for reversibly concealing an
anti-CD3 antigen binding site of a molecule.
25-32. (canceled)
33. A pharmaceutical composition comprising the
protease-activatable T cell activating bispecific molecule of claim
1 and a pharmaceutically acceptable carrier.
34-37. (canceled)
38. A method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the protease-activatable T cell
activating bispecific molecule of claim 1.
39. The method of claim 38 for treating or delaying progression of
cancer, treating or delaying progression of an immune related
disease, or enhancing or stimulating an immune response or function
in an individual.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/138,417, filed on Sep. 21, 2018, which is a
continuation of International Patent Application No.
PCT/EP2017/056556, filed on Mar. 20, 2017, which claims priority to
European Patent Application No. 16161740.2, filed on Mar. 22, 2016,
and to U.S. Patent Application No. 62/433,327, filed on Dec. 13,
2016, the disclosures of which are incorporated herein by reference
in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 14, 2022, is named
51177-025002_Sequence_Listing_2_14_22_ST25 and is 309,161 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention generally relates to novel
protease-activatable antigen-binding molecules that comprise an
anti-idiotype-binding moiety which reversibly masks antigen binding
of the molecule. Specifically, the invention relates to T cell
binding molecules having an anti-idiotype-binding moiety that masks
the CD3-binding moiety until cleaved by a protease. This allows the
CD3-binding moiety to be inaccessible or "masked" until it is in
proximity to a target tissue, such as a tumor, e.g.,
tumor-infiltrating T cells. In addition, the present invention
relates to polynucleotides encoding such protease-activated T cell
binding molecules and idiotype-specific polypeptides, and vectors
and host cells comprising such polynucleotides. The invention
further relates to methods for producing the protease-activated T
cell binding molecules of the invention, and to methods of using
the same, e.g., in the treatment of disease.
BACKGROUND
[0004] The selective destruction of an individual target cell or a
specific target cell type is often desirable in a variety of
clinical settings. For example, it is a primary goal of cancer
therapy to specifically destroy tumor cells, while leaving healthy
cells and tissues intact and undamaged.
[0005] An attractive way of achieving this is by inducing an immune
response against the tumor, to make immune effector cells such as
natural killer (NK) cells or cytotoxic T lymphocytes (CTLs) attack
and destroy tumor cells. In this regard, bispecific antibodies
designed to bind with one "arm" to a surface antigen on target
cells, and with the second "arm" to an activating, invariant
component of the T cell receptor (TCR) complex, have become of
interest in recent years. The simultaneous binding of such an
antibody to both of its targets will force a temporary interaction
between target cell and T cell, causing activation of any cytotoxic
T cell and subsequent lysis of the target cell. Hence, the immune
response is re-directed to the target cells and is independent of
peptide antigen presentation by the target cell or the specificity
of the T cell as would be relevant for normal MHC-restricted
activation of CTLs.
[0006] In this context it is crucial that CTLs are activated only
when in close proximity to a target cell, i.e., the immunological
synapse is mimicked. Particularly desirable are T cell activating
bispecific molecules that do not require lymphocyte preconditioning
or co-stimulation in order to elicit efficient lysis of target
cells. Several bispecific antibody formats have been developed and
their suitability for T cell mediated immunotherapy investigated.
These include BiTE (bispecific T cell engager) molecules (Nagorsen
and Bauerle, Exp Cell Res 317, 1255-1260 (2011)), diabodies
(Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives
thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol
293, 41-66 (1999)), DART (dual affinity retargeting) molecules,
(Moore et al., Blood 117, 4542-51 (2011)), and triomabs (Seimetz et
al., Cancer Treat Rev 36, 458-467 (2010)).
[0007] The task of generating bispecific molecules suitable for
treatment provides several technical challenges related to
efficacy, toxicity, applicability and producibility that have to be
met. In instances where the bispecific molecule targets an antigen
on a target cell, e.g., a cancer cell, that is also expressed in
non-target tissue, toxicity can occur. There is thus a need for
efficacious T cell activating bispecific molecules that unleash
full T cell activation in the presence of target cells but not in
the presence of normal cells or tissue.
SUMMARY OF THE INVENTION
[0008] The invention generally relates to T cell activating
bispecific molecules that are activated selectively in the presence
of a target cell.
[0009] In one aspect, the invention relates to a
protease-activatable T cell activating bispecific molecule
comprising [0010] (a) a first antigen binding moiety capable of
specific binding to CD3; [0011] (b) a second antigen binding moiety
capable of specific binding to a target cell antigen; and [0012]
(c) a masking moiety covalently attached to the T cell bispecific
binding molecule through a protease-cleavable linker, wherein the
masking moiety is capable of specific binding to the idiotype of
the first or the second antigen binding moiety thereby reversibly
concealing the first or second antigen binding moiety.
[0013] In one embodiment, the masking moiety of the
protease-activatable T cell activating bispecific molecule is
covalently attached to the first antigen binding moiety. In one
embodiment the masking moiety is covalently attached to the heavy
chain variable region of the first antigen binding moiety.
[0014] In one embodiment the masking moiety is covalently attached
to the light chain variable region of the first antigen binding
moiety. In one embodiment the masking moiety is an anti-idiotype
scFv.
[0015] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a second masking moiety reversibly
concealing the second antigen binding moiety.
[0016] In one embodiment the protease capable of cleaving the
protease-cleavable linker is expressed by the target cell. In one
embodiment the second antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions of the
Fab light chain and the Fab heavy chain are exchanged. In one
embodiment the second antigen binding moiety is a crossover Fab
molecule wherein the constant regions of the Fab light chain and
the Fab heavy chain are exchanged. In one embodiment the first
antigen binding moiety is a conventional Fab molecule. In one
embodiment the protease-activatable T cell activating bispecific
molecule comprises not more than one antigen binding moiety capable
of specific binding to CD3. In one embodiment the
protease-activatable T cell activating bispecific molecule
comprises a third antigen binding moiety which is a Fab molecule
capable of specific binding to a target cell antigen. In one
particular embodiment the third antigen binding moiety is identical
to the second antigen binding moiety. In one particular embodiment
the third antigen binding moiety is not identical to the second
antigen binding moiety. In one embodiment the second antigen
binding moiety is capable of specific binding to FolR1 or HER1. In
one embodiment the second antigen binding moiety is capable of
specific binding to FolR1, HER1 or Mesothelin. In one embodiment
the second antigen binding moiety is capable of specific binding to
FolR1, HER1, HER2 or Mesothelin.
[0017] In one embodiment the first and the second antigen binding
moiety are fused to each other, optionally via a peptide linker. In
one particular embodiment the second antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the first antigen binding moiety. In one
particular embodiment the first antigen binding moiety is fused at
the C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the second antigen binding moiety. In one particular
embodiment the Fab light chain of the first antigen binding moiety
and the Fab light chain of the second antigen binding moiety are
fused to each other, optionally via a peptide linker.
[0018] In one embodiment the protease-activatable T cell activating
bispecific molecule additionally comprises an Fc domain composed of
a first and a second subunit capable of stable association. In one
embodiment the Fc domain is an IgG, specifically an IgG.sub.1 or
IgG.sub.4, Fc domain. In one embodiment the Fc domain is a human Fc
domain. In one embodiment the Fc domain exhibits reduced binding
affinity to an Fc receptor and/or reduced effector function, as
compared to a native IgG.sub.1 Fc domain. In one embodiment the Fc
domain comprises one or more amino acid substitution that reduces
binding to an Fc receptor and/or effector function. In one
particular embodiment the one or more amino acid substitution is at
one or more position selected from the group of L234, L235, and
P329 (Kabat numbering). In one particular embodiment each subunit
of the Fc domain comprises three amino acid substitutions that
reduce binding to an activating Fc receptor and/or effector
function wherein said amino acid substitutions are L234A, L235A and
P329G. In one particular embodiment the Fc receptor is an Fc.gamma.
receptor. In one embodiment the effector function is
antibody-dependent cell-mediated cytotoxicity (ADCC). In one
embodiment, the target cell is a human cell.
[0019] In one embodiment the masking moiety comprises a heavy chain
variable region comprising at least one of: [0020] (a) a heavy
chain complementarity determining region (CDR H) 1 amino acid
sequence of DYSIH (SEQ ID NO:20); [0021] (b) a CDR H2 amino acid
sequence of WINTETGEPAYADDFKG (SEQ ID NO:21); and [0022] (c) a CDR
H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22).
[0023] In one embodiment the masking moiety comprises a light chain
variable region comprising at least one of: [0024] (a) a light
chain (CDR L)1 amino acid sequence of RASKSVSTSNYSYIH (SEQ ID
NO:23); [0025] (b) a CDR L2 amino acid sequence of YVSYLES (SEQ ID
NO:24); and [0026] (c) a CDR L3 amino acid sequence of QHSREFPWT
(SEQ ID NO:25).
[0027] In one embodiment the masking moiety comprises a heavy chain
variable region comprising: [0028] (a) a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
DYSIH (SEQ ID NO:20); [0029] (b) a CDR H2 amino acid sequence of
WINTETGEPAYADDFKG (SEQ ID NO:21); [0030] (c) a CDR H3 amino acid
sequence of PYDYDVLDY (SEQ ID NO:22); and a light chain variable
region comprising: [0031] (d) a light chain (CDR L)1 amino acid
sequence of RASKSVSTSNYSYIH (SEQ ID NO:23); [0032] (e) a CDR L2
amino acid sequence of YVSYLES (SEQ ID NO:24); and [0033] (f) a CDR
L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).
[0034] In one embodiment the masking moiety comprises a heavy chain
variable region comprising at least one of: [0035] (a) a heavy
chain complementarity determining region (CDR H) 1 amino acid
sequence of SYGVS (SEQ ID NO:26); [0036] (b) a CDR H2 amino acid
sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); and [0037] (c) a CDR
H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO:28).
[0038] In one embodiment the masking moiety comprises a light chain
variable region comprising at least one of: [0039] (a) a light
chain (CDR L)1 amino acid sequence of RASENIDSYLA (SEQ ID NO:29);
[0040] (b) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30);
and [0041] (c) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID
NO:31).
[0042] In one embodiment the masking moiety comprises a heavy chain
variable region comprising: [0043] (a) a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
SYGVS (SEQ ID NO:26); [0044] (b) a CDR H2 amino acid sequence of
IIWGDGSTNYHSALIS (SEQ ID NO:27); [0045] (c) a CDR H3 amino acid
sequence of GITTVVDDYYAMDY (SEQ ID NO:28); and a light chain
variable region comprising: [0046] (d) a light chain (CDR L)1 amino
acid sequence of RASENIDSYLA (SEQ ID NO:29); [0047] (e) a CDR L2
amino acid sequence of AATFLAD (SEQ ID NO:30); and [0048] (f) a CDR
L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).
[0049] In one embodiment the protease cleavable linker comprises at
least one protease recognition sequence. In one embodiment the
protease cleavable linker comprises a protease recognition
sequence. In one embodiment the protease recognition sequence is
selected from the group consisting of:
TABLE-US-00001 (a) (SEQ ID NO: 36) RQARVVNG; (b) (SEQ ID NO: 37)
VHMPLGFLGPGRSRGSFP; (c) (SEQ ID NO: 38) RQARVVNGXXXXXVPLSLYSG; and
(d) (SEQ ID NO: 39) RQARVVNGVPLSLYSG (e) (SEQ ID NO: 40) PLGLWSQ,
wherein X is any amino acid.
[0050] In one embodiment the protease cleavable linker comprises a
protease recognition sequence. In one embodiment the protease
recognition sequence is selected from the group consisting of:
TABLE-US-00002 (a) (SEQ ID NO: 36) RQARVVNG; (b) (SEQ ID NO: 37)
VHMPLGFLGPGRSRGSFP; (c) (SEQ ID NO: 38) RQARVVNGXXXXXVPLSLYSG; (d)
(SEQ ID NO: 39) RQARVVNGVPLSLYSG; (e) (SEQ ID NO: 40) PLGLWSQ; (f)
(SEQ ID NO: 97) VHMPLGFLGPRQARVVNG; (g) (SEQ ID NO: 98) FVGGTG; (h)
(SEQ ID NO: 99) KKAAPVNG; (i) (SEQ ID NO: 100) PMAKKVNG; (j) (SEQ
ID NO: 101) QARAKVNG; (k) (SEQ ID NO: 102) VHMPLGFLGP; (l) (SEQ ID
NO: 103) QARAK; (m) (SEQ ID NO: 104) VHMPLGFLGPPMAKK; (n) (SEQ ID
NO: 105) KKAAP; and (o) (SEQ ID NO: 106) PMAKK, wherein X is any
amino acid.
[0051] In one embodiment the protease capable of cleaving the
protease-cleavable linker is selected from the group consisting of
metalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and A
Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30
and 33, serine protease, e.g., urokinase-type plasminogen activator
and Matriptase, cysteine protease, aspartic protease, and cathepsin
protease. In one specific embodiment the protease is MMP9 or MMP2.
In a further specific embodiment, the protease is Matriptase. In
one embodiment the protease cleavable linker comprises the protease
recognition sequence RQARVVNG (SEQ ID NO:36).
[0052] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the first antigen
binding moiety comprises at least one heavy chain complementarity
determining region (CDR) comprising an amino acid sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at least one light
chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18
and SEQ ID NO: 19.
[0053] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the first antigen
binding moiety comprises the heavy chain complementarity
determining region (CDRs) of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ
ID NO: 46 and the light chain CDRs of SEQ ID NO: 17, SEQ ID NO: 18
and SEQ ID NO: 19.
[0054] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the first antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 43
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 55.
[0055] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the first antigen
binding moiety comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 43 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 55.
[0056] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to FolR1 and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO: 14, SEQ
ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ
ID NO: 19.
[0057] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to FolR1 and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO: 151, SEQ
ID NO: 152 and SEQ ID NO: 153 and at least one light chain CDR
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ
ID NO: 156.
[0058] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to FolR1 and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16
and at least one light chain CDR selected from the group of SEQ ID
NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0059] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to FolR1 and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO:
153 and at least one light chain CDR selected from the group of SEQ
ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156.
[0060] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 47
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 55.
[0061] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 157
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 158.
[0062] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 47 and a light chain variable
region comprising the amino acid sequence of SEQ ID NO: 55.
[0063] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 157 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO:
158.
[0064] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to Mesothelin and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO: 107, SEQ
ID NO: 108 and SEQ ID NO: 109 and at least one light chain CDR
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ
ID NO: 112.
[0065] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to Mesothelin and
comprises at least one heavy chain complementarity determining
region (CDR) comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO:
109 and at least one light chain CDR selected from the group of SEQ
ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112.
[0066] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 113
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 114.
[0067] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain variable region comprising
the amino acid sequence of SEQ ID NO: 113 and a light chain
variable region comprising the amino acid sequence of SEQ ID NO:
114.
[0068] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to HER1 and comprises
at least one heavy chain complementarity determining region (CDR)
of any one of the antibodies disclosed in WO/2006/082515
incorporated herein by reference in its entirety.
[0069] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to HER1 and comprises
at least one heavy chain complementarity determining region (CDR)
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57 and SEQ
ID NO: 58 and at least one light chain CDR comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0070] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety is capable of specific binding to HER1 and comprises
at least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0071] In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain comprising an amino acid
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to the amino acid sequence of SEQ ID NO: 32 and a light
chain comprising an amino acid sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of
SEQ ID NO: 33. In one embodiment of the protease-activatable T cell
activating bispecific molecule described herein the second antigen
binding moiety comprises a heavy chain comprising the amino acid
sequence of SEQ ID NO: 32 and a light chain comprising the amino
acid sequence of SEQ ID NO: 33.
[0072] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 43 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 55, and the second and third antigen binding moieties are
capable of specific binding to HER2, wherein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 160
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 161, wherein the third
antigen binding moiety comprises a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 159 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 161.
[0073] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0074]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:2; [0075] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; and [0076] (c) a light chain comprising
the amino acid sequence of SEQ ID NO:1.
[0077] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0078]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:4; [0079] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; and [0080] (c) a light chain comprising
the amino acid sequence of SEQ ID NO:1.
[0081] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0082]
(a) at least one heavy chain comprising the amino acid sequence of
SEQ ID NO:32; [0083] (b) at least one light chain comprising the
amino acid sequence of SEQ ID NO:34.
[0084] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0085]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:72; [0086] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; and [0087] (c) a light chain comprising an
amino acid sequence of SEQ ID NO:1.
[0088] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0089]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:85; [0090] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; and [0091] (c) a light chain comprising an
amino acid sequence of SEQ ID NO:1.
[0092] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0093]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:73; [0094] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; [0095] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:1; and [0096] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO: 74.
[0097] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0098]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:77; [0099] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:82; [0100] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:78; and [0101] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO:81.
[0102] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0103]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:76; [0104] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:77; [0105] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:78; and [0106] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO:79.
[0107] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0108]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:132; [0109] (b) a second heavy chain comprising the amino
acid sequence of SEQ ID NO:136; [0110] (c) a first light chain
comprising an amino acid sequence of SEQ ID NO:81; and [0111] (d) a
second light chain comprising an amino acid sequence of SEQ ID
NO:133.
[0112] In one particular embodiment the protease-activatable T cell
activating bispecific molecule described herein comprises [0113]
(a) a first heavy chain comprising the amino acid sequence of SEQ
ID NO:137; [0114] (b) a second heavy chain comprising the amino
acid sequence of SEQ ID NO:139; [0115] (c) a first light chain
comprising an amino acid sequence of SEQ ID NO:81; and [0116] (d) a
second light chain comprising an amino acid sequence of SEQ ID
NO:138.
[0117] In one aspect, the invention relates to an idiotype-specific
polypeptide for reversibly concealing an anti-CD3 antigen binding
site of a molecule. In one embodiment the idiotype-specific
polypeptide is an anti-idiotype scFv. In one embodiment the
idiotype-specific polypeptide is covalently attached to the
molecule through a linker. In one embodiment the linker is a
peptide linker. In one embodiment the linker is a
protease-cleavable linker. In one embodiment the peptide linker
comprises at least one protease recognition site. In one embodiment
the protease is selected from the group consisting of
metalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and A
Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23, 28-30
and 33, serine protease, e.g., urokinase-type plasminogen activator
and Matriptase, cysteine protease, aspartic protease, and cathepsin
protease. In one specific embodiment the protease is MMP9 or MMP2.
In a further specific embodiment, the protease is Matriptase. In
one embodiment the idiotype-specific polypeptide is covalently
attached to the molecule through more than one linker. In one
embodiment the idiotype-specific polypeptide is covalently attached
to the molecule through two linkers.
[0118] In one embodiment the molecule which comprises the anti-CD3
antigen binding site is a T-cell activating bispecific molecule. In
one particular embodiment the idiotype-specific polypeptide
comprises a heavy chain variable region comprising at least one of:
[0119] (a) a heavy chain complementarity determining region (CDR H)
1 amino acid sequence of DYSIH (SEQ ID NO:20); [0120] (b) a CDR H2
amino acid sequence of WINTETGEPAYADDFKG (SEQ ID NO:21); and [0121]
(c) a CDR H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22).
[0122] In one particular embodiment the idiotype-specific
polypeptide comprises a light chain variable region comprising at
least one of: [0123] (a) a light chain (CDR L)1 amino acid sequence
of RASKSVSTSNYSYIH (SEQ ID NO:23); [0124] (b) a CDR L2 amino acid
sequence of YVSYLES (SEQ ID NO:24); and [0125] (c) a CDR L3 amino
acid sequence of QHSREFPWT (SEQ ID NO:25).
[0126] In one particular embodiment the idiotype-specific
polypeptide comprises: [0127] (a) a heavy chain complementarity
determining region (CDR H) 1 amino acid sequence of DYSIH (SEQ ID
NO:20); [0128] (b) a CDR H2 amino acid sequence of
WINTETGEPAYADDFKG (SEQ ID NO:21); [0129] (c) a CDR H3 amino acid
sequence of PYDYDVLDY (SEQ ID NO:22); and a light chain variable
region comprising: [0130] (d) a light chain (CDR L)1 amino acid
sequence of RASKSVSTSNYSYIH (SEQ ID NO:23); [0131] (e) a CDR L2
amino acid sequence of YVSYLES (SEQ ID NO:24); and [0132] (f) a CDR
L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25).
[0133] In one particular embodiment the idiotype-specific
polypeptide comprises a heavy chain variable region comprising at
least one of: [0134] (a) a heavy chain complementarity determining
region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO:26);
[0135] (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID
NO:27); and [0136] (c) a CDR H3 amino acid sequence of
GITTVVDDYYAMDY (SEQ ID NO:28).
[0137] In one particular embodiment the idiotype-specific
polypeptide comprises a light chain variable region comprising at
least one of: [0138] (a) a light chain (CDR L)1 amino acid sequence
of RASENIDSYLA (SEQ ID NO:29); [0139] (b) a CDR L2 amino acid
sequence of AATFLAD (SEQ ID NO:30); and [0140] (c) a CDR L3 amino
acid sequence of QHYYSTPYT (SEQ ID NO:31).
[0141] In one particular embodiment the idiotype-specific
polypeptide comprises a heavy chain variable region comprising:
[0142] (a) a heavy chain complementarity determining region (CDR H)
1 amino acid sequence of SYGVS (SEQ ID NO:26); [0143] (b) a CDR H2
amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); [0144] (c)
a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO:28); and
a light chain variable region comprising: [0145] (d) a light chain
(CDR L)1 amino acid sequence of RASENIDSYLA (SEQ ID NO:29); [0146]
(e) a CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and
[0147] (f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID
NO:31).
[0148] According to another aspect of the invention, an isolated
polynucleotide encoding a protease-activatable T cell activating
bispecific molecule of the invention or a fragment thereof is
provided. The invention also encompasses polypeptides encoded by
the polynucleotides of the invention. The invention further
provides an expression vector comprising the isolated
polynucleotide of the invention, and a host cell comprising the
isolated polynucleotide or the expression vector of the invention.
In some embodiments the host cell is a eukaryotic cell,
particularly a mammalian cell. In another aspect is provided a
method of producing the protease-activated T cell molecule of the
invention, comprising the steps of a) culturing the host cell of
the invention under conditions suitable for the expression of the
protease-activated T cell molecule and b) recovering the
protease-activated T cell molecule. The invention also encompasses
a protease-activated T cell molecule produced by the method of the
invention.
[0149] In another aspect is provided a method of producing the
idiotype-specific polypeptide of the invention, comprising the
steps of a) culturing the host cell of the invention under
conditions suitable for the expression of the protease-activated T
cell molecule and b) recovering the idiotype-specific polypeptide.
The invention also encompasses a idiotype-specific polypeptide
produced by the method of the invention.
[0150] The invention further provides a pharmaceutical composition
comprising the protease-activatable T cell activating bispecific
molecule of the invention and a pharmaceutically acceptable
carrier.
[0151] Also encompassed by the invention are methods of using the
protease-activated T cell molecule and pharmaceutical composition
of the invention. In one aspect the invention provides a
protease-activated T cell molecule or a pharmaceutical composition
of the invention for use as a medicament. In one aspect is provided
a protease-activated T cell molecule or a pharmaceutical
composition according to the invention for use in the treatment of
a disease in an individual in need thereof. In a specific
embodiment the disease is cancer.
[0152] Also provided is the use of a protease-activated T cell
molecule of the invention for the manufacture of a medicament for
the treatment of a disease in an individual in need thereof; as
well as a method of treating a disease in an individual, comprising
administering to said individual a therapeutically effective amount
of a composition comprising the protease-activated T cell molecule
according to the invention in a pharmaceutically acceptable form.
In a specific embodiment the disease is cancer. In any of the above
embodiments the individual preferably is a mammal, particularly a
human.
[0153] The invention also provides a method for inducing lysis of a
target cell, particularly a tumor cell, comprising contacting a
target cell with a protease-activated T cell molecule of the
invention in the presence of a T cell, particularly a cytotoxic T
cell.
[0154] In another aspect the invention also provides a composition
comprising a protease-activatable T cell activating bispecific
molecule described herein and a pharmaceutically acceptable
carrier.
[0155] In another aspect the invention also provides a composition
comprising an idiotype-specific polypeptide as described herein and
a pharmaceutically acceptable carrier.
[0156] In another aspect the invention also provides a
protease-activatable T cell activating bispecific molecule or an
idiotype-specific polypeptide as described herein, or the
composition described herein, for use as a medicament. In one
embodiment the medicament is for treating or delaying progression
of cancer, treating or delaying progression of an immune related
disease, and/or enhancing or stimulating an immune response or
function in an individual.
[0157] In another aspect the invention also provides a
protease-activatable T cell activating bispecific molecule or
idiotype-specific polypeptide as described herein for use in the
treatment of a disease in an individual in need thereof. In one
embodiment, the disease is a proliferative disorder, particularly
cancer.
[0158] In another aspect the invention also provides a method of
treating a disease in an individual, comprising administering to
said individual a therapeutically effective amount of a composition
comprising the protease-activatable T cell activating bispecific
molecule or composition as described herein.
[0159] In another aspect the invention also provides a method for
inducing lysis of a target cell, comprising contacting a target
cell with the protease-activatable T cell activating bispecific
molecule or composition as described herein in the presence of a T
cell. In one embodiment the method for inducing lysis of a target
cell is an in vitro method. In one embodiment the target cell is a
cancer cell. In one embodiment the target cell expresses a protease
capable of activating the protease-activatable T cell activating
bispecific molecule.
[0160] In another aspect the invention also provides an
anti-idiotype CD3 antibody or antigen-binding fragment thereof
specific for an idiotype of an anti-CD3 antigen-binding molecule,
wherein the anti-idiotype CD3 antibody or fragment thereof when
bound to the anti-CD3 antigen-binding molecule specifically blocks
binding of the anti-CD3 antigen-binding molecule to CD3.
[0161] In one embodiment, the anti-idiotype CD3 antibody or
antigen-binding fragment thereof is reversibly associated with the
anti-CD3 antigen-binding molecule through a peptide linker
comprising a protease recognition site. In one embodiment, the CD3
is a mouse, monkey or human CD3.
[0162] In another aspect the invention provides a method of
reducing in vivo toxicity of a T cell activating bispecific
molecule comprising attaching an idiotype-specific polypeptide as
described herein to the T cell activating bispecific molecule with
a protease-cleavable linker to form a protease-activatable T cell
activating bispecific molecule, wherein the protease-activatable T
cell activating bispecific molecule has reduced in vivo toxicity
compared to the T cell activating bispecific molecule.
SHORT DESCRIPTION OF THE FIGURES
[0163] FIGS. 1A-1E depict schematics of different CD3 binders with
masking moieties. FIG. 1A: 7859 anti-ID CH2527 scFv 4.15.64 MK062
Matriptase site CD3 Fc. FIG. 1B: 7860 anti-ID CH2527 scFv 4.32.63
MK062 Matriptase site CD3 Fc. FIG. 1C: 7857 anti-ID CH2527 scFv
4.15.64 non-cleavable linker CD3 Fc. FIG. 1D: ID 7858 anti-ID
CH2527 scFv 4.32.63 non-cleavable linker CD3 Fc. FIG. 1E: 7861
monovalent CD3 Fc.
[0164] FIG. 2 shows a table summarizing the affinities of the
anti-idiotypic masking moieties to the CD3 binder (CH2527).
[0165] FIGS. 3A-3D shows Capillary Electrophoresis-SDS analysis of
the molecules depicted in FIGS. 1A and 1B. FIGS. 3A and 3B:
Capillary Electrophoresis-SDS analysis of the molecule depicted in
FIG. 1A under non reducing (FIG. 3A) and reducing conditions (FIG.
3B). Comparison of the untreated (I) and treated molecule (III)
shows complete cleavage of the anti-ID scFv after rhMatriptase/ST14
treatment for 48 h at 37.degree. C. One sample (II) was untreated
but incubated at 37.degree. C. for 48 h. FIGS. 3C and 3D: Capillary
Electrophoresis-SDS analysis of the molecule depicted in FIG. 1B
under non-reducing (FIG. 3C) and reducing conditions (FIG. 3D).
Comparison of the untreated (I) and treated molecule (III) shows
complete cleavage of the anti-ID scFv after rhMatriptase/ST14
treatment for 48 h at 37.degree. C. One sample (II) was untreated
but incubated at 37.degree. C. for 48 h.
[0166] FIGS. 4A-4C show the effect of anti-idiotypic masking of CD3
binding. FIGS. 4A and 4B depict results of Jurkat NFAT reporter
assays to show the masking effect of anti-idiotypic CD3 scFv
4.15.64 (FIG. 4A) or anti-idiotypic CD3 scFv 4.32.63 (FIG. 4B).
Monovalent CD3 IgGs were crosslinked via an anti-human Fc antibody
(coated on assay plate) before Jurkat NFAT (acute lymphatic
leukemia reporter cell line with a NFAT promoter, expressing human
CD3.epsilon.) were added. The Jurkat-NFAT reporter cell line
(Promega) is a human acute lymphatic leukemia reporter cell line
with a NFAT promoter, expressing human CD3.epsilon.. If CD3 binder
binds CD3.epsilon. Luciferase is expressed and this can be measured
in Luminescence after addition of One-Glo substrate (Promega). FIG.
4C shows a comparison of EC50 values of CD3.epsilon. binding for
masked and unmasked monovalent CD3 binder.
[0167] FIGS. 5A-5H depict schematics of different T cell bispecific
molecules with masking moieties. FIG. 5A: 7344 anti-ID CH2527 scFv
4.15.64 MK062 Matriptase site CD3 16D5 Fc. FIG. 5B: 7676 anti-ID
CH2527 scFv 4.15.64 non-cleavable linker CD3 16D5 Fc. FIG. 5C: 7496
anti-ID CH2527 scFv 4.32.63 MK062 Matriptase site CD3 16D5 Fc. FIG.
5D: 7611 anti-ID CH2527 scFv 4.32.63 non-cleavable linker CD3 16D5
Fc. FIG. 5E: 6298 GA916-D-16D5-02 sf W(1). FolR1 16D5 classic 2+1
TCB with common light chain. FIG. 5F: 6100 GA916-D-16D5 sf W(3a).
FolR1 16D5 inverted 2+1 TCB with common light chain. FIG. 5G: ID
6182 DP47GS TCB sf CHO W(9a). DP47 inverted 2+1 TCB. FIG. 5H: 7494
anti-ID CH2527 Fab 4.15.64 MK062 Matriptase site CD3 16D5 Fc.
[0168] FIG. 6 shows a first plasmid ratios used for transfection by
size exclusion chromatography (1(hole): 1 (knob): 3 (CLC)).
[0169] FIG. 7 shows a second plasmid ratios used for transfection
by size exclusion chromatography. (1(hole): 2 (knob): 3 (CLC)).
[0170] FIG. 8 shows CE-SDS analysis of the TCB molecule depicted in
FIG. 5A (ID 7344) (final purified preparation): Lane A=non-reduced,
lane B=reduced, lane C=Protein standard.
[0171] FIG. 9 shows CE-SDS analysis of the TCB molecule depicted in
FIG. 5B (ID 7676) (final purified preparation): Lane A=non-reduced,
lane B=reduced, lane C=Protein standard.
[0172] FIG. 10 shows CE-SDS analysis of the TCB molecule depicted
in FIG. 5C (ID 7496) (final purified preparation): Lane
A=non-reduced, lane B=reduced, lane C=Protein standard.
[0173] FIG. 11 shows CE-SDS analysis of the TCB molecule depicted
in FIG. 5D (ID 7611) (final purified preparation): Lane
A=non-reduced, lane B=reduced, lane C=Protein standard.
[0174] FIG. 12A-12D show shows Capillary Electrophoresis-SDS
analysis of the molecules depicted in FIGS. 5A and 5C. FIGS. 12A
and 12B show Capillary Electrophoresis of the molecules depicted in
FIG. 5A (ID 7344) anti-ID CH2527 scFv 4.15.64 MK062 CD3 16D6 Fc
under non reducing (FIG. 12A) and reducing conditions (FIG. 12B).
Comparison of the untreated (I) and treated molecule (III) shows
complete cleavage of the anti-ID scFv after rhMatriptase/ST14
treatment for 48 h at 37.degree. C. One sample (II) was untreated
but incubated at 37.degree. C. for 48 h. Pre-stained protein Marker
(IV) Mark 12 (Invitrogen) was used for estimation of correct
molecule weight. FIGS. 12C and 12D shows Capillary Electrophoresis
of the molecule depicted in FIG. 5C (ID 7496) anti-ID CH2527 scFv
4.32.63 MK062 CD3 16D6 Fc under non reducing (FIG. 12C) and
reducing conditions (FIG. 12D). Comparison of the untreated (I) and
treated molecule (III) shows complete cleavage of the anti-ID scFv
after rhMatriptase/ST14 treatment for 48 h at 37.degree. C. One
sample (II) was untreated but incubated at 37.degree. C. for 48 h.
Pre-stained protein Marker (IV) Mark 12 (Invitrogen) was used for
estimation of correct molecule weight.
[0175] FIG. 13 shows FolR1 expression level quantification done by
Qifikit (Dako). Antibody for FolR1: #LS-C125620-100 (LifeSpan
BioSciences Inc); used at 20 .mu.g/ml; mouse IgG1 isotype: #554121
(BD).
[0176] FIGS. 14A and 14B show T cell activation by protease
activated TCBs. FIG. 14A shows killing of Skov3 cells induced by
protease-activated TCB molecules at a concentration of 10 nM (TCBs
with different anti-idiotypic CD3 masks, cleavable and
non-cleavable linker, molecules pre-treated with purified
rhMatriptase/ST14) and human PBMCs after 48 h of incubation
(E:T=7:1, effectors are human PBMCs). Pre-treatment with
rhMatriptase/ST14 (R&D Systems) was done for 24 h at 37.degree.
C. FIG. 14B shows T cell activation of human PBMCs induced by
protease activated TCB binding of 10 nM (TCBs with different
anti-idiotypic CD3 masks, cleavable and non-cleavable linker,
treated molecules) on Skov3 cells after 48 h of incubation
(E:T=7:1, effectors are human PBMCs). T cell activation markers
CD25 (left panels) and CD69 (right panels). CD4+ and CD8+ T cells
as indicated.
[0177] FIGS. 15A and 15B show T cell activation by protease
activated TCBs. FIG. 15A shows killing of Mkn-45 cells induced by
protease activated TCB molecules at a concentration of 100 nM (TCBs
with different anti-idiotypic CD3 masks, cleavable and
non-cleavable linker, treated molecules) and human PBMCs after 48 h
of incubation (E:T=7:1, effectors are human PBMCs). Pre-treatment
with rhMatriptase/ST14 (R&D Systems) was done for 24 h at
37.degree. C. FIG. 15B shows T cell activation of human PBMCs
induced by protease activated TCB binding of 100 nM (TCBs with
different anti-idiotypic CD3 masks, cleavable and non-cleavable
linker, treated molecules) on Mkn-45 cells after 48 h of incubation
(E:T=7:1, effectors are human PBMCs). T cell activation markers
CD25 (left panels) and CD69 (right panels). CD4+ and CD8+ T cells
as indicated.
[0178] FIG. 16 shows killing of HT29 cells induced by protease
activated TCB molecules at a concentration of 10 nM (TCBs with
different anti-idiotypic CD3 masks, cleavable and non-cleavable
linker, treated molecules) and human PBMCs after 48 h of incubation
(E:T=10:1, effectors are human PBMCs). Pre-treatment with
rhMatriptase/ST14 (R&D Systems) was done for 24 h at 37.degree.
C. Bars from left to right are 7344: anti-ID CH2527 scFv 4.15.64
MK062 CD3 16D6Fc; 7344: anti-ID CH2527 scFv 4.15.64 MK062 CD3
16D6Fc_treated; 7676: anti-ID CH2527 scFv 4.15.64 non-cleavable CD3
16D6Fc; 7496 anti-ID CH2527 scFv 4.32.63 MK062 CD3 16D6 Fc; 7496
anti-ID CH2527 scFv 4.32.63 MK062 CD3 16D6 Fc_treated; 7611: ID
anti CH2527 scFv 4.32.63 non-cleavable linker CD3 16D6 Fc; 6298
GA916-D-16D5-02 sf W(1); 6182 DP47GS TCB sf CHO W(9a).
[0179] FIG. 17 shows killing of Skov3 cells induced by protease
activated TCB molecules at a concentration of 10 nM (TCBs with
different anti-idiotypic CD3 masks, cleavable and non-cleavable
linker, treated molecules) and human PBMCs (from a different donor
than PBMCs used for FIG. 14A) after 48 h of incubation (E:T=10:1,
effectors are human PBMCs). Pre-treatment with rhMatriptase/ST14
(R&D Systems) was done for 24 h at 37.degree. C. Bars from left
to right are 7344: anti-ID CH2527 scFv 4.15.64 MK062 CD3 16D6Fc;
7344: anti-ID CH2527 scFv 4.15.64 MK062 CD3 16D6Fc_treated; 7676:
anti-ID CH2527 scFv 4.15.64 non-cleavable CD3 16D6Fc; 7496 anti-ID
CH2527 scFv 4.32.63 MK062 CD3 16D6 Fc; 7496 anti-ID CH2527 scFv
4.32.63 MK062 CD3 16D6 Fc_treated; 7611: ID anti CH2527 scFv
4.32.63 non-cleavable linker CD3 16D6 Fc; 6298 GA916-D-16D5-02 sf
W(1); 6182 DP47GS TCB sf CHO W(9a).
[0180] FIGS. 18A and 18B show T cell activation by protease
activated TCBs. FIG. 18A shows dose-dependent killing of HeLa cells
induced by protease activated TCB molecules (TCB with
anti-idiotypic CD3 4.15.64 mask, cleavable and non-cleavable
linker, treated molecule) and human PBMCs (isolated from buffy
coat) after 48 h of incubation (E:T=10:1, effectors are human
PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) was
done for 24 h at 37.degree. C. FIG. 18B shows dose-dependent T cell
activation of human PBMCs induced by protease activated TCB binding
(TCB with anti-idiotypic CD3 4.15.64 mask, cleavable and
non-cleavable linker, treated molecule) on HeLa cells after 48 h of
incubation (E:T=10:1, effectors are human PBMCs). T cell activation
markers CD25 (left panels) and CD69 (right panels). CD4+ and CD8+ T
cells as indicated.
[0181] FIGS. 19A and 19B show T cell activation by protease
activated TCBs. FIG. 19A shows dose-dependent killing of HeLa cells
induced by protease activated TCB molecules (TCB with
anti-idiotypic CD3 4.32.63 mask, cleavable and non-cleavable
linker, treated molecule) and human PBMCs (isolated from buffy
coat) after 48 h of incubation (E:T=10:1, effectors are human
PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) was
done for 24 h at 37.degree. C. FIG. 19B shows dose-dependent T cell
activation of human PBMCs induced by protease activated TCB binding
(TCB with anti-idiotypic CD3 4.32.63 mask, cleavable and
non-cleavable linker, treated molecule) on HeLa cells after 48 h of
incubation (E:T=10:1, effectors are human PBMCs). T cell activation
markers CD25 (left panels) and CD69 (right panels). CD4+ and CD8+ T
cells as indicated.
[0182] FIGS. 20A and 20B show T cell activation by protease
activated TCBs. FIG. 20A shows dose-dependent killing of Skov3
cells induced by protease activated TCB molecules (TCB with
anti-idiotypic CD3 4.15.64 mask, cleavable and non-cleavable
linker, treated molecule) and human PBMCs (isolated from buffy
coat) after 48 h of incubation (E:T=10:1, effectors are human
PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) was
done for 24 h at 37.degree. C. FIG. 20B shows dose-dependent T cell
activation of human PBMCs induced by protease activated TCB binding
(TCB with anti-idiotypic CD3 4.15.64 mask, cleavable and
non-cleavable linker, treated molecule) on Skov3 cells after 48 h
of incubation (E:T=10:1, effectors are human PBMCs). T cell
activation markers CD25 (left panels) and CD69 (right panels). CD4+
and CD8+ T cells as indicated.
[0183] FIGS. 21A and 21B show T cell activation by protease
activated TCBs. FIG. 21A shows dose-dependent killing of Skov3
cells induced by protease activated TCB molecules (TCB with
anti-idiotypic CD3 4.32.63 mask, cleavable and non-cleavable
linker, treated molecule) and human PBMCs (isolated from buffy
coat) after 48 h of incubation (E:T=10:1, effectors are human
PBMCs). Pre-treatment with rhMatriptase/ST14 (R&D Systems) was
done for 24 h at 37.degree. C. FIG. 21B shows dose-dependent T cell
activation of human PBMCs induced by protease activated TCB binding
(TCB with anti-idiotypic CD3 4.32.63 mask, cleavable and
non-cleavable linker, treated molecule) on Skov3 cells after 48 h
of incubation (E:T=10:1, effectors are human PBMCs). T cell
activation markers CD25 (left panels) and CD69 (right panels). CD4+
and CD8+ T cells as indicated.
[0184] FIGS. 22A and 22B show T cell activation by protease
activated TCBs. FIG. 22A shows dose-dependent T cell activation of
human PBMCs (different donor than in experiments described above)
induced by protease activated TCB binding (TCB with anti-idiotypic
CD3 4.15.64 mask, cleavable and non-cleavable linker, treated
molecule) on HT29 cells after 48 h of incubation (E:T=10:1,
effectors are human PBMCs). T cell activation markers CD25 (left
panels) and CD69 (right panels). CD4+ and CD8+ T cells as
indicated. FIG. 22B shows dose-dependent T cell activation of human
PBMCs (different donor than in FIG. 16) induced by protease
activated TCB binding (TCB with anti-idiotypic CD3 4.32.63 mask,
cleavable and non-cleavable linker, treated molecule) on HT29 cells
after 48 h of incubation (E:T=10:1, effectors are human PBMCs). T
cell activation markers CD25 (left panels) and CD69 (right panels).
CD4+ and CD8+ T cells as indicated.
[0185] FIG. 23 shows dose-dependent T cell activation of human
PBMCs (different donor than in experiments described above) induced
by protease activated TCB binding (TCB with anti-idiotypic CD3
4.15.64 mask, cleavable and non-cleavable linker, treated molecule)
on HRCEpiC cells after 48 h of incubation (E:T=10:1, effectors are
human PBMCs). T cell activation markers CD25 (left panels) and CD69
(right panels). CD4+ and CD8+ T cells as indicated.
[0186] FIG. 24 shows killing of Ovcar3 cells induced by protease
activated TCB molecules at a concentration of 50 nM (TCBs with
different anti-idiotypic CD3 masks, cleavable and non-cleavable
linker, treated molecules) and human PBMCs after 48 h of incubation
(E:T=10:1, effectors are human PBMCs). Pre-treatment with
rhMatriptase/ST14 (R&D Systems) was done for 10 min at
37.degree. C. (not fully cleaved).
[0187] FIG. 25 shows killing of Skov3 cells induced by 10 nM of
protease activated TCB molecules (TCB with anti-idiotypic CD3
4.15.64 mask, cleavable and non-cleavable linker, treated molecule)
and human PBMCs (isolated from buffy coat) after 48 h of incubation
(E:T=10:1, effectors are three different Donors for human PBMCs).
Pre-treatment with rhMatriptase/ST14 (R&D Systems) was done for
24 h at 37.degree. C.
[0188] FIG. 26 shows killing of Skov3 cells induced by 10 nM of
protease activated TCB molecules (TCB with anti-idiotypic CD3
4.32.63 mask, cleavable and non-cleavable linker, treated molecule)
and human PBMCs (isolated from buffy coat) after 48 h of incubation
(E:T=10:1, effectors are three different Donors for human PBMCs).
Pre-treatment with rhMatriptase/ST14 (R&D Systems) was done for
24 h at 37.degree. C.
[0189] FIG. 27 shows killing of HeLa cells induced by 100 pM of
protease activated TCB molecules (TCB with anti-idiotypic CD3
4.32.63 mask, cleavable and non-cleavable linker, treated molecule)
and human PBMCs (isolated from buffy coat) after 48 h of incubation
(E:T=10:1, effectors are three different Donors for human PBMCs).
Pre-treatment with rhMatriptase/ST14 (R&D Systems) was done for
24 h at 37.degree. C.
[0190] FIG. 28 depicts a schematic of anti-ID GA201 scFv Matrix
Metalloprotease site GA201 Fc (GA201-anti-GA201-scFv).
[0191] FIG. 29 depicts a schematics of the anti HER1 antibody
GA201.
[0192] FIGS. 30A and 30B show capillary Electrophoresis-SDS
analysis of the molecule depicted in FIG. 28 under non-reducing
(FIG. 30A) and reducing conditions (FIG. 30B). The molecule
depicted in FIG. 28 was purified to homogeneity by Protein A and
Size Exclusion chromatography and subjected to Capillary
electrophoresis-SDS analysis.
[0193] FIG. 31 shows FACS analysis of GA201-anti-GA201-scFv and
GA201 binding to HER1 expressed on H322M cells to confirm masking
effect of anti-idiotypic GA201 scFv. GA201-anti-GA201-scFv was
incubated overnight with the Matrix Metalloprotease MMP-2 and
binding to H322M cells was compared to uncleaved
GA201-anti-GA201-scFv, GA201 and an isotype IgG1 control antibody.
Binding to HER1 on H322M cells was detected with a F(ab')2-goat
anti-human IgG Fc secondary antibody FITC conjugate and analyzed by
FACS using the BD FACS Canto II. The median fluorescence intensity
(MFI) was used for analysis.
[0194] FIG. 32 shows surface plasmon resonance analysis of HER1
binding of masked and unmasked GA201, before and after MMP2
cleavage.
[0195] FIGS. 33A-33J depict schematics of different T cell
bispecific molecules with masking moieties. FIG. 33A: ID 8364. 16D5
TCB, classic format, anti ID CH2527 scFv 4.32.63 MMP9-MK062
Matriptase site N-terminally fused to CD3. FIG. 33B: ID 8363. 16D5
TCB, classic format, anti ID CH2527 scFv 4.32.63 Cathepsin S/B site
N-terminally fused to CD3. FIG. 33C: ID 8365. 16D5 TCB, inverted
format, anti ID CH2527 scFv 4.32.63 MK062 Matriptase site
N-terminally fused to common light chain. FIG. 33D: ID 8366. 16D5
TCB, inverted format, anti ID CH2527 scFv 4.32.63 non-cleavable
linker N-terminally fused to common light chain. FIG. 33E: ID 8672.
aMesothelin RG7787 charged residues TCB, classic format, anti ID
CH2527 scFv 4.32.63 MMP9-MK062 Matriptase site N-terminally fused
to CD3.times.Fab. FIG. 33F: ID 8673. aMesothelin RG7787 charged
residues TCB, classic format, anti ID CH2527 scFv 4.32.63
non-cleavable linker N-terminally fused to CD3.times.Fab. FIG. 33G:
ID 8674. aMesothelin RG7787 charged residues TCB, inverted format,
anti ID CH2527 scFv 4.32.63 MMP9-MK062 Matriptase site N-terminally
fused to CD3 XFab. FIG. 33H: 8675. aMesothelin RG7787 charged
residues TCB, inverted format, anti ID CH2527 scFv 4.32.63
non-cleavable linker N-terminally fused to CD3 XFab. FIG. 33I: ID
8505. aMesothelin RG7787 charged residues CD3 XFab TCB, inverted
format. FIG. 33J: ID 8676. CD3 XFab aMesothelin RG7787 charged
residues TCB, classic format.
[0196] FIG. 34 depicts CE-SDS analysis of the TCB ID 8365 and TCB
ID 8366 (final purified preparation): Lane A=Protein standard, lane
B=protein stored at 4.degree. C., lane C=protein pretreated with
rhMatriptase/ST14 (R&D Systems), lane D=protein incubated for
72 h at 37.degree. C. and lane E=molecule 3.
[0197] FIGS. 35A and 35B. depicts CE-SDS analysis of the TCB
depicted in FIG. 33A (ID 8364) and the TCB depicted in FIG. 33B (ID
8363). FIG. 35A: CE-SDS analysis of the TCB 8364 (final purified
preparation): Lane A=Protein standard, lane B=protein stored at
4.degree. C., lane C=protein pretreated with rhMatriptase/ST14
(R&D Systems), lane D=protein incubated for 72 h at 37.degree.
C. and lane E=non-cleavable linker construct. FIG. 35B: CE-SDS
analysis of the TCB 8363 (final purified preparation): Lane
A=Protein standard, lane B=protein stored at 4.degree. C., lane
C=protein pretreated with rhCathepsin B (R&D Systems), lane
D=protein pretreated with rhCathepsin S (R&D Systems), lane
E=protein incubated for 72 h at 37.degree. C. and lane
F=non-cleavable linker construct.
[0198] FIGS. 36A and 36B. depicts Jurkat NFAT activation assay
using HeLa and Skov-3 cells as target cells. Each point represents
the mean value of triplicates. Standard deviation is indicated by
error bars. Jurkat-NFAT reporter cell line (Promega) is a human
acute lymphatic leukemia reporter cell line with a NFAT promoter,
expressing human CD3c. If the CD3 binder of the TCB binds the tumor
target and the CD3 (cross-linkage is necessary) binds CD3.epsilon.
the Luciferase expression can be measured in Luminescence after
addition of One-Glo substrate (Promega). The FolR1 TCB (black
triangles pointing down) and the pretreated protease activated TCB
(8364, grey filled squares) with N-terminally fused anti ID CD3
4.32.63 scFv and MMP9-Matriptase MK062 site were compared. The
molecule was treated with rhMatriptase/ST14 (R&D Systems) for
about 20 h at 37.degree. C. The masked TCB (containing a GS
non-cleavable linker, grey triangles pointing up) and the
non-targeted TCB control (empty triangle pointing down) are shown
as well. The dotted line shows the Luminescence of target cells and
effector cells without any TCB.
[0199] FIG. 36A shows a Jurkat NFAT activation assay using HeLa
cells as target cells.
[0200] FIG. 36B shows a Jurkat NFAT activation assay using Skov-3
cells as target cells.
[0201] FIGS. 37A-37D depict tumor cell cytotoxicity mediated by
FolR1 TCBs and human PBMCs (Effector:Target=10:1). Each point
represents the mean value of triplicates. Standard deviation is
indicated by error bars. FIG. 37A: HeLa target cell cytotoxicity.
Comparison of two different formats of the Protease activated TCBs
both containing an anti idiotypic CD3 scFv linked with a MK062
Matriptase linker. FIG. 37B: Skov-3 target cell cytotoxicity.
Comparison of two different formats of the Protease activated TCBs
both containing an anti idiotypic CD3 scFv linked with a MK062
Matriptase linker. FIG. 37C: HeLa target cell cytotoxicity.
Comparison of classic Protease activated TCB containing an anti
idiotypic CD3 scFv and GS linkers with different protease sites.
Protease activated TCB containing the MMP9-Matriptase MK062 linker
(8364, grey squares), FolR1 TCB (light grey triangles pointing
down), protease activated TCB containing only Matriptase MK062
(light grey rhomb)/Cathepsin site (grey circles) or non-cleavable
linker (black triangles pointing down). FIG. 37D: Skov-3 target
cell cytotoxicity. Comparison of classic Protease activated TCB
containing an anti idiotypic CD3 scFv and GS linkers with different
protease sites. Protease activated TCB containing the
MMP9-Matriptase MK062 linker (8364, grey squares), FolR1 TCB (light
grey triangles pointing down), protease activated TCB containing
only Matriptase MK062 (light grey rhomb)/Cathepsin site (grey
circles) or non-cleavable linker (black triangles pointing
down).
[0202] FIGS. 38A and 38B depict quantification of CD69 of CD8
positive cells after co-incubation of primary human renal
epithelial cortical cells (FIG. 38A) or human bronchial epithelial
cells (FIG. 38B) with 200 nM of the different TCBs and three
different donors of human PBMCs. T cells were stained after 48 h of
incubation. (E:T=10:1, effectors are human PBMCs). Median
fluorescence intensity of T cell activation marker CD69 for
CD8.sup.+ T cells is shown. Each point represents the mean value of
triplicates of three different human PBMC donors. Standard
deviation is indicated in error bars. Unpaired t test was used for
statistical analysis.
[0203] FIGS. 39A and 39B depict tumor cell cytotoxicity mediated by
MSLN TCBs and human PBMCs (Effector:Target=10:1). Maximal lysis of
the target cells (=100%) was achieved by incubation of target cells
with 1% Triton X-100 20 h before LDH readout. Minimal lysis (=0%)
refers to target cells co-incubated with effector cells without any
TCB. Each point represents the mean value of triplicates. Standard
deviation is indicated by error bars. FIG. 39A: NCI H596 target
cell cytotoxicity. Protease activated MSLN TCB containing an anti
idiotypic CD3 scFv linked with a MMP9-MK062 Matriptase linker. The
protease activated TCB (8672, light grey circles), the MSLN TCB
(dark grey triangles pointing down) and the protease activated TCB
containing a non-cleavable linker (8673, grey triangles pointing
up) are compared. FIG. 39B: AsPC-1 target cell cytotoxicity.
Protease activated MSLN TCB containing an anti idiotypic CD3 scFv
linked with a MMP9-MK062 Matriptase linker. The protease activated
TCB (8672, light grey circles), the MSLN TCB (dark grey triangles
pointing down) and the protease activated TCB containing a
non-cleavable linker (8673, grey triangles pointing up) are
compared.
[0204] FIG. 40 depicts a Jurkat-NFAT activation assay with primary
tumor samples and Protease activated FolR1 TCBs. Jurkat NFAT
reporter cells are activated after co-incubation with FolR1 TCB
(6298) and Protease activated FolR1 TCB containing MMP9-Matriptase
cleavage site (8364). Protease activated FolR1 TCBs (8363, 8408)
and control TCBs (8409, 7235) do not induce Luciferase expression.
The dotted line indicates the baseline Luminescence for Jurkat NFAT
cells co-incubated with tumor.
[0205] FIGS. 41A-41C: Capillary electrophoresis of protease
activated TCBs after incubation in human serum. Molecules were
incubated for 0 or 14 days in human IgG depleted serum at
37.degree. C. in a humidified incubator (5% CO.sub.2). All
molecules were purified by affinity chromatography (ProteinA) and
then analyzed by Capillary electrophoresis. FIG. 41A: CE-SDS
analysis of serum, FolR1 TCB (6298) in serum at day 0 and day 14.
FIG. 41B: CE-SDS analysis of serum, Protease activated FolR1 TCB
with MMP9-Matriptase linker (8364) in serum at day 0 and day 14.
FIG. 41C: CE-SDS analysis of serum, Protease activated FolR1 TCB
with Matriptase linker (8408) in serum at day 0 and day 14 and the
precleaved molecule in serum.
[0206] FIGS. 42A-42F depict schematics of different T cell
bispecific molecules with masking moieties. FIG. 42A: ID 8955.
Herceptarg TCB, classic format, anti ID CH2527 scFv 4.32.63 MK062
MMP9 linker N-terminally fused to VH. FIG. 42B: ID 8957. Herceptarg
TCB, classic format, anti ID CH2527 scFv 4.32.63 non cleavable
linker N-terminally fused to VH. FIG. 42C: ID 8959. Herceptarg TCB,
classic format. FIG. 42D: ID 8997. FolR1 36F2 TCB, classic format,
anti ID CH2527 scFv 4.32.63 MK062 MMP9 linker N-terminally fused to
VH. FIG. 42E: ID 8998. FolR1 36F2 TCB, classic format, anti ID
CH2527 scFv 4.32.63 non cleavable linker N-terminally fused to VH.
FIG. 42F: ID 8996. FolR1 36F2 TCB, classic format.
[0207] FIG. 43 depicts Human Bronchial Epithelial Cell toxicity
mediated by human PBMCs and 100 nM or 10 nM of TCBs. Maximal lysis
of the target cells (=100%) was achieved by incubation of target
cells with 1% Triton X-100 20 h before LDH readout. Minimal lysis
(=0%) refers to target cells co-incubated with effector cells
without any TCB. Each point represents the mean value of
triplicates. Standard deviation is indicated by error bars.
[0208] FIG. 44 depicts FolR1 negative target cell (Mkn-45)
cytotoxicity mediated by 100 nM of FolR1 TCBs and human PBMCs.
Maximal lysis of the target cells (=100%) was achieved by
incubation of target cells with 1% Triton X-100 20 h before LDH
readout. Minimal lysis (=0%) refers to target cells co-incubated
with effector cells without any TCB. Each point represents the mean
value of triplicates. Standard deviation is indicated by error
bars.
[0209] FIG. 45A to FIG. 45N depict schematic diagrams of exemplary
antibody constructs.
DETAILED DESCRIPTION
Definitions
[0210] Terms are used herein as generally used in the art, unless
otherwise defined in the following. As used herein, the term
"antigen binding molecule" refers in its broadest sense to a
molecule that specifically binds an antigenic determinant. Examples
of antigen binding molecules are immunoglobulins and derivatives,
e.g., fragments, thereof.
[0211] The term "bispecific" means that the antigen binding
molecule is able to specifically bind to at least two distinct
antigenic determinants. Typically, a bispecific antigen binding
molecule comprises two antigen binding sites, each of which is
specific for a different antigenic determinant. In certain
embodiments the bispecific antigen binding molecule is capable of
simultaneously binding two antigenic determinants, particularly two
antigenic determinants expressed on two distinct cells.
[0212] The term "valent" as used herein denotes the presence of a
specified number of antigen binding sites in an antigen binding
molecule. As such, the term "monovalent binding to an antigen"
denotes the presence of one (and not more than one) antigen binding
site specific for the antigen in the antigen binding molecule.
[0213] An "antigen binding site" refers to the site, i.e. one or
more amino acid residues, of an antigen binding molecule which
provides interaction with the antigen. For example, the antigen
binding site of an antibody comprises amino acid residues from the
complementarity determining regions (CDRs). A native immunoglobulin
molecule typically has two antigen binding sites, a Fab molecule
typically has a single antigen binding site.
[0214] As used herein, the term "antigen binding moiety" refers to
a polypeptide molecule that specifically binds to an antigenic
determinant. In one embodiment, an antigen binding moiety is able
to direct the entity to which it is attached (e.g., a second
antigen binding moiety) to a target site, for example to a specific
type of tumor cell or tumor stroma bearing the antigenic
determinant. In another embodiment an antigen binding moiety is
able to activate signaling through its target antigen, for example
a T cell receptor complex antigen. Antigen binding moieties include
antibodies and fragments thereof as further defined herein.
Particular antigen binding moieties include an antigen binding
domain of an antibody, comprising an antibody heavy chain variable
region and an antibody light chain variable region. In certain
embodiments, the antigen binding moieties may comprise antibody
constant regions as further defined herein and known in the art.
Useful heavy chain constant regions include any of the five
isotypes: .alpha., .delta., .epsilon., .gamma., or .mu.. Useful
light chain constant regions include any of the two isotypes:
.kappa. and .lamda..
[0215] As used herein, the term "antigenic determinant" is
synonymous with "antigen" and "epitope," and refers to a site
(e.g., a contiguous stretch of amino acids or a conformational
configuration made up of different regions of non-contiguous amino
acids) on a polypeptide macromolecule to which an antigen binding
moiety binds, forming an antigen binding moiety-antigen complex.
Useful antigenic determinants can be found, for example, on the
surfaces of tumor cells, on the surfaces of virus-infected cells,
on the surfaces of other diseased cells, on the surface of immune
cells, free in blood serum, and/or in the extracellular matrix
(ECM). The proteins referred to as antigens herein (e.g., FolR1,
HER1, HER2, CD3, Mesothelin) can be any native form of the proteins
from any vertebrate source, including mammals such as primates
(e.g., humans) and rodents (e.g., mice and rats), unless otherwise
indicated. In a particular embodiment the antigen is a human
protein. Where reference is made to a specific protein herein, the
term encompasses the "full-length", unprocessed protein as well as
any form of the protein that results from processing in the cell.
The term also encompasses naturally occurring variants of the
protein, e.g., splice variants or allelic variants. Exemplary human
proteins useful as antigens include, but are not limited to: FolR1,
HER1 and CD3, particularly the epsilon subunit of CD3 (see UniProt
no. P07766 (version 130), NCBI RefSeq no. NP_000724.1, SEQ ID NO:
54 for the human sequence; or UniProt no. Q95LI5 (version 49), NCBI
GenBank no. BAB71849.1 for the cynomolgus [Macaca fascicularis]
sequence). In certain embodiments the protease-activatable T cell
activating bispecific molecule of the invention binds to an epitope
of CD3 or a target cell antigen that is conserved among the CD3 or
target antigen from different species. In certain embodiments the
protease-activatable T cell activating bispecific molecule of the
invention binds to CD3 and FolR1, but does not bind to FolR2 or
FolR3. In certain embodiments the protease-activatable T cell
activating bispecific molecule of the invention binds to CD3 and
HER1. In certain embodiments the protease-activatable T cell
activating bispecific molecule of the invention binds to CD3 and
Mesothelin. In certain embodiments the protease-activatable T cell
activating bispecific molecule of the invention binds to CD3 and
HER2. By "specific binding" is meant that the binding is selective
for the antigen and can be discriminated from unwanted or
non-specific interactions. The ability of an antigen binding moiety
to bind to a specific antigenic determinant can be measured either
through an enzyme-linked immunosorbent assay (ELISA) or other
techniques familiar to one of skill in the art, e.g., surface
plasmon resonance (SPR) technique (analyzed on a BIAcore
instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
In one embodiment, the extent of binding of an antigen binding
moiety to an unrelated protein is less than about 10% of the
binding of the antigen binding moiety to the antigen as measured,
e.g., by SPR. In certain embodiments, an antigen binding moiety
that binds to the antigen, or an antigen binding molecule
comprising that antigen binding moiety, has a dissociation constant
(K.sub.D) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM
(e.g., 10.sup.-8M or less, e.g., from 10.sup.-8M to 10.sup.-13M,
e.g., from 10.sup.-9M to 10.sup.-13 M). "Affinity" refers to the
strength of the sum total of non-covalent interactions between a
single binding site of a molecule (e.g., a receptor) and its
binding partner (e.g., a ligand). Unless indicated otherwise, as
used herein, "binding affinity" refers to intrinsic binding
affinity which reflects a 1:1 interaction between members of a
binding pair (e.g., an antigen binding moiety and an antigen, or a
receptor and its ligand). The affinity of a molecule X for its
partner Y can generally be represented by the dissociation constant
(K.sub.D), which is the ratio of dissociation and association rate
constants (k.sub.off and k.sub.on, respectively). Thus, equivalent
affinities may comprise different rate constants, as long as the
ratio of the rate constants remains the same. Affinity can be
measured by well-established methods known in the art, including
those described herein. A particular method for measuring affinity
is Surface Plasmon Resonance (SPR).
[0216] "Reduced binding", for example reduced binding to an Fc
receptor, refers to a decrease in affinity for the respective
interaction, as measured for example by SPR. For clarity the term
includes also reduction of the affinity to zero (or below the
detection limit of the analytic method), i.e. complete abolishment
of the interaction. Conversely, "increased binding" refers to an
increase in binding affinity for the respective interaction.
[0217] "T cell activation" as used herein refers to one or more
cellular response of a T lymphocyte, particularly a cytotoxic T
lymphocyte, selected from: proliferation, differentiation, cytokine
secretion, cytotoxic effector molecule release, cytotoxic activity,
and expression of activation markers. The protease-activatable T
cell activating bispecific molecules of the invention are capable
of inducing T cell activation. Suitable assays to measure T cell
activation are known in the art described herein.
[0218] A "target cell antigen" as used herein refers to an
antigenic determinant presented on the surface of a target cell,
for example a cell in a tumor such as a cancer cell or a cell of
the tumor stroma. As used herein, the terms "first" and "second"
with respect to antigen binding moieties etc., are used for
convenience of distinguishing when there is more than one of each
type of moiety. Use of these terms is not intended to confer a
specific order or orientation of the protease-activatable T cell
activating bispecific molecule unless explicitly so stated.
[0219] A "Fab molecule" refers to a protein consisting of the VH
and CH1 domain of the heavy chain (the "Fab heavy chain") and the
VL and CL domain of the light chain (the "Fab light chain") of an
immunoglobulin.
[0220] By "fused" is meant that the components (e.g., a Fab
molecule and an Fc domain subunit) are linked by peptide bonds,
either directly or via one or more peptide linkers.
[0221] As used herein, the term "single-chain" refers to a molecule
comprising amino acid monomers linearly linked by peptide bonds. In
certain embodiments, one of the antigen binding moieties is a
single-chain Fab molecule, i.e. a Fab molecule wherein the Fab
light chain and the Fab heavy chain are connected by a peptide
linker to form a single peptide chain. In a particular such
embodiment, the C-terminus of the Fab light chain is connected to
the N-terminus of the Fab heavy chain in the single-chain Fab
molecule.
[0222] By a "crossover" Fab molecule (also termed "Crossfab") is
meant a Fab molecule wherein either the variable regions or the
constant regions of the Fab heavy and light chain are exchanged,
i.e. the crossover Fab molecule comprises a peptide chain composed
of the light chain variable region and the heavy chain constant
region, and a peptide chain composed of the heavy chain variable
region and the light chain constant region. For clarity, in a
crossover Fab molecule wherein the variable regions of the Fab
light chain and the Fab heavy chain are exchanged, the peptide
chain comprising the heavy chain constant region is referred to
herein as the "heavy chain" of the crossover Fab molecule.
Conversely, in a crossover Fab molecule wherein the constant
regions of the Fab light chain and the Fab heavy chain are
exchanged, the peptide chain comprising the heavy chain variable
region is referred to herein as the "heavy chain" of the crossover
Fab molecule.
[0223] In contrast thereto, by a "conventional" Fab molecule is
meant a Fab molecule in its natural format, i.e. comprising a heavy
chain composed of the heavy chain variable and constant regions
(VH-CH1), and a light chain composed of the light chain variable
and constant regions (VL-CL).
[0224] The term "immunoglobulin molecule" refers to a protein
having the structure of a naturally occurring antibody. For
example, immunoglobulins of the IgG class are heterotetrameric
glycoproteins of about 150,000 daltons, composed of two light
chains and two heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3), also called
a heavy chain constant region. Similarly, from N- to C-terminus,
each light chain has a variable region (VL), also called a variable
light domain or a light chain variable domain, followed by a
constant light (CL) domain, also called a light chain constant
region. The heavy chain of an immunoglobulin may be assigned to one
of five types, called .alpha. (IgA), .delta. (IgD), .epsilon.
(IgE), .gamma. (IgG), or .mu. (IgM), some of which may be further
divided into subtypes, e.g., .gamma..sub.1 (IgG.sub.1),
.gamma..sub.2 (IgG.sub.2), .gamma..sub.3 (IgG.sub.3), .gamma..sub.4
(IgG.sub.4), .alpha..sub.1 (IgA.sub.1) and .alpha..sub.2
(IgA.sub.2). The light chain of an immunoglobulin may be assigned
to one of two types, called kappa (.kappa.) and lambda (.lamda.),
based on the amino acid sequence of its constant domain. An
immunoglobulin essentially consists of two Fab molecules and an Fc
domain, linked via the immunoglobulin hinge region.
[0225] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, and antibody
fragments so long as they exhibit the desired antigen-binding
activity.
[0226] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2, diabodies, linear antibodies, single-chain
antibody molecules (e.g., scFv), and single-domain antibodies. For
a review of certain antibody fragments, see Hudson et al., Nat Med
9, 129-134 (2003). For a review of scFv fragments, see e.g.,
Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and
5,587,458. For discussion of Fab and F(ab')2 fragments comprising
salvage receptor binding epitope residues and having increased in
vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibody
fragments with two antigen-binding sites that may be bivalent or
bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et
al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl
Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are
also described in Hudson et al., Nat Med 9, 129-134 (2003).
Single-domain antibodies are antibody fragments comprising all or a
portion of the heavy chain variable domain or all or a portion of
the light chain variable domain of an antibody. In certain
embodiments, a single-domain antibody is a human single-domain
antibody (Domantis, Inc., Waltham, Mass.; see e.g., U.S. Pat. No.
6,248,516 B 1). Antibody fragments can be made by various
techniques, including but not limited to proteolytic digestion of
an intact antibody as well as production by recombinant host cells
(e.g., E. coli or phage), as described herein.
[0227] The term "antigen binding domain" refers to the part of an
antibody that comprises the area which specifically binds to and is
complementary to part or all of an antigen. An antigen binding
domain may be provided by, for example, one or more antibody
variable domains (also called antibody variable regions).
Particularly, an antigen binding domain comprises an antibody light
chain variable region (VL) and an antibody heavy chain variable
region (VH).
[0228] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007). A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. The term "hypervariable region" or "HVR", as used
herein, refers to each of the regions of an antibody variable
domain which are hypervariable in sequence and/or form structurally
defined loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
complementarity determining regions (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. With the exception of CDR1 in VH, CDRs generally
comprise the amino acid residues that form the hypervariable loops.
Hypervariable regions (HVRs) are also referred to as
"complementarity determining regions" (CDRs), and these terms are
used herein interchangeably in reference to portions of the
variable region that form the antigen binding regions. This
particular region has been described by Kabat et al., U.S. Dept. of
Health and Human Services, Sequences of Proteins of Immunological
Interest (1983) and by Chothia et al., J Mol Biol 196:901-917
(1987), where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table 1 as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00003 TABLE 1 CDR Definitions.sup.1 CDR Kabat Chothia
AbM.sup.2 V.sub.H CDRI 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-58
50-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDRI 24-34 26-32
24-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97
.sup.1 Numbering of all CDR definitions in Table 1 is according to
the numbering conventions set forth by Kabat et al. (see below).
.sup.2"AbM" with a lowercase "b" as used in Table 1 refers to the
CDRs as defined by Oxford Molecular`s "AbM" antibody modeling
software.
[0229] Kabat et al. also defined a numbering system for variable
region sequences that is applicable to any antibody. One of
ordinary skill in the art can unambiguously assign this system of
"Kabat numbering" to any variable region sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in an antibody variable region are according to
the Kabat numbering system.
[0230] The polypeptide sequences of the sequence listing are not
numbered according to the Kabat numbering system. However, it is
well within the ordinary skill of one in the art to convert the
numbering of the sequences of the Sequence Listing to Kabat
numbering.
[0231] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0232] The "class" of an antibody or immunoglobulin refers to the
type of constant domain or constant region possessed by its heavy
chain. There are five major classes of antibodies: IgA, IgD, IgE,
IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
IgG.sub.4, IgA.sub.1, and IgA.sub.2. The heavy chain constant
domains that correspond to the different classes of immunoglobulins
are called .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively.
[0233] The term "Fc domain" or "Fc region" herein is used to define
a C-terminal region of an immunoglobulin heavy chain that contains
at least a portion of the constant region. The term includes native
sequence Fc regions and variant Fc regions. Although the boundaries
of the Fc region of an IgG heavy chain might vary slightly, the
human IgG heavy chain Fc region is usually defined to extend from
Cys226, or from Pro230, to the carboxyl-terminus of the heavy
chain. However, the C-terminal lysine (Lys447) of the Fc region may
or may not be present. Unless otherwise specified herein, numbering
of amino acid residues in the Fc region or constant region is
according to the EU numbering system, also called the EU index, as
described in Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md., 1991. A "subunit" of an Fc domain as used
herein refers to one of the two polypeptides forming the dimeric Fc
domain, i.e. a polypeptide comprising C-terminal constant regions
of an immunoglobulin heavy chain, capable of stable
self-association. For example, a subunit of an IgG Fc domain
comprises an IgG CH2 and an IgG CH3 constant domain.
[0234] A "modification promoting the association of the first and
the second subunit of the Fc domain" is a manipulation of the
peptide backbone or the post-translational modifications of an Fc
domain subunit that reduces or prevents the association of a
polypeptide comprising the Fc domain subunit with an identical
polypeptide to form a homodimer. A modification promoting
association as used herein particularly includes separate
modifications made to each of the two Fc domain subunits desired to
associate (i.e. the first and the second subunit of the Fc domain),
wherein the modifications are complementary to each other so as to
promote association of the two Fc domain subunits. For example, a
modification promoting association may alter the structure or
charge of one or both of the Fc domain subunits so as to make their
association sterically or electrostatically favorable,
respectively. Thus, (hetero)dimerization occurs between a
polypeptide comprising the first Fc domain subunit and a
polypeptide comprising the second Fc domain subunit, which might be
non-identical in the sense that further components fused to each of
the subunits (e.g., antigen binding moieties) are not the same. In
some embodiments the modification promoting association comprises
an amino acid mutation in the Fc domain, specifically an amino acid
substitution. In a particular embodiment, the modification
promoting association comprises a separate amino acid mutation,
specifically an amino acid substitution, in each of the two
subunits of the Fc domain.
[0235] The term "effector functions" refers to those biological
activities attributable to the Fc region of an antibody, which vary
with the antibody isotype. Examples of antibody effector functions
include: C1q binding and complement dependent cytotoxicity (CDC),
Fc receptor binding, antibody-dependent cell-mediated cytotoxicity
(ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine
secretion, immune complex-mediated antigen uptake by antigen
presenting cells, down regulation of cell surface receptors (e.g.,
B cell receptor), and B cell activation.
[0236] As used herein, the terms "engineer, engineered,
engineering", are considered to include any manipulation of the
peptide backbone or the post-translational modifications of a
naturally occurring or recombinant polypeptide or fragment thereof.
Engineering includes modifications of the amino acid sequence, of
the glycosylation pattern, or of the side chain group of individual
amino acids, as well as combinations of these approaches.
[0237] The term "amino acid mutation" as used herein is meant to
encompass amino acid substitutions, deletions, insertions, and
modifications. Any combination of substitution, deletion,
insertion, and modification can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., reduced binding to an Fc receptor, or
increased association with another peptide. Amino acid sequence
deletions and insertions include amino- and/or carboxy-terminal
deletions and insertions of amino acids. Particular amino acid
mutations are amino acid substitutions. For the purpose of altering
e.g., the binding characteristics of an Fc region, non-conservative
amino acid substitutions, i.e. replacing one amino acid with
another amino acid having different structural and/or chemical
properties, are particularly preferred. Amino acid substitutions
include replacement by non-naturally occurring amino acids or by
naturally occurring amino acid derivatives of the twenty standard
amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine,
homoserine, 5-hydroxylysine). Amino acid mutations can be generated
using genetic or chemical methods well known in the art. Genetic
methods may include site-directed mutagenesis, PCR, gene synthesis
and the like. It is contemplated that methods of altering the side
chain group of an amino acid by methods other than genetic
engineering, such as chemical modification, may also be useful.
Various designations may be used herein to indicate the same amino
acid mutation. For example, a substitution from proline at position
329 of the Fc domain to glycine can be indicated as 329G, G329,
G.sub.329, P329G, or Pro329Gly.
[0238] As used herein, term "polypeptide" refers to a molecule
composed of monomers (amino acids) linearly linked by amide bonds
(also known as peptide bonds). The term "polypeptide" refers to any
chain of two or more amino acids, and does not refer to a specific
length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein," "amino acid chain," or any other term
used to refer to a chain of two or more amino acids, are included
within the definition of "polypeptide," and the term "polypeptide"
may be used instead of, or interchangeably with any of these terms.
The term "polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology, but is not necessarily
translated from a designated nucleic acid sequence. It may be
generated in any manner, including by chemical synthesis. A
polypeptide of the invention may be of a size of about 3 or more, 5
or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or
more, 100 or more, 200 or more, 500 or more, 1,000 or more, or
2,000 or more amino acids. Polypeptides may have a defined
three-dimensional structure, although they do not necessarily have
such structure. Polypeptides with a defined three-dimensional
structure are referred to as folded, and polypeptides which do not
possess a defined three-dimensional structure, but rather can adopt
a large number of different conformations, and are referred to as
unfolded.
[0239] By an "isolated" polypeptide or a variant, or derivative
thereof is intended a polypeptide that is not in its natural
milieu. No particular level of purification is required. For
example, an isolated polypeptide can be removed from its native or
natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for the
purpose of the invention, as are native or recombinant polypeptides
which have been separated, fractionated, or partially or
substantially purified by any suitable technique.
[0240] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary. In situations where ALIGN-2 is employed
for amino acid sequence comparisons, the % amino acid sequence
identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which can alternatively be phrased as
a given amino acid sequence A that has or comprises a certain %
amino acid sequence identity to, with, or against a given amino
acid sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0241] The term "polynucleotide" refers to an isolated nucleic acid
molecule or construct, e.g., messenger RNA (mRNA), virally-derived
RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.,
an amide bond, such as found in peptide nucleic acids (PNA). The
term "nucleic acid molecule" refers to any one or more nucleic acid
segments, e.g., DNA or RNA fragments, present in a
polynucleotide.
[0242] By "isolated" nucleic acid molecule or polynucleotide is
intended a nucleic acid molecule, DNA or RNA, which has been
removed from its native environment. For example, a recombinant
polynucleotide encoding a polypeptide contained in a vector is
considered isolated for the purposes of the present invention.
Further examples of an isolated polynucleotide include recombinant
polynucleotides maintained in heterologous host cells or purified
(partially or substantially) polynucleotides in solution. An
isolated polynucleotide includes a polynucleotide molecule
contained in cells that ordinarily contain the polynucleotide
molecule, but the polynucleotide molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the present
invention, as well as positive and negative strand forms, and
double-stranded forms. Isolated polynucleotides or nucleic acids
according to the present invention further include such molecules
produced synthetically. In addition, a polynucleotide or a nucleic
acid may be or may include a regulatory element such as a promoter,
ribosome binding site, or a transcription terminator. By a nucleic
acid or polynucleotide having a nucleotide sequence at least, for
example, 95% "identical" to a reference nucleotide sequence of the
present invention, it is intended that the nucleotide sequence of
the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to five point
mutations per each 100 nucleotides of the reference nucleotide
sequence. In other words, to obtain a polynucleotide having a
nucleotide sequence at least 95% identical to a reference
nucleotide sequence, up to 5% of the nucleotides in the reference
sequence may be deleted or substituted with another nucleotide, or
a number of nucleotides up to 5% of the total nucleotides in the
reference sequence may be inserted into the reference sequence.
These alterations of the reference sequence may occur at the 5' or
3' terminal positions of the reference nucleotide sequence or
anywhere between those terminal positions, interspersed either
individually among residues in the reference sequence or in one or
more contiguous groups within the reference sequence. As a
practical matter, whether any particular polynucleotide sequence is
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
nucleotide sequence of the present invention can be determined
conventionally using known computer programs, such as the ones
discussed above for polypeptides (e.g., ALIGN-2).
[0243] The term "expression cassette" refers to a polynucleotide
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a target cell. The recombinant
expression cassette can be incorporated into a plasmid, chromosome,
mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
Typically, the recombinant expression cassette portion of an
expression vector includes, among other sequences, a nucleic acid
sequence to be transcribed and a promoter. In certain embodiments,
the expression cassette of the invention comprises polynucleotide
sequences that encode bispecific antigen binding molecules of the
invention or fragments thereof.
[0244] The term "vector" or "expression vector" is synonymous with
"expression construct" and refers to a DNA molecule that is used to
introduce and direct the expression of a specific gene to which it
is operably associated in a target cell. The term includes the
vector as a self-replicating nucleic acid structure as well as the
vector incorporated into the genome of a host cell into which it
has been introduced. The expression vector of the present invention
comprises an expression cassette. Expression vectors allow
transcription of large amounts of stable mRNA. Once the expression
vector is inside the target cell, the ribonucleic acid molecule or
protein that is encoded by the gene is produced by the cellular
transcription and/or translation machinery. In one embodiment, the
expression vector of the invention comprises an expression cassette
that comprises polynucleotide sequences that encode bispecific
antigen binding molecules of the invention or fragments
thereof.
[0245] The terms "host cell", "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein. A host cell is any
type of cellular system that can be used to generate the bispecific
antigen binding molecules of the present invention. Host cells
include cultured cells, e.g., mammalian cultured cells, such as CHO
cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63
mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells,
yeast cells, insect cells, and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue.
[0246] An "activating Fc receptor" is an Fc receptor that following
engagement by an Fc domain of an antibody elicits signaling events
that stimulate the receptor-bearing cell to perform effector
functions. Human activating Fc receptors include Fc.gamma.RIIIa
(CD16a), Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI
(CD89).
[0247] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an
immune mechanism leading to the lysis of antibody-coated target
cells by immune effector cells. The target cells are cells to which
antibodies or derivatives thereof comprising an Fc region
specifically bind, generally via the protein part that is
N-terminal to the Fc region. As used herein, the term "reduced
ADCC" is defined as either a reduction in the number of target
cells that are lysed in a given time, at a given concentration of
antibody in the medium surrounding the target cells, by the
mechanism of ADCC defined above, and/or an increase in the
concentration of antibody in the medium surrounding the target
cells, required to achieve the lysis of a given number of target
cells in a given time, by the mechanism of ADCC. The reduction in
ADCC is relative to the ADCC mediated by the same antibody produced
by the same type of host cells, using the same standard production,
purification, formulation and storage methods (which are known to
those skilled in the art), but that has not been engineered. For
example the reduction in ADCC mediated by an antibody comprising in
its Fc domain an amino acid substitution that reduces ADCC, is
relative to the ADCC mediated by the same antibody without this
amino acid substitution in the Fc domain. Suitable assays to
measure ADCC are well known in the art (see e.g., PCT publication
no. WO 2006/082515 or PCT publication no. WO 2012/130831).
[0248] An "effective amount" of an agent refers to the amount that
is necessary to result in a physiological change in the cell or
tissue to which it is administered.
[0249] A "therapeutically effective amount" of an agent, e.g., a
pharmaceutical composition, refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic or prophylactic result. A therapeutically effective
amount of an agent for example eliminates, decreases, delays,
minimizes or prevents adverse effects of a disease.
[0250] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). Particularly, the individual or subject is a human.
[0251] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0252] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical composition, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0253] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of a disease
in the individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments,
protease-activatable T cell activating bispecific molecules of the
invention are used to delay development of a disease or to slow the
progression of a disease.
[0254] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0255] An "idiotype-specific polypeptide" as used herein refers to
a polypeptide that recognizes the idiotype of an antigen-binding
moiety, e.g., an antigen-binding moiety specific for CD3. The
idiotype-specific polypeptide is capable of specifically binding to
the variable region of the antigen-binding moiety and thereby
reducing or preventing specific binding of the antigen-binding
moiety to its cognate antigen. When associated with a molecule that
comprises the antigen-binding moiety, the idiotype-specific
polypeptide can function as a masking moiety of the molecule.
Specifically disclosed herein are anti-idiotype antibodies or
anti-idiotype-binding antibody fragments specific for the idiotype
of anti-CD3 binding molecules.
[0256] "Protease" or "proteolytic enzyme" as used herein refers to
any proteolytic enzyme that cleaves the linker at a recognition
site and that is expressed by a target cell. Such proteases might
be secreted by the target cell or remain associated with the target
cell, e.g., on the target cell surface. Examples of proteases
include but are not limited to metalloproteinases, e.g., matrix
metalloproteinase 1-28 and A Disintegrin And Metalloproteinase
(ADAM) 2, 7-12, 15, 17-23, 28-30 and 33, serine proteases, e.g.,
urokinase-type plasminogen activator and Matriptase, cysteine
protease, aspartic proteases, and members of the cathepsin
family.
[0257] "Protease activatable" as used herein, with respect to the T
cell activating bispecific molecule, refers to a T cell activating
bispecific molecule having reduced or abrogated ability to activate
T cells due to a masking moiety that reduces or abrogates the T
cell activating bispecific molecule's ability to bind to CD3. Upon
dissociation of the masking moiety by proteolytic cleavage, e.g.,
by proteolytic cleavage of a linker connecting the masking moiety
to the T cell activating bispecific molecule, binding to CD3 is
restored and the T cell activating bispecific molecule is thereby
activated.
[0258] "Reversibly concealing" as used herein refers to the binding
of a masking moiety or idiotype-specific polypeptide to an
antigen-binding moiety or molecule such as to prevent the
antigen-binding moiety or molecule from its antigen, e.g., CD3.
This concealing is reversible in that the idiotype-specific
polypeptide can be released from the antigen-binding moiety or
molecule, e.g., by protease cleavage, and thereby freeing the
antigen-binding moiety or molecule to bind to its antigen.
DETAILED DESCRIPTION
[0259] In one aspect, the invention relates to a
protease-activatable T cell activating bispecific molecule
comprising [0260] (a) a first antigen binding moiety capable of
specific binding to CD3; [0261] (b) a second antigen binding moiety
capable of specific binding to a target cell antigen; and [0262]
(c) a masking moiety covalently attached to the T cell bispecific
binding molecule through a protease-cleavable linker, wherein the
masking moiety is capable of specific binding to the idiotype of
the first or the second antigen binding moiety thereby reversibly
concealing the first or second antigen binding moiety.
[0263] The first antigen binding moiety capable of specific binding
to CD3 comprises an idiotype. In one embodiment, the masking moiety
of the protease-activatable T cell activating bispecific molecule
is covalently attached to the first antigen binding moiety. In one
embodiment the masking moiety is covalently attached to the heavy
chain variable region of the first antigen binding moiety. In one
embodiment the masking moiety is covalently attached to the light
chain variable region of the first antigen binding moiety. This
covalent bond is separate from the specific binding, which is
preferably non-covalent, of the masking moiety to the idiotype
first antigen binding site. The idiotype of the first antigen
binding moiety comprises its variable region. In one embodiment the
masking moiety binds to amino acid residues that make contact with
CD3 when the first antigen biding moiety is bound to CD3. In a
preferred embodiment, the masking moiety is not the cognate antigen
or fragments thereof of the first antigen binding moiety, i.e., the
masking moiety is not a CD3 or fragments thereof. In one embodiment
the masking moiety is an anti-idiotypic antibody or fragment
thereof. In one embodiment, the masking moiety is an anti-idiotypic
scFv. Exemplary embodiments of masking moieties which are
anti-idiotypic scFv, and protease activatable T cell activating
molecules comprising such masking moieties, are described in detail
in the examples. In one embodiment the protease-activatable T cell
activating bispecific molecule comprises a second masking moiety
reversibly concealing the second antigen binding moiety.
[0264] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, and which comprises at least one heavy
chain complementarity determining region (CDR) selected from the
group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46
and at least one light chain CDR selected from the group of SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; (ii) a second antigen binding
moiety which is a Fab molecule capable of specific binding to a
target cell antigen.
[0265] In one embodiment the first antigen binding moiety comprises
a heavy chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
an amino acid sequence of SEQ ID NO: 43 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence
of SEQ ID NO: 55.
[0266] In one embodiment the first antigen binding moiety comprises
the heavy chain variable region comprising an amino acid sequence
of SEQ ID NO: 43 and the light chain variable region comprising an
amino acid sequence of SEQ ID NO: 55.
[0267] In a specific embodiment the second antigen binding moiety
is capable of specific binding to FolR1 and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:
16 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0268] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to FolR1 and comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 47 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 55.
[0269] In a specific embodiment the second antigen binding moiety
is capable of specific binding to FolR1 and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID
NO: 153 and at least one light chain CDR selected from the group of
SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO: 156.
[0270] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to FolR1 and comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 157 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 158.
[0271] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to HER1 and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0272] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to HER1 and comprises a heavy
chain comprising an amino acid sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of
SEQ ID NO: 32, and a light chain comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
an amino acid sequence of SEQ ID NO: 33.
[0273] In another specific embodiment, the second antigen binding
moiety is capable of specific binding to HER1 and comprises a heavy
chain variable region comprising an amino acid sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino
acid sequence of SEQ ID NO: 115 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 116.
[0274] In a specific embodiment the second antigen binding moiety
is capable of specific binding to Mesothelin and comprises at least
one heavy chain complementarity determining region (CDR) selected
from the group consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ
ID NO: 109 and at least one light chain CDR selected from the group
of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ ID NO: 112.
[0275] In another specific embodiment, the second antigen binding
moiety is capable of specific binding Mesothelin and comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 113 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 114.
[0276] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at
least one light chain CDR selected from the group of SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to FolR1
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 14,
SEQ ID NO: 15 and SEQ ID NO: 16 and at least one light chain CDR
selected from the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID
NO: 19.
[0277] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, (ii) a
second antigen binding moiety which is a Fab molecule capable of
specific binding to FolR1 comprising heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 47 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55.
[0278] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at
least one light chain CDR selected from the group of SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to FolR1
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 151,
SEQ ID NO: 152 and SEQ ID NO: 153 and at least one light chain CDR
selected from the group of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ
ID NO: 156.
[0279] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, (ii) a
second antigen binding moiety which is a Fab molecule capable of
specific binding to FolR1 comprising heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 157 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 158.
[0280] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at
least one light chain CDR selected from the group of SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to HER1
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 56,
SEQ ID NO: 57 and SEQ ID NO: 58 and at least one light chain CDR
selected from the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID
NO: 61.
[0281] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55. (ii) a
second antigen binding moiety which is a Fab molecule capable of
specific binding to HER1 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 115 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 116.
[0282] In a particular embodiment, the first antigen binding moiety
is a crossover Fab molecule wherein either the variable or the
constant regions of the Fab light chain and the Fab heavy chain are
exchanged.
[0283] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at
least one light chain CDR selected from the group of SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19; (ii) a second antigen binding moiety
which is a Fab molecule capable of specific binding to Mesothelin
comprising at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 107,
SEQ ID NO: 108 and SEQ ID NO: 109 and at least one light chain CDR
selected from the group of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ
ID NO: 112.
[0284] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, (ii) a
second antigen binding moiety which is a Fab molecule capable of
specific binding to Mesothelin comprising heavy chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 113 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 114.
[0285] In one embodiment, the second antigen binding moiety is a
conventional Fab molecule.
[0286] In a particular embodiment, the first antigen binding moiety
is a crossover Fab molecule wherein the constant regions of the Fab
light chain and the Fab heavy chain are exchanged, and the second
antigen binding moiety is a conventional Fab molecule. In a further
particular embodiment, the first and the second antigen binding
moiety are fused to each other, optionally through a peptide
linker.
[0287] In particular embodiments, the protease-activatable T cell
activating bispecific molecule further comprises an Fc domain
composed of a first and a second subunit capable of stable
association. In a further particular embodiment, not more than one
antigen binding moiety capable of specific binding to CD3 is
present in the protease-activatable T cell activating bispecific
molecule (i.e. the protease-activatable T cell activating
bispecific molecule provides monovalent binding to CD3).
Protease-Activatable T Cell Activating Bispecific Molecule
Formats
[0288] The components of the protease-activatable T cell activating
bispecific molecule can be fused to each other in a variety of
configurations. Exemplary configurations are depicted in FIGS.
1A-1E and 5A-5H. Further exemplary configurations are depicted in
FIGS. 33A-33K.
[0289] In particular embodiments, the protease-activatable T cell
activating bispecific molecule comprises an Fc domain composed of a
first and a second subunit capable of stable association. In some
embodiments, the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the first or
the second subunit of the Fc domain.
[0290] In one such embodiment, the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the Fab heavy chain of the second antigen binding moiety. In a
specific such embodiment, the protease-activatable T cell
activating bispecific molecule essentially consists of a first and
a second antigen binding moiety, an Fc domain composed of a first
and a second subunit, and optionally one or more peptide linkers,
wherein the first antigen binding moiety is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the Fab heavy chain of
the second antigen binding moiety, and the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the first or the second subunit of the Fc domain.
Optionally, the Fab light chain of the first antigen binding moiety
and the Fab light chain of the second antigen binding moiety may
additionally be fused to each other.
[0291] In another such embodiment, the first antigen binding moiety
is fused at the C-terminus of the Fab heavy chain to the N-terminus
of the first or second subunit of the Fc domain. In a specific such
embodiment, the protease-activatable T cell activating bispecific
molecule essentially consists of a first and a second antigen
binding moiety, an Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
first and the second antigen binding moiety are each fused at the
C-terminus of the Fab heavy chain to the N-terminus of one of the
subunits of the Fc domain.
[0292] In other embodiments, the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the first or second subunit of the Fc domain.
[0293] In a particular such embodiment, the second antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the first antigen binding
moiety. In a specific such embodiment, the protease-activatable T
cell activating bispecific molecule essentially consists of a first
and a second antigen binding moiety, an Fc domain composed of a
first and a second subunit, and optionally one or more peptide
linkers, wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the first or the second subunit of the
Fc domain. Optionally, the Fab light chain of the first antigen
binding moiety and the Fab light chain of the second antigen
binding moiety may additionally be fused to each other.
[0294] The antigen binding moieties may be fused to the Fc domain
or to each other directly or through a peptide linker, comprising
one or more amino acids, typically about 2-20 amino acids. Peptide
linkers are known in the art and are described herein. Suitable,
non-immunogenic peptide linkers include, for example,
(G.sub.4S).sub.n, (SG.sub.4).sub.n, (G.sub.4S).sub.n or
G.sub.4(SG.sub.4).sub.n peptide linkers. "n" is generally a number
between 1 and 10, typically between 2 and 4. A particularly
suitable peptide linker for fusing the Fab light chains of the
first and the second antigen binding moiety to each other is
(G.sub.4S).sub.2. An exemplary peptide linker suitable for
connecting the Fab heavy chains of the first and the second antigen
binding moiety is EPKSC(D)-(G.sub.4S).sub.2 (SEQ ID NOs 105 and
106). Additionally, linkers may comprise (a portion of) an
immunoglobulin hinge region. Particularly where an antigen binding
moiety is fused to the N-terminus of an Fc domain subunit, it may
be fused via an immunoglobulin hinge region or a portion thereof,
with or without an additional peptide linker.
[0295] A protease-activatable T cell activating bispecific molecule
with a single antigen binding moiety capable of specific binding to
a target cell antigen is useful, particularly in cases where
internalization of the target cell antigen is to be expected
following binding of a high affinity antigen binding moiety. In
such cases, the presence of more than one antigen binding moiety
specific for the target cell antigen may enhance internalization of
the target cell antigen, thereby reducing its availability.
[0296] In many other cases, however, it will be advantageous to
have a protease-activatable T cell activating bispecific molecule
comprising two or more antigen binding moieties specific for a
target cell antigen (see examples in shown in FIGS. 5A-5H), for
example to optimize targeting to the target site or to allow
crosslinking of target cell antigens.
[0297] Accordingly, in certain embodiments, the
protease-activatable T cell activating bispecific molecule of the
invention further comprises a third antigen binding moiety which is
a Fab molecule capable of specific binding to a target cell
antigen. In one embodiment, the third antigen binding moiety is a
conventional Fab molecule. In one embodiment, the third antigen
binding moiety is capable of specific binding to the same target
cell antigen as the second antigen binding moiety. In a particular
embodiment, the first antigen binding moiety is capable of specific
binding to CD3, and the second and third antigen binding moieties
are capable of specific binding to a target cell antigen. In a
particular embodiment, the second and the third antigen binding
moiety are identical (i.e. they comprise the same amino acid
sequences).
[0298] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to FolR1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0299] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the second and third
antigen binding moieties are capable of specific binding to FolR1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0300] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the second and third
antigen binding moieties are capable of specific binding to FolR1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0301] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 43 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 55, and the second and third antigen binding moieties are
capable of specific binding to FolR1, wherein the second and third
antigen binding moieties comprise a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 47 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55.
[0302] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to HER1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0303] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the second and third
antigen binding moieties are capable of specific binding to HER1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.
[0304] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; and the second and
third antigen binding moieties are capable of specific binding to
HER1, wherein the second and third antigen binding moieties
comprise at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 56,
SEQ ID NO: 57 and SEQ ID NO: 58 and at least one light chain CDR
selected from the group of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID
NO: 61.
[0305] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 43 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 55, and the second and third antigen binding moieties are
capable of specific binding to HER1, wherein the second and third
antigen binding moieties comprise a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 115 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 116.
[0306] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to HER2,
wherein the second antigen binding moiety comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID
NO: 144 and at least one light chain CDR selected from the group of
SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150, and wherein the
third antigen binding moiety comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and
at least one light chain CDR selected from the group of SEQ ID NO:
148, SEQ ID NO: 149 and SEQ ID NO: 150.
[0307] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the second and third
antigen binding moieties are capable of specific binding to HER2,
wherein the second antigen binding moiety comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 142, SEQ ID NO: 143 and SEQ ID
NO: 144 and at least one light chain CDR selected from the group of
SEQ ID NO: 148, SEQ ID NO: 149 and SEQ ID NO: 150, and wherein the
third antigen binding moiety comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and
at least one light chain CDR selected from the group of SEQ ID NO:
148, SEQ ID NO: 149 and SEQ ID NO: 150.
[0308] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 43 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 55, and the second and third antigen binding moieties are
capable of specific binding to HER2, wherein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 160
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 161, wherein the third
antigen binding moiety comprises a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 159 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 161.
[0309] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to
Mesothelin, wherein the second and third antigen binding moieties
comprise at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 107,
SEQ ID NO: 108 and SEQ ID NO: 109 and at least one light chain CDR
selected from the group of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ
ID NO: 112.
[0310] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group of SEQ
ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; and the second and third
antigen binding moieties are capable of specific binding to
Mesothelin, wherein the second and third antigen binding moieties
comprise at least one heavy chain complementarity determining
region (CDR) selected from the group consisting of SEQ ID NO: 107,
SEQ ID NO: 108 and SEQ ID NO: 109 and at least one light chain CDR
selected from the group of SEQ ID NO: 110, SEQ ID NO: 111 and SEQ
ID NO: 112.
[0311] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises a heavy chain
variable region comprising an amino acid sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 43, and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 55, and the second and third antigen binding moieties are
capable of specific binding to Mesothelin, wherein the second and
third antigen binding moieties comprise a heavy chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 113 and a light chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 114.
[0312] In one embodiment, the first antigen binding moiety is
capable of specific binding to CD3, and the second and third
antigen binding moieties are capable of specific binding to HER1,
wherein the second and third antigen binding moieties comprise at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group consisting of SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO:
61.
[0313] In a particular embodiment, the first antigen binding moiety
is capable of specific binding to CD3, and comprises at least one
heavy chain complementarity determining region (CDR) selected from
the group consisting of SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO:
46 and at least one light chain CDR selected from the group
consisting of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19; and
the second and third antigen binding moieties are capable of
specific binding to HER1, wherein the second and third antigen
binding moieties comprise at least one heavy chain complementarity
determining region (CDR) selected from the group consisting of SEQ
ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 58 and at least one light
chain CDR selected from the group consisting of SEQ ID NO: 59, SEQ
ID NO: 60 and SEQ ID NO: 61.
[0314] In one embodiment, the third antigen binding moiety is fused
at the C-terminus of the Fab heavy chain to the N-terminus of the
first or second subunit of the Fc domain. In a more specific
embodiment, the second and the third antigen binding moiety are
each fused at the C-terminus of the Fab heavy chain to the
N-terminus of one of the subunits of the Fc domain, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the Fab heavy chain of the second
antigen binding moiety. Optionally, the Fab light chain of the
first antigen binding moiety and the Fab light chain of the second
antigen binding moiety may additionally be fused to each other.
[0315] The second and the third antigen binding moiety may be fused
to the Fc domain directly or through a peptide linker. In a
particular embodiment the second and the third antigen binding
moiety are each fused to the Fc domain through an immunoglobulin
hinge region. In a specific embodiment, the immunoglobulin hinge
region is a human IgG.sub.1 hinge region. In one embodiment the
second and the third antigen binding moiety and the Fc domain are
part of an immunoglobulin molecule. In a particular embodiment the
immunoglobulin molecule is an IgG class immunoglobulin. In an even
more particular embodiment the immunoglobulin is an IgG.sub.1
subclass immunoglobulin. In another embodiment the immunoglobulin
is an IgG.sub.4 subclass immunoglobulin. In a further particular
embodiment the immunoglobulin is a human immunoglobulin. In other
embodiments the immunoglobulin is a chimeric immunoglobulin or a
humanized immunoglobulin. In one embodiment, the
protease-activatable T cell activating bispecific molecule
essentially consists of an immunoglobulin molecule capable of
specific binding to a target cell antigen, and an antigen binding
moiety capable of specific binding to CD3 wherein the antigen
binding moiety is a Fab molecule, particularly a crossover Fab
molecule, fused to the N-terminus of one of the immunoglobulin
heavy chains, optionally via a peptide linker.
[0316] In a particular embodiment, the first and the third antigen
binding moiety are each fused at the C-terminus of the Fab heavy
chain to the N-terminus of one of the subunits of the Fc domain,
and the second antigen binding moiety is fused at the C-terminus of
the Fab heavy chain to the N-terminus of the Fab heavy chain of the
first antigen binding moiety. In a specific such embodiment, the
protease-activatable T cell activating bispecific molecule
essentially consists of a first, a second and a third antigen
binding moiety, an Fc domain composed of a first and a second
subunit, and optionally one or more peptide linkers, wherein the
second antigen binding moiety is fused at the C-terminus of the Fab
heavy chain to the N-terminus of the Fab heavy chain of the first
antigen binding moiety, and the first antigen binding moiety is
fused at the C-terminus of the Fab heavy chain to the N-terminus of
the first subunit of the Fc domain, and wherein the third antigen
binding moiety is fused at the C-terminus of the Fab heavy chain to
the N-terminus of the second subunit of the Fc domain. Optionally,
the Fab light chain of the first antigen binding moiety and the Fab
light chain of the second antigen binding moiety may additionally
be fused to each other.
[0317] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 44, the
heavy chain CDR 2 of SEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID
NO: 46, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR
2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to FolR1 comprising the heavy chain CDR 1 of SEQ ID NO: 14,
the heavy chain CDR 2 of SEQ ID NO: 15, the heavy chain CDR 3 of
SEQ ID NO: 16, the light chain CDR 1 of SEQ ID NO: 17, the light
chain CDR 2 of SEQ ID NO: 18 and the light chain CDR3 of SEQ ID NO:
19.
[0318] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to FolR1 comprising heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 47 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 55.
[0319] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 44, the
heavy chain CDR 2 of SEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID
NO: 46, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR
2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to FolR1 comprising the heavy chain CDR 1 of SEQ ID NO:
151, the heavy chain CDR 2 of SEQ ID NO: 152, the heavy chain CDR 3
of SEQ ID NO: 153, the light chain CDR 1 of SEQ ID NO: 154, the
light chain CDR 2 of SEQ ID NO: 155 and the light chain CDR3 of SEQ
ID NO: 156.
[0320] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to FolR1 comprising heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 157 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 158.
[0321] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 44, the
heavy chain CDR 2 of SEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID
NO: 46, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR
2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to HER1 comprising the heavy chain CDR 1 of SEQ ID NO: 56,
the heavy chain CDR 2 of SEQ ID NO: 57, the heavy chain CDR 3 of
SEQ ID NO: 58, the light chain CDR 1 of SEQ ID NO: 59, the light
chain CDR 2 of SEQ ID NO: 60 and the light chain CDR3 of SEQ ID NO:
61.
[0322] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to HER1 comprising a heavy chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to the amino acid sequence of SEQ ID NO: 115 and
a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 116.
[0323] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 44, the
heavy chain CDR 2 of SEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID
NO: 46, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR
2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to HER2, wherein the second antigen binding moiety
comprises the heavy chain CDR 1 of SEQ ID NO: 142, the heavy chain
CDR 2 of SEQ ID NO: 143, the heavy chain CDR 3 of SEQ ID NO: 144,
the light chain CDR 1 of SEQ ID NO: 148, the light chain CDR 2 of
SEQ ID NO: 149 and the light chain CDR3 of SEQ ID NO: 150, and
wherein the third antigen binding moiety comprises the heavy chain
CDR 1 of SEQ ID NO: 145, the heavy chain CDR 2 of SEQ ID NO: 146,
the heavy chain CDR 3 of SEQ ID NO: 148, the light chain CDR 1 of
SEQ ID NO: 148, the light chain CDR 2 of SEQ ID NO: 149 and the
light chain CDR3 of SEQ ID NO: 150.
[0324] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to HER2, wherein the second antigen binding moiety comprises a
heavy chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 160 and a light chain variable
region comprising an amino acid sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid
sequence of SEQ ID NO: 161, and wherein the third antigen binding
moiety comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 159 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 161.
[0325] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3, comprising the heavy chain
complementarity determining region (CDR) 1 of SEQ ID NO: 44, the
heavy chain CDR 2 of SEQ ID NO: 45, the heavy chain CDR 3 of SEQ ID
NO: 46, the light chain CDR 1 of SEQ ID NO: 17, the light chain CDR
2 of SEQ ID NO: 18 and the light chain CDR 3 of SEQ ID NO: 19,
wherein the first antigen binding moiety is a crossover Fab
molecule wherein either the variable or the constant regions,
particularly the constant regions, of the Fab light chain and the
Fab heavy chain are exchanged; (ii) a second and a third antigen
binding moiety each of which is a Fab molecule capable of specific
binding to Mesothelin comprising the heavy chain CDR 1 of SEQ ID
NO: 107, the heavy chain CDR 2 of SEQ ID NO: 108, the heavy chain
CDR 3 of SEQ ID NO: 109, the light chain CDR 1 of SEQ ID NO: 110,
the light chain CDR 2 of SEQ ID NO: 111 and the light chain CDR3 of
SEQ ID NO: 112.
[0326] In one embodiment the present invention provides a
protease-activatable T cell activating bispecific molecule
comprising
(i) a first antigen binding moiety which is a Fab molecule capable
of specific binding to CD3 comprising a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 43 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 55, wherein
the first antigen binding moiety is a crossover Fab molecule
wherein either the variable or the constant regions, particularly
the constant regions, of the Fab light chain and the Fab heavy
chain are exchanged; (ii) a second and a third antigen binding
moiety each of which is a Fab molecule capable of specific binding
to Mesothelin comprising heavy chain variable region comprising an
amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to the amino acid sequence of SEQ ID NO: 113 and
a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 114. The protease-activatable
T cell activating bispecific molecule according to any of the ten
above embodiments may further comprise (iii) an Fc domain composed
of a first and a second subunit capable of stable association,
wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety, and the first
antigen binding moiety is fused at the C-terminus of the Fab heavy
chain to the N-terminus of the first subunit of the Fc domain, and
wherein the third antigen binding moiety is fused at the C-terminus
of the Fab heavy chain to the N-terminus of the second subunit of
the Fc domain. In some of the protease-activatable T cell
activating bispecific molecule of the invention, the Fab light
chain of the first antigen binding moiety and the Fab light chain
of the second antigen binding moiety are fused to each other,
optionally via a linker peptide. Depending on the configuration of
the first and the second antigen binding moiety, the Fab light
chain of the first antigen binding moiety may be fused at its
C-terminus to the N-terminus of the Fab light chain of the second
antigen binding moiety, or the Fab light chain of the second
antigen binding moiety may be fused at its C-terminus to the
N-terminus of the Fab light chain of the first antigen binding
moiety. Fusion of the Fab light chains of the first and the second
antigen binding moiety further reduces mispairing of unmatched Fab
heavy and light chains, and also reduces the number of plasmids
needed for expression of some of the protease-activatable T cell
activating bispecific molecule of the invention. In certain
embodiments the protease-activatable T cell activating bispecific
molecule comprises a polypeptide wherein the Fab light chain
variable region of the first antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the first antigen binding moiety (i.e. a the first
antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain variable region is replaced by a light
chain variable region), which in turn shares a carboxy-terminal
peptide bond with an Fc domain subunit
(VL.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)), and a polypeptide wherein a
the Fab heavy chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In some embodiments the
protease-activatable T cell activating bispecific molecule further
comprises a polypeptide wherein the Fab heavy chain variable region
of the first antigen binding moiety shares a carboxy-terminal
peptide bond with the Fab light chain constant region of the first
antigen binding moiety (VH.sub.(1)-CL.sub.(1)) and the Fab light
chain polypeptide of the second antigen binding moiety
(VL.sub.(2)-CL.sub.(2)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0327] In alternative embodiments the protease-activatable T cell
activating bispecific molecule comprises a polypeptide wherein the
Fab heavy chain variable region of the first antigen binding moiety
shares a carboxy-terminal peptide bond with the Fab light chain
constant region of the first antigen binding moiety (i.e. the first
antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain constant region is replaced by a light
chain constant region), which in turn shares a carboxy-terminal
peptide bond with an Fc domain subunit
(VH.sub.(1)-CL.sub.(1)-CH2-CH3(-CH4)), and a polypeptide wherein
the Fab heavy chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In some embodiments the
protease-activatable T cell activating bispecific molecule further
comprises a polypeptide wherein the Fab light chain variable region
of the first antigen binding moiety shares a carboxy-terminal
peptide bond with the Fab heavy chain constant region of the first
antigen binding moiety (VL.sub.(1)-CH1.sub.(1)) and the Fab light
chain polypeptide of the second antigen binding moiety
(VL.sub.(2)-CL.sub.(2)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0328] In some embodiments, the protease-activatable T cell
activating bispecific molecule comprises a polypeptide wherein the
Fab light chain variable region of the first antigen binding moiety
shares a carboxy-terminal peptide bond with the Fab heavy chain
constant region of the first antigen binding moiety (i.e. the first
antigen binding moiety comprises a crossover Fab heavy chain,
wherein the heavy chain variable region is replaced by a light
chain variable region), which in turn shares a carboxy-terminal
peptide bond with the Fab heavy chain of the second antigen binding
moiety, which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit
(VL.sub.(1)-CH1.sub.(1)-VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In
other embodiments, the protease-activatable T cell activating
bispecific molecule comprises a polypeptide wherein the Fab heavy
chain variable region of the first antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the first antigen binding moiety (i.e. the first antigen
binding moiety comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
the Fab heavy chain of the second antigen binding moiety, which in
turn shares a carboxy-terminal peptide bond with an Fc domain
subunit
(VH.sub.(1)-CL.sub.(1)-VH.sub.(2)-CH1.sub.(2)-CH2-CH3(-CH4)). In
still other embodiments, the protease-activatable T cell activating
bispecific molecule comprises a polypeptide wherein the Fab heavy
chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab light chain variable
region of the first antigen binding moiety which in turn shares a
carboxy-terminal peptide bond with the Fab heavy chain constant
region of the first antigen binding moiety (i.e. the first antigen
binding moiety comprises a crossover Fab heavy chain, wherein the
heavy chain variable region is replaced by a light chain variable
region), which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-VL.sub.(1)-CH1.sub.(1)-CH2-CH3(-CH4)). In
other embodiments, the protease-activatable T cell activating
bispecific molecule comprises a polypeptide wherein the Fab heavy
chain of the second antigen binding moiety shares a
carboxy-terminal peptide bond with the Fab heavy chain variable
region of the first antigen binding moiety which in turn shares a
carboxy-terminal peptide bond with the Fab light chain constant
region of the first antigen binding moiety (i.e. the first antigen
binding moiety comprises a crossover Fab heavy chain, wherein the
heavy chain constant region is replaced by a light chain constant
region), which in turn shares a carboxy-terminal peptide bond with
an Fc domain subunit
(VH.sub.(2)-CH1.sub.(2)-VH.sub.(1)-CL.sub.(1)-CH2-CH3(-CH4)).
[0329] In some of these embodiments the protease-activatable T cell
activating bispecific molecule further comprises a crossover Fab
light chain polypeptide of the first antigen binding moiety,
wherein the Fab heavy chain variable region of the first antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
(VH.sub.(1)-CL.sub.(1)), and the Fab light chain polypeptide of the
second antigen binding moiety (VL.sub.(2)-CL.sub.(2)). In others of
these embodiments the protease-activatable T cell activating
bispecific molecule further comprises a crossover Fab light chain
polypeptide, wherein the Fab light chain variable region of the
first antigen binding moiety shares a carboxy-terminal peptide bond
with the Fab heavy chain constant region of the first antigen
binding moiety (VL.sub.(1)-CH1.sub.(1)), and the Fab light chain
polypeptide of the second antigen binding moiety
(VL.sub.(2)-CL.sub.(2)). In still others of these embodiments the
protease-activatable T cell activating bispecific molecule further
comprises a polypeptide wherein the Fab light chain variable region
of the first antigen binding moiety shares a carboxy-terminal
peptide bond with the Fab heavy chain constant region of the first
antigen binding moiety which in turn shares a carboxy-terminal
peptide bond with the Fab light chain polypeptide of the second
antigen binding moiety
(VL.sub.(1)-CH1.sub.(1)-VL.sub.(2)-CL.sub.(2)), a polypeptide
wherein the Fab heavy chain variable region of the first antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain polypeptide of the second antigen binding moiety
(VH.sub.(1)-CL.sub.(1)-VL.sub.(2)-CL.sub.(2)), a polypeptide
wherein the Fab light chain polypeptide of the second antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
light chain variable region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
heavy chain constant region of the first antigen binding moiety
(VL.sub.(2)-CL.sub.(2)-VL.sub.(1)-CH1.sub.(1)), or a polypeptide
wherein the Fab light chain polypeptide of the second antigen
binding moiety shares a carboxy-terminal peptide bond with the Fab
heavy chain variable region of the first antigen binding moiety
which in turn shares a carboxy-terminal peptide bond with the Fab
light chain constant region of the first antigen binding moiety
(VL.sub.(2)-CL.sub.(2)-VH.sub.(1)-CL.sub.(1)). The
protease-activatable T cell activating bispecific molecule
according to these embodiments may further comprise (i) an Fc
domain subunit polypeptide (CH2-CH3(-CH4)), or (ii) a polypeptide
wherein the Fab heavy chain of a third antigen binding moiety
shares a carboxy-terminal peptide bond with an Fc domain subunit
(VH.sub.(3)-CH1.sub.(3)-CH2-CH3(-CH4)) and the Fab light chain
polypeptide of a third antigen binding moiety
(VL.sub.(3)-CL.sub.(3)). In certain embodiments the polypeptides
are covalently linked, e.g., by a disulfide bond.
[0330] According to any of the above embodiments, components of the
protease-activatable T cell activating bispecific molecule (e.g.,
antigen binding moiety, Fc domain) may be fused directly or through
various linkers, particularly peptide linkers comprising one or
more amino acids, typically about 2-20 amino acids, that are
described herein or are known in the art. Suitable, non-immunogenic
peptide linkers include, for example, (G.sub.4S).sub.n,
(SG.sub.4).sub.n, (G.sub.4S).sub.n or G.sub.4(SG.sub.4).sub.n
peptide linkers, wherein n is generally a number between 1 and 10,
typically between 2 and 4.
Fc Domain
[0331] The Fc domain of the protease-activatable T cell activating
bispecific molecule consists of a pair of polypeptide chains
comprising heavy chain domains of an immunoglobulin molecule. For
example, the Fc domain of an immunoglobulin G (IgG) molecule is a
dimer, each subunit of which comprises the CH2 and CH3 IgG heavy
chain constant domains. The two subunits of the Fc domain are
capable of stable association with each other. In one embodiment
the protease-activatable T cell activating bispecific molecule of
the invention comprises not more than one Fc domain.
[0332] In one embodiment according the invention the Fc domain of
the protease-activatable T cell activating bispecific molecule is
an IgG Fc domain. In a particular embodiment the Fc domain is an
IgG.sub.1 Fc domain. In another embodiment the Fc domain is an
IgG.sub.4 Fc domain. In a more specific embodiment, the Fc domain
is an IgG.sub.4 Fc domain comprising an amino acid substitution at
position S228 (Kabat numbering), particularly the amino acid
substitution S228P. This amino acid substitution reduces in vivo
Fab arm exchange of IgG.sub.4 antibodies (see Stubenrauch et al.,
Drug Metabolism and Disposition 38, 84-91 (2010)). In a further
particular embodiment the Fc domain is human.
Fc Domain Modifications Promoting Heterodimerization
[0333] Protease-activatable T cell activating bispecific molecules
according to the invention comprise different antigen binding
moieties, fused to one or the other of the two subunits of the Fc
domain, thus the two subunits of the Fc domain are typically
comprised in two non-identical polypeptide chains. Recombinant
co-expression of these polypeptides and subsequent dimerization
leads to several possible combinations of the two polypeptides. To
improve the yield and purity of protease-activatable T cell
activating bispecific molecules in recombinant production, it will
thus be advantageous to introduce in the Fc domain of the
protease-activatable T cell activating bispecific molecule a
modification promoting the association of the desired
polypeptides.
[0334] Accordingly, in particular embodiments the Fc domain of the
protease-activatable T cell activating bispecific molecule
according to the invention comprises a modification promoting the
association of the first and the second subunit of the Fc domain.
The site of most extensive protein-protein interaction between the
two subunits of a human IgG Fc domain is in the CH3 domain of the
Fc domain. Thus, in one embodiment said modification is in the CH3
domain of the Fc domain.
[0335] In a specific embodiment said modification is a so-called
"knob-into-hole" modification, comprising a "knob" modification in
one of the two subunits of the Fc domain and a "hole" modification
in the other one of the two subunits of the Fc domain.
[0336] The knob-into-hole technology is described e.g., in U.S.
Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621
(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the
method involves introducing a protuberance ("knob") at the
interface of a first polypeptide and a corresponding cavity
("hole") in the interface of a second polypeptide, such that the
protuberance can be positioned in the cavity so as to promote
heterodimer formation and hinder homodimer formation. Protuberances
are constructed by replacing small amino acid side chains from the
interface of the first polypeptide with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino acid side chains
with smaller ones (e.g., alanine or threonine). Accordingly, in a
particular embodiment, in the CH3 domain of the first subunit of
the Fc domain of the protease-activatable T cell activating
bispecific molecule an amino acid residue is replaced with an amino
acid residue having a larger side chain volume, thereby generating
a protuberance within the CH3 domain of the first subunit which is
positionable in a cavity within the CH3 domain of the second
subunit, and in the CH3 domain of the second subunit of the Fc
domain an amino acid residue is replaced with an amino acid residue
having a smaller side chain volume, thereby generating a cavity
within the CH3 domain of the second subunit within which the
protuberance within the CH3 domain of the first subunit is
positionable.
[0337] The protuberance and cavity can be made by altering the
nucleic acid encoding the polypeptides, e.g., by site-specific
mutagenesis, or by peptide synthesis.
[0338] In a specific embodiment, in the CH3 domain of the first
subunit of the Fc domain the threonine residue at position 366 is
replaced with a tryptophan residue (T366W), and in the CH3 domain
of the second subunit of the Fc domain the tyrosine residue at
position 407 is replaced with a valine residue (Y407V). In one
embodiment, in the second subunit of the Fc domain additionally the
threonine residue at position 366 is replaced with a serine residue
(T366S) and the leucine residue at position 368 is replaced with an
alanine residue (L368A).
[0339] In yet a further embodiment, in the first subunit of the Fc
domain additionally the serine residue at position 354 is replaced
with a cysteine residue (S354C), and in the second subunit of the
Fc domain additionally the tyrosine residue at position 349 is
replaced by a cysteine residue (Y349C). Introduction of these two
cysteine residues results in formation of a disulfide bridge
between the two subunits of the Fc domain, further stabilizing the
dimer (Carter, J Immunol Methods 248, 7-15 (2001)).
[0340] In a particular embodiment the antigen binding moiety
capable of binding to CD3 is fused (optionally via the antigen
binding moiety capable of binding to a target cell antigen) to the
first subunit of the Fc domain (comprising the "knob"
modification). Without wishing to be bound by theory, fusion of the
antigen binding moiety capable of binding to CD3 to the
knob-containing subunit of the Fc domain will (further) minimize
the generation of antigen binding molecules comprising two antigen
binding moieties capable of binding to CD3 (steric clash of two
knob-containing polypeptides).
[0341] In an alternative embodiment a modification promoting
association of the first and the second subunit of the Fc domain
comprises a modification mediating electrostatic steering effects,
e.g., as described in PCT publication WO 2009/089004. Generally,
this method involves replacement of one or more amino acid residues
at the interface of the two Fc domain subunits by charged amino
acid residues so that homodimer formation becomes electrostatically
unfavorable but heterodimerization electrostatically favorable.
Fc Domain Modifications Reducing Fc Receptor Binding and/or
Effector Function
[0342] The Fc domain confers to the protease-activatable T cell
activating bispecific molecule favorable pharmacokinetic
properties, including a long serum half-life which contributes to
good accumulation in the target tissue and a favorable tissue-blood
distribution ratio. At the same time it may, however, lead to
undesirable targeting of the protease-activatable T cell activating
bispecific molecule to cells expressing Fc receptors rather than to
the preferred antigen-bearing cells. Moreover, the co-activation of
Fc receptor signaling pathways may lead to cytokine release which,
in combination with the T cell activating properties and the long
half-life of the antigen binding molecule, results in excessive
activation of cytokine receptors and severe side effects upon
systemic administration. Activation of (Fc receptor-bearing) immune
cells other than T cells may even reduce efficacy of the
protease-activatable T cell activating bispecific molecule due to
the potential destruction of T cells e.g., by NK cells.
[0343] Accordingly, in particular embodiments the Fc domain of the
protease-activatable T cell activating bispecific molecules
according to the invention exhibits reduced binding affinity to an
Fc receptor and/or reduced effector function, as compared to a
native IgG.sub.1 Fc domain. In one such embodiment the Fc domain
(or the protease-activatable T cell activating bispecific molecule
comprising said Fc domain) exhibits less than 50%, preferably less
than 20%, more preferably less than 10% and most preferably less
than 5% of the binding affinity to an Fc receptor, as compared to a
native IgG.sub.1 Fc domain (or a protease-activatable T cell
activating bispecific molecule comprising a native IgG.sub.1 Fc
domain), and/or less than 50%, preferably less than 20%, more
preferably less than 10% and most preferably less than 5% of the
effector function, as compared to a native IgG.sub.1 Fc domain
domain (or a protease-activatable T cell activating bispecific
molecule comprising a native IgG.sub.1 Fc domain). In one
embodiment, the Fc domain domain (or the protease-activatable T
cell activating bispecific molecule comprising said Fc domain) does
not substantially bind to an Fc receptor and/or induce effector
function. In a particular embodiment the Fc receptor is an
Fc.gamma. receptor. In one embodiment the Fc receptor is a human Fc
receptor. In one embodiment the Fc receptor is an activating Fc
receptor. In a specific embodiment the Fc receptor is an activating
human Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. In one embodiment the effector function is one or
more selected from the group of CDC, ADCC, ADCP, and cytokine
secretion. In a particular embodiment the effector function is
ADCC. In one embodiment the Fc domain domain exhibits substantially
similar binding affinity to neonatal Fc receptor (FcRn), as
compared to a native IgG.sub.1 Fc domain domain. Substantially
similar binding to FcRn is achieved when the Fc domain (or the
protease-activatable T cell activating bispecific molecule
comprising said Fc domain) exhibits greater than about 70%,
particularly greater than about 80%, more particularly greater than
about 90% of the binding affinity of a native IgG.sub.1 Fc domain
(or the protease-activatable T cell activating bispecific molecule
comprising a native IgG.sub.1 Fc domain) to FcRn.
[0344] In certain embodiments the Fc domain is engineered to have
reduced binding affinity to an Fc receptor and/or reduced effector
function, as compared to a non-engineered Fc domain. In particular
embodiments, the Fc domain of the protease-activatable T cell
activating bispecific molecule comprises one or more amino acid
mutation that reduces the binding affinity of the Fc domain to an
Fc receptor and/or effector function. Typically, the same one or
more amino acid mutation is present in each of the two subunits of
the Fc domain. In one embodiment the amino acid mutation reduces
the binding affinity of the Fc domain to an Fc receptor. In one
embodiment the amino acid mutation reduces the binding affinity of
the Fc domain to an Fc receptor by at least 2-fold, at least
5-fold, or at least 10-fold. In embodiments where there is more
than one amino acid mutation that reduces the binding affinity of
the Fc domain to the Fc receptor, the combination of these amino
acid mutations may reduce the binding affinity of the Fc domain to
an Fc receptor by at least 10-fold, at least 20-fold, or even at
least 50-fold. In one embodiment the protease-activatable T cell
activating bispecific molecule comprising an engineered Fc domain
exhibits less than 20%, particularly less than 10%, more
particularly less than 5% of the binding affinity to an Fc receptor
as compared to a protease-activatable T cell activating bispecific
molecule comprising a non-engineered Fc domain. In a particular
embodiment the Fc receptor is an Fc.gamma. receptor. In some
embodiments the Fc receptor is a human Fc receptor. In some
embodiments the Fc receptor is an activating Fc receptor. In a
specific embodiment the Fc receptor is an activating human
Fc.gamma. receptor, more specifically human Fc.gamma.RIIIa,
Fc.gamma.RI or Fc.gamma.RIIa, most specifically human
Fc.gamma.RIIIa. Preferably, binding to each of these receptors is
reduced. In some embodiments binding affinity to a complement
component, specifically binding affinity to C1q, is also reduced.
In one embodiment binding affinity to neonatal Fc receptor (FcRn)
is not reduced. Substantially similar binding to FcRn, i.e.
preservation of the binding affinity of the Fc domain to said
receptor, is achieved when the Fc domain (or the
protease-activatable T cell activating bispecific molecule
comprising said Fc domain) exhibits greater than about 70% of the
binding affinity of a non-engineered form of the Fc domain (or the
protease-activatable T cell activating bispecific molecule
comprising said non-engineered form of the Fc domain) to FcRn. The
Fc domain, or protease-activatable T cell activating bispecific
molecules of the invention comprising said Fc domain, may exhibit
greater than about 80% and even greater than about 90% of such
affinity. In certain embodiments the Fc domain of the
protease-activatable T cell activating bispecific molecule is
engineered to have reduced effector function, as compared to a
non-engineered Fc domain. The reduced effector function can
include, but is not limited to, one or more of the following:
reduced complement dependent cytotoxicity (CDC), reduced
antibody-dependent cell-mediated cytotoxicity (ADCC), reduced
antibody-dependent cellular phagocytosis (ADCP), reduced cytokine
secretion, reduced immune complex-mediated antigen uptake by
antigen-presenting cells, reduced binding to NK cells, reduced
binding to macrophages, reduced binding to monocytes, reduced
binding to polymorphonuclear cells, reduced direct signaling
inducing apoptosis, reduced crosslinking of target-bound
antibodies, reduced dendritic cell maturation, or reduced T cell
priming. In one embodiment the reduced effector function is one or
more selected from the group of reduced CDC, reduced ADCC, reduced
ADCP, and reduced cytokine secretion. In a particular embodiment
the reduced effector function is reduced ADCC. In one embodiment
the reduced ADCC is less than 20% of the ADCC induced by a
non-engineered Fc domain (or a protease-activatable T cell
activating bispecific molecule comprising a non-engineered Fc
domain). In one embodiment the amino acid mutation that reduces the
binding affinity of the Fc domain to an Fc receptor and/or effector
function is an amino acid substitution. In one embodiment the Fc
domain comprises an amino acid substitution at a position selected
from the group of E233, L234, L235, N297, P331 and P329. In a more
specific embodiment the Fc domain comprises an amino acid
substitution at a position selected from the group of L234, L235
and P329. In some embodiments the Fc domain comprises the amino
acid substitutions L234A and L235A. In one such embodiment, the Fc
domain is an IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc
domain. In one embodiment the Fc domain comprises an amino acid
substitution at position P329. In a more specific embodiment the
amino acid substitution is P329A or P329G, particularly P329G. In
one embodiment the Fc domain comprises an amino acid substitution
at position P329 and a further amino acid substitution at a
position selected from E233, L234, L235, N297 and P331. In a more
specific embodiment the further amino acid substitution is E233P,
L234A, L235A, L235E, N297A, N297D or P331S. In particular
embodiments the Fc domain comprises amino acid substitutions at
positions P329, L234 and L235. In more particular embodiments the
Fc domain comprises the amino acid mutations L234A, L235A and P329G
("P329G LALA"). In one such embodiment, the Fc domain is an
IgG.sub.1 Fc domain, particularly a human IgG.sub.1 Fc domain. The
"P329G LALA" combination of amino acid substitutions almost
completely abolishes Fc.gamma. receptor (as well as complement)
binding of a human IgG.sub.1 Fc domain, as described in PCT
publication no. WO 2012/130831, incorporated herein by reference in
its entirety. WO 2012/130831 also describes methods of preparing
such mutant Fc domains and methods for determining its properties
such as Fc receptor binding or effector functions. IgG.sub.4
antibodies exhibit reduced binding affinity to Fc receptors and
reduced effector functions as compared to IgG.sub.1 antibodies.
Hence, in some embodiments the Fc domain of the
protease-activatable T cell activating bispecific molecules of the
invention is an IgG.sub.4 Fc domain, particularly a human IgG.sub.4
Fc domain. In one embodiment the IgG.sub.4 Fc domain comprises
amino acid substitutions at position 5228, specifically the amino
acid substitution S228P. To further reduce its binding affinity to
an Fc receptor and/or its effector function, in one embodiment the
IgG.sub.4 Fc domain comprises an amino acid substitution at
position L235, specifically the amino acid substitution L235E. In
another embodiment, the IgG.sub.4 Fc domain comprises an amino acid
substitution at position P329, specifically the amino acid
substitution P329G. In a particular embodiment, the IgG.sub.4 Fc
domain comprises amino acid substitutions at positions 5228, L235
and P329, specifically amino acid substitutions S228P, L235E and
P329G. Such IgG.sub.4 Fc domain mutants and their Fc.gamma.
receptor binding properties are described in PCT publication no. WO
2012/130831, incorporated herein by reference in its entirety.
[0345] In a particular embodiment the Fc domain exhibiting reduced
binding affinity to an Fc receptor and/or reduced effector
function, as compared to a native IgG.sub.1 Fc domain, is a human
IgG.sub.1 Fc domain comprising the amino acid substitutions L234A,
L235A and optionally P329G, or a human IgG.sub.4 Fc domain
comprising the amino acid substitutions S228P, L235E and optionally
P329G.
[0346] In certain embodiments N-glycosylation of the Fc domain has
been eliminated. In one such embodiment the Fc domain comprises an
amino acid mutation at position N297, particularly an amino acid
substitution replacing asparagine by alanine (N297A) or aspartic
acid (N297D).
[0347] In addition to the Fc domains described hereinabove and in
PCT publication no. WO 2012/130831, Fc domains with reduced Fc
receptor binding and/or effector function also include those with
substitution of one or more of Fc domain residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0348] Mutant Fc domains can be prepared by amino acid deletion,
substitution, insertion or modification using genetic or chemical
methods well known in the art. Genetic methods may include
site-specific mutagenesis of the encoding DNA sequence, PCR, gene
synthesis, and the like. The correct nucleotide changes can be
verified for example by sequencing.
[0349] Binding to Fc receptors can be easily determined e.g., by
ELISA, or by Surface Plasmon Resonance (SPR) using standard
instrumentation such as a BIAcore instrument (GE Healthcare), and
Fc receptors such as may be obtained by recombinant expression. A
suitable such binding assay is described herein. Alternatively,
binding affinity of Fc domains or cell activating bispecific
antigen binding molecules comprising an Fc domain for Fc receptors
may be evaluated using cell lines known to express particular Fc
receptors, such as human NK cells expressing Fc.gamma.IIIa
receptor.
[0350] Effector function of an Fc domain, or a protease-activatable
T cell activating bispecific molecule comprising an Fc domain, can
be measured by methods known in the art. A suitable assay for
measuring ADCC is described herein. Other examples of in vitro
assays to assess ADCC activity of a molecule of interest are
described in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl
Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl
Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;
Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,
non-radioactive assays methods may be employed (see, for example,
ACTI.TM. non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.)).
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656
(1998).
[0351] In some embodiments, binding of the Fc domain to a
complement component, specifically to C1q, is reduced. Accordingly,
in some embodiments wherein the Fc domain is engineered to have
reduced effector function, said reduced effector function includes
reduced CDC. C1q binding assays may be carried out to determine
whether the protease-activatable T cell activating bispecific
molecule is able to bind Clq and hence has CDC activity. See e.g.,
C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J Immunol Methods 202, 163
(1996); Cragg et al., Blood 101, 1045-1052 (2003); and Cragg and
Glennie, Blood 103, 2738-2743 (2004)).
Antigen Binding Moieties
[0352] The antigen binding molecule of the invention is bispecific,
i.e. it comprises at least two antigen binding moieties capable of
specific binding to two distinct antigenic determinants. According
to the invention, the antigen binding moieties are Fab molecules
(i.e. antigen binding domains composed of a heavy and a light
chain, each comprising a variable and a constant region). In one
embodiment said Fab molecules are human. In another embodiment said
Fab molecules are humanized. In yet another embodiment said Fab
molecules comprise human heavy and light chain constant
regions.
[0353] At least one of the antigen binding moieties is a crossover
Fab molecule. Such modification prevent mispairing of heavy and
light chains from different Fab molecules, thereby improving the
yield and purity of the protease-activatable T cell activating
bispecific molecule of the invention in recombinant production. In
a particular crossover Fab molecule useful for the
protease-activatable T cell activating bispecific molecule of the
invention, the constant regions of the Fab light chain and the Fab
heavy chain are exchanged. In another crossover Fab molecule useful
for the protease-activatable T cell activating bispecific molecule
of the invention, the variable regions of the Fab light chain and
the Fab heavy chain are exchanged.
[0354] In a particular embodiment according to the invention, the
protease-activatable T cell activating bispecific molecule is
capable of simultaneous binding to a target cell antigen,
particularly a tumor cell antigen, and CD3. In one embodiment, the
protease-activatable T cell activating bispecific molecule is
capable of crosslinking a T cell and a target cell by simultaneous
binding to a target cell antigen and CD3. In an even more
particular embodiment, such simultaneous binding results in lysis
of the target cell, particularly a tumor cell. In one embodiment,
such simultaneous binding results in activation of the T cell. In
other embodiments, such simultaneous binding results in a cellular
response of a T lymphocyte, particularly a cytotoxic T lymphocyte,
selected from the group of: proliferation, differentiation,
cytokine secretion, cytotoxic effector molecule release, cytotoxic
activity, and expression of activation markers. In one embodiment,
binding of the protease-activatable T cell activating bispecific
molecule to CD3 without simultaneous binding to the target cell
antigen does not result in T cell activation.
[0355] In one embodiment, the protease-activatable T cell
activating bispecific molecule is capable of re-directing cytotoxic
activity of a T cell to a target cell. In a particular embodiment,
said re-direction is independent of MHC-mediated peptide antigen
presentation by the target cell and and/or specificity of the T
cell.
[0356] Particularly, a T cell according to any of the embodiments
of the invention is a cytotoxic T cell. In some embodiments the T
cell is a CD4.sup.+ or a CD8.sup.+ T cell, particularly a CD8.sup.+
T cell.
CD3 Binding Moiety
[0357] The protease-activatable T cell activating bispecific
molecule of the invention comprises at least one antigen binding
moiety capable of binding to CD3 (also referred to herein as an
"CD3 antigen binding moiety" or "first antigen binding moiety"). In
a particular embodiment, the protease-activatable T cell activating
bispecific molecule comprises not more than one antigen binding
moiety capable of specific binding to CD3. In one embodiment the
protease-activatable T cell activating bispecific molecule provides
monovalent binding to CD3. The CD3 antigen binding is a crossover
Fab molecule, i.e. a Fab molecule wherein either the variable or
the constant regions of the Fab heavy and light chains are
exchanged. In embodiments where there is more than one antigen
binding moiety capable of specific binding to a target cell antigen
comprised in the protease-activatable T cell activating bispecific
molecule, the antigen binding moiety capable of specific binding to
CD3 preferably is a crossover Fab molecule and the antigen binding
moieties capable of specific binding to a target cell antigen are
conventional Fab molecules.
[0358] In a particular embodiment CD3 is human CD3 or cynomolgus
CD3, most particularly human CD3. In a particular embodiment the
CD3 antigen binding moiety is cross-reactive for (i.e. specifically
binds to) human and cynomolgus CD3. In some embodiments, the first
antigen binding moiety is capable of specific binding to the
epsilon subunit of CD3.
[0359] The CD3 antigen binding moiety comprises at least one heavy
chain complementarity determining region (CDR) selected from the
group consisting of SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13
and at least one light chain CDR selected from the group of SEQ ID
NO: 17, SEQ ID NO: 18, SEQ ID NO: 19.
[0360] In one embodiment the CD3 antigen binding moiety comprises
the heavy chain CDR1 of SEQ ID NO: 11, the heavy chain CDR2 of SEQ
ID NO: 12, the heavy chain CDR3 of SEQ ID NO: 13, the light chain
CDR1 of SEQ ID NO: 17, the light chain CDR2 of SEQ ID NO: 18, and
the light chain CDR3 of SEQ ID NO: 19.
[0361] In one embodiment the CD3 antigen binding moiety comprises
the heavy chain CDR1 of SEQ ID NO: 44, the heavy chain CDR2 of SEQ
ID NO: 45, the heavy chain CDR3 of SEQ ID NO: 46, the light chain
CDR1 of SEQ ID NO: 17, the light chain CDR2 of SEQ ID NO: 18, and
the light chain CDR3 of SEQ ID NO: 19.
[0362] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region sequence that is at least about 95%,
96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of
SEQ ID NO: 43, and a light chain variable region sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 55.
[0363] In one embodiment the CD3 antigen binding moiety comprises a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 43 and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 55.
[0364] In one embodiment the CD3 antigen binding moiety comprises
the heavy chain variable region sequence of SEQ ID NO: 43 and the
light chain variable region sequence of SEQ ID NO: 55.
Target Cell Antigen Binding Moiety
[0365] The protease-activatable T cell activating bispecific
molecule of the invention comprises at least one antigen binding
moiety capable of binding to a target cell antigen (also referred
to herein as an "target cell antigen binding moiety" or "second" or
"third" antigen binding moiety). In certain embodiments, the
protease-activatable T cell activating bispecific molecule
comprises two antigen binding moieties capable of binding to a
target cell antigen. In a particular such embodiment, each of these
antigen binding moieties specifically binds to the same antigenic
determinant. In an even more particular embodiment, all of these
antigen binding moieties are identical. In one embodiment, the
protease-activatable T cell activating bispecific molecule
comprises an immunoglobulin molecule capable of specific binding to
a target cell antigen. In one embodiment the protease-activatable T
cell activating bispecific molecule comprises not more than two
antigen binding moieties capable of binding to a target cell
antigen.
[0366] In a preferred embodiment, the target cell antigen binding
moiety is a Fab molecule, particularly a conventional Fab molecule
that binds to a specific antigenic determinant and is able to
direct the Protease-activatable T cell activating bispecific
molecule to a target site, for example to a specific type of tumor
cell that bears the antigenic determinant.
[0367] In certain embodiments the target cell antigen binding
moiety specifically binds to a cell surface antigen. In a
particular embodiment the target cell antigen binding moiety
specifically binds to a Folate Receptor 1 (FolR1) on the surface of
a target cell. In another specific such embodiment the target cell
antigen binding moiety specifically binds to an epidermal growth
factor receptor (EGFR), specifically, a human EGFR, e.g., HER1. In
another specific such embodiment the target cell antigen binding
moiety specifically binds to HER2. In another specific such
embodiment the target cell antigen binding moiety specifically
binds to Mesothelin, specifically, to human Mesothelin.
[0368] In certain embodiments the target cell antigen binding
moiety is directed to an antigen associated with a pathological
condition, such as an antigen presented on a tumor cell or on a
virus-infected cell. Suitable antigens are cell surface antigens,
for example, but not limited to, cell surface receptors. In
particular embodiments the antigen is a human antigen. In a
specific embodiment the target cell antigen is selected from Folate
Receptor 1 (FolR1) and epidermal growth factor receptor (EGFR),
specifically, a human EGFR, e.g., HER1. In a further specific
embodiment the target cell antigen is HER2. In a further specific
embodiment the target cell antigen is Mesothelin.
[0369] In some embodiments the protease-activatable T cell
activating bispecific molecule comprises at least one antigen
binding moiety that is specific for HER1. In one embodiment, the
antigen binding moiety that is specific for HER1 comprises at least
one heavy chain complementarity determining region (CDR) of
selected from the group consisting of SEQ ID NO: 56, SEQ ID NO: 57
and SEQ ID NO: 58 and at least one light chain CDR selected from
the group of SEQ ID NO: 59, SEQ ID NO: 60, and SEQ ID NO: 61.
[0370] In one embodiment, the antigen binding moiety that is
specific for HER1 comprises the heavy chain CDR1 of SEQ ID NO: 56,
the heavy chain CDR2 of SEQ ID NO: 57, the heavy chain CDR3 of SEQ
ID NO: 58, the light chain CDR1 of SEQ ID NO: 59, the light chain
CDR2 of SEQ ID NO: 60, and the light chain CDR3 of SEQ ID NO:
61.
[0371] In one embodiment, the antigen binding moiety that is
specific for HER1 comprises the heavy chain and light chain CDR
sequences of an anti-HER1 antibody disclosed in PCT Application
Publication Number WO2006/082515.
[0372] In one embodiment the protease-activatable T cell activating
bispecific molecule comprising at least one antigen binding moiety
that is specific for HER1 comprises at least one of a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 32, a polypeptide sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33, a
polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to SEQ ID NO: 34. In one embodiment the
protease-activatable T cell activating bispecific molecule
comprising at least one antigen binding moiety that is specific for
HER1 comprises the polypeptide sequence of SEQ ID NO: 32, the
polypeptide sequence of SEQ ID NO: 33, and the polypeptide sequence
of SEQ ID NO: 34.
[0373] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER1 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the
heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID
NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain
CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain
CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the
light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID
NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
[0374] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER1 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the
heavy chain CDR3 of SEQ ID NO: 28, the light chain CDR1 of SEQ ID
NO: 29, the light chain CDR2 of SEQ ID NO: 30, and the light chain
CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain
CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the
light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID
NO: 30, and the light chain CDR3 of SEQ ID NO: 31.
[0375] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER1 further comprises an
anti-idiotypic CD3 scFv comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 41 or 42. In one embodiment, the anti-idiotypic scFv comprises
the polypeptide sequence of SEQ ID NO: 41 or 42.
[0376] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER1 further comprises an
anti-idiotypic HER1 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 48, the heavy chain CDR2 of SEQ ID NO: 49, the
heavy chain CDR3 of SEQ ID NO: 50, the light chain CDR1 of SEQ ID
NO: 51, the light chain CDR2 of SEQ ID NO: 52, and the light chain
CDR3 of SEQ ID NO: 53. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 48, the heavy chain
CDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the
light chain CDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID
NO: 52, and the light chain CDR3 of SEQ ID NO: 53.
[0377] In one embodiments the protease-activatable T cell
activating bispecific molecule that comprises at least one antigen
binding moiety that is specific for HER1 further comprises a linker
comprising a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 35.
[0378] In one embodiment the protease-activatable T cell activating
bispecific molecule comprising at least one antigen binding moiety
that is specific for HER1 further comprises a linker having a
protease recognition site comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or
106. In one embodiment, the protease recognition site comprises the
polypeptide sequence of SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,
100, 101, 102, 103, 104, 105 or 106. In one embodiment, the
protease recognition site comprises the polypeptide sequence of SEQ
ID NO: 36. In one embodiment, the protease recognition site
comprises the polypeptide sequence of SEQ ID NO: 97. In one
embodiments the protease-activatable T cell activating bispecific
molecule comprising at least one antigen binding moiety that is
specific for HER1 further comprises a linker comprising a
polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95 or 96. In one embodiment, the linker comprises
the polypeptide sequence of SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95 or 96. In one embodiment, the linker
comprises the polypeptide sequence of SEQ ID NO: 7. In one
embodiment, the linker comprises the polypeptide sequence of SEQ ID
NO: 86.
[0379] In some embodiments the protease-activatable T cell
activating bispecific molecule comprises at least one antigen
binding moiety that is specific for HER2. In one embodiment, the
antigen binding moiety that is specific for HER2 comprises at least
one heavy chain complementarity determining region (CDR) of
selected from the group consisting of SEQ ID NO: 142, SEQ ID NO:
143 and SEQ ID NO: 144 and at least one light chain CDR selected
from the group of SEQ ID NO: 148, SEQ ID NO: 149, and SEQ ID NO:
150. In a further one embodiment, the antigen binding moiety that
is specific for HER2 comprises at least one heavy chain
complementarity determining region (CDR) of selected from the group
consisting of SEQ ID NO: 145, SEQ ID NO: 146 and SEQ ID NO: 147 and
at least one light chain CDR selected from the group of SEQ ID NO:
148, SEQ ID NO: 149, and SEQ ID NO: 150.
[0380] In one embodiment, the antigen binding moiety that is
specific for HER2 comprises the heavy chain CDR1 of SEQ ID NO: 142,
the heavy chain CDR2 of SEQ ID NO: 143, the heavy chain CDR3 of SEQ
ID NO: 144, the light chain CDR1 of SEQ ID NO: 148, the light chain
CDR2 of SEQ ID NO: 149, and the light chain CDR3 of SEQ ID NO: 150.
In a further embodiment, the antigen binding moiety that is
specific for HER2 comprises the heavy chain CDR1 of SEQ ID NO: 145,
the heavy chain CDR2 of SEQ ID NO: 146, the heavy chain CDR3 of SEQ
ID NO: 147, the light chain CDR1 of SEQ ID NO: 148, the light chain
CDR2 of SEQ ID NO: 149, and the light chain CDR3 of SEQ ID NO:
150.
[0381] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER2 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the
heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID
NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain
CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain
CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the
light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID
NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
[0382] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER2 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the
heavy chain CDR3 of SEQ ID NO: 28, the light chain CDR1 of SEQ ID
NO: 29, the light chain CDR2 of SEQ ID NO: 30, and the light chain
CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain
CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the
light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID
NO: 30, and the light chain CDR3 of SEQ ID NO: 31.
[0383] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER2 further comprises an
anti-idiotypic CD3 scFv comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 41 or 42. In one embodiment, the anti-idiotypic scFv comprises
the polypeptide sequence of SEQ ID NO: 41 or 42.
[0384] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for HER2 further comprises an
anti-idiotypic HER2 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 48, the heavy chain CDR2 of SEQ ID NO: 49, the
heavy chain CDR3 of SEQ ID NO: 50, the light chain CDR1 of SEQ ID
NO: 51, the light chain CDR2 of SEQ ID NO: 52, and the light chain
CDR3 of SEQ ID NO: 53. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 48, the heavy chain
CDR2 of SEQ ID NO: 49, the heavy chain CDR3 of SEQ ID NO: 50, the
light chain CDR1 of SEQ ID NO: 51, the light chain CDR2 of SEQ ID
NO: 52, and the light chain CDR3 of SEQ ID NO: 53.
[0385] In one embodiments the protease-activatable T cell
activating bispecific molecule that comprises at least one antigen
binding moiety that is specific for HER2 further comprises a linker
comprising a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 35.
[0386] In one embodiment the protease-activatable T cell activating
bispecific molecule comprising at least one antigen binding moiety
that is specific for HER2 further comprises a linker having a
protease recognition site comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or
106. In one embodiment, the protease recognition site comprises the
polypeptide sequence of SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,
100, 101, 102, 103, 104, 105 or 106. In one embodiment, the
protease recognition site comprises the polypeptide sequence of SEQ
ID NO: 36. In one embodiment, the protease recognition site
comprises the polypeptide sequence of SEQ ID NO: 97. In one
embodiments the protease-activatable T cell activating bispecific
molecule comprising at least one antigen binding moiety that is
specific for HER2 further comprises a linker comprising a
polypeptide sequence that is at least about 95%, 96%, 97%, 98%, 99%
or 100% identical to SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95 or 96. In one embodiment, the linker comprises
the polypeptide sequence of SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95 or 96. In one embodiment, the linker
comprises the polypeptide sequence of SEQ ID NO: 7. In one
embodiment, the linker comprises the polypeptide sequence of SEQ ID
NO: 86.
[0387] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
132, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 136, a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 81 and a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
133.
[0388] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 132, the polypeptide sequence of SEQ ID NO: 136, the
polypeptide sequence of SEQ ID NO: 81 and the polypeptide sequence
of SEQ ID NO: 133.
[0389] In particular embodiments the protease-activatable T cell
activating bispecific molecule comprises at least one antigen
binding moiety that is specific for FolR1. In one embodiment the
FolR1 is a human FolR1. In one embodiment, the protease-activatable
T cell activating bispecific molecule comprises at least one
antigen binding moiety that is specific for human FolR1 and does
not bind to human FolR2 or human FolR3. In one embodiment, the
antigen binding moiety that is specific for FolR1 comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15
and SEQ ID NO: 16 and at least one light chain CDR selected from
the group of SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.
[0390] In one embodiment, the antigen binding moiety that is
specific for FolR1 comprises the heavy chain CDR1 of SEQ ID NO: 14,
the heavy chain CDR2 of SEQ ID NO: 15, the heavy chain CDR3 of SEQ
ID NO: 16, the light chain CDR1 of SEQ ID NO: 17, the light chain
CDR2 of SEQ ID NO: 18, and the light chain CDR3 of SEQ ID NO:
19.
[0391] In a further embodiment, the antigen binding moiety that is
specific for FolR1 comprises a heavy chain variable region sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 47 and a light chain variable region sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
55, or variants thereof that retain functionality.
[0392] In one embodiment, the antigen binding moiety that is
specific for FolR1 comprises the heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 47 and the light
chain variable region comprising an amino acid sequence of SEQ ID
NO: 55.
[0393] In one embodiment, the antigen binding moiety that is
specific for FolR1 comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 151, SEQ ID NO: 152 and SEQ ID NO: 153 and
at least one light chain CDR selected from the group of SEQ ID NO:
154, SEQ ID NO: 155 and SEQ ID NO: 156.
[0394] In one embodiment, the antigen binding moiety that is
specific for FolR1 comprises the heavy chain CDR1 of SEQ ID NO:
151, the heavy chain CDR2 of SEQ ID NO: 152, the heavy chain CDR3
of SEQ ID NO: 153, the light chain CDR1 of SEQ ID NO: 154, the
light chain CDR2 of SEQ ID NO: 155, and the light chain CDR3 of SEQ
ID NO: 156.
[0395] In a further embodiment, the antigen binding moiety that is
specific for FolR1 comprises a heavy chain variable region sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 157 and a light chain variable region sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 158, or variants thereof that retain functionality.
[0396] In one embodiment, the antigen binding moiety that is
specific for FolR1 comprises the heavy chain variable region
comprising an amino acid sequence of SEQ ID NO: 157 and the light
chain variable region comprising an amino acid sequence of SEQ ID
NO: 158.
[0397] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
2, and a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 1.
[0398] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 2, and the polypeptide sequence of SEQ ID NO: 1.
[0399] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for FolR1 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the
heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID
NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain
CDR3 of SEQ ID NO: 25. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 20, the heavy chain
CDR2 of SEQ ID NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the
light chain CDR1 of SEQ ID NO: 23, the light chain CDR2 of SEQ ID
NO: 24, and the light chain CDR3 of SEQ ID NO: 25.
[0400] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for FolR1 further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the
heavy chain CDR3 of SEQ ID NO: 28, the light chain CDR1 of SEQ ID
NO: 29, the light chain CDR2 of SEQ ID NO: 30, and the light chain
CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain
CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the
light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID
NO: 30, and the light chain CDR3 of SEQ ID NO: 31.
[0401] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for FolR1 further comprises an
anti-idiotypic CD3 scFv comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 41 or 42. In one embodiment, the anti-idiotypic scFv comprises
the polypeptide sequence of SEQ ID NO: 41 or 42.
[0402] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for FolR1 further comprises a
linker having a protease recognition site comprising a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101,
102, 103, 104, 105 or 106. In one embodiment, the protease
recognition site comprises the polypeptide sequence of SEQ ID NO:
36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or
106. In one embodiment, the protease recognition site comprises the
polypeptide sequence of SEQ ID NO: 36. In one embodiment, the
protease recognition site comprises the polypeptide sequence of SEQ
ID NO: 97. In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for ForR1 further comprises a
linker comprising a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7, 8, 9,
10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In one
embodiment, the linker comprises the polypeptide sequence of SEQ ID
NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In
one embodiment, the linker comprises the polypeptide sequence of
SEQ ID NO: 7. In one embodiment, the linker comprises the
polypeptide sequence of SEQ ID NO: 86.
[0403] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
1, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 3 and a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 72.
[0404] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
1, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 3 and a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 72.
[0405] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
1, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 3 and a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 85.
[0406] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
1, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 3, a polypeptide sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 73 and a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 74.
[0407] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 1, the polypeptide sequence of SEQ ID NO: 3 and the polypeptide
sequence of SEQ ID NO: 72.
[0408] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 1, the polypeptide sequence of SEQ ID NO: 3 and the polypeptide
sequence of SEQ ID NO: 85.
[0409] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 1, the polypeptide sequence of SEQ ID NO: 3, the polypeptide
sequence of SEQ ID NO: 73 and the polypeptide sequence of SEQ ID
NO: 74.
[0410] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
137, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 139, a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 81 and a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
138.
[0411] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 137, the polypeptide sequence of SEQ ID NO: 139, the
polypeptide sequence of SEQ ID NO: 81 and the polypeptide sequence
of SEQ ID NO: 138.
[0412] In particular embodiments the protease-activatable T cell
activating bispecific molecule comprises at least one antigen
binding moiety that is specific for Mesothelin. In one embodiment
the Mesothelin is human Mesothelin. In one embodiment, the
protease-activatable T cell activating bispecific molecule
comprises at least one antigen binding moiety that is specific for
human Mesothelin. In one embodiment, the antigen binding moiety
that is specific for Mesothelin comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and
at least one light chain CDR selected from the group of SEQ ID NO:
110, SEQ ID NO: 111 and SEQ ID NO: 112.
[0413] In one embodiment, the antigen binding moiety that is
specific for Mesothelin comprises the heavy chain CDR1 of SEQ ID
NO: 107, the heavy chain CDR2 of SEQ ID NO: 108, the heavy chain
CDR3 of SEQ ID NO: 109, the light chain CDR1 of SEQ ID NO: 110, the
light chain CDR2 of SEQ ID NO: 111, and the light chain CDR3 of SEQ
ID NO: 112.
[0414] In a further embodiment, the antigen binding moiety that is
specific for Mesothelin comprises a heavy chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 113 and a light chain variable region
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 114, or variants thereof that retain
functionality.
[0415] In one embodiment, the antigen binding moiety that is
specific for Mesothelin comprises the heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 113 and the light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 114.
[0416] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for Mesothelin further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the
heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID
NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain
CDR3 of SEQ ID NO: 25.
[0417] In one embodiment, the anti-idiotypic scFv comprises the
heavy chain CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID
NO: 21, the heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1
of SEQ ID NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the
light chain CDR3 of SEQ ID NO: 25.
[0418] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for Mesothelin further comprises an
anti-idiotypic CD3 scFv comprising at least one of the heavy chain
CDR1 of SEQ ID NO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the
heavy chain CDR3 of SEQ ID NO: 28, the light chain CDR1 of SEQ ID
NO: 29, the light chain CDR2 of SEQ ID NO: 30, and the light chain
CDR3 of SEQ ID NO: 31. In one embodiment, the anti-idiotypic scFv
comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain
CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the
light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID
NO: 30, and the light chain CDR3 of SEQ ID NO: 31.
[0419] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for Mesothelin further comprises an
anti-idiotypic CD3 scFv comprising a polypeptide sequence that is
at least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID
NO: 41 or 42. In one embodiment, the anti-idiotypic scFv comprises
the polypeptide sequence of SEQ ID NO: 41 or 42.
[0420] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for Mesothelin further comprises a
linker having a protease recognition site comprising a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99, 100, 101,
102, 103, 104, 105 or 106. In one embodiment, the protease
recognition site comprises the polypeptide sequence of SEQ ID NO:
36, 37, 38, 39, 40, 97, 98, 99, 100, 101, 102, 103, 104, 105 or
106. In one embodiment, the protease recognition site comprises the
polypeptide sequence of SEQ ID NO: 36. In one embodiment, the
protease recognition site comprises the polypeptide sequence of SEQ
ID NO: 97.
[0421] In one embodiments the protease-activatable T cell
activating bispecific molecule comprising at least one antigen
binding moiety that is specific for Mesothelin further comprises a
linker comprising a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7, 8, 9,
10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In one
embodiment, the linker comprises the polypeptide sequence of SEQ ID
NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96. In
one embodiment, the linker comprises the polypeptide sequence of
SEQ ID NO: 7. In one embodiment, the linker comprises the
polypeptide sequence of SEQ ID NO: 86.
[0422] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
77, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 78, a polypeptide sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 81 and a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 82.
[0423] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
76, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 77, a polypeptide sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 78 and a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 79.
[0424] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 77, the polypeptide sequence of SEQ ID NO: 78, the polypeptide
sequence of SEQ ID NO: 81 and the polypeptide sequence of SEQ ID
NO: 82.
[0425] In one embodiment the protease-activatable T cell activating
bispecific molecule comprises the polypeptide sequence of SEQ ID
NO: 76, the polypeptide sequence of SEQ ID NO: 77, the polypeptide
sequence of SEQ ID NO: 78 and the polypeptide sequence of SEQ ID
NO: 79.
[0426] In one embodiment, provided is a T cell activating
bispecific molecule comprises a polypeptide sequence that is at
least about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
76, a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 77, a polypeptide sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
SEQ ID NO: 78 and a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81.
[0427] In one embodiment the T cell activating bispecific molecule
comprises a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 77, a polypeptide
sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%
identical to SEQ ID NO: 78, a polypeptide sequence that is at least
about 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 81
and a polypeptide sequence that is at least about 95%, 96%, 97%,
98%, 99% or 100% identical to SEQ ID NO: 84.
[0428] In one embodiment the T cell activating bispecific molecule
comprises the polypeptide sequence of SEQ ID NO: 76, the
polypeptide sequence of SEQ ID NO: 77, the polypeptide sequence of
SEQ ID NO: 78 and the polypeptide sequence of SEQ ID NO: 81.
[0429] In one embodiment the T cell activating bispecific molecule
comprises the polypeptide sequence of SEQ ID NO: 77, the
polypeptide sequence of SEQ ID NO: 78, the polypeptide sequence of
SEQ ID NO: 81 and the polypeptide sequence of SEQ ID NO: 84.
Masking Moiety
[0430] The protease-activatable T cell activating bispecific
molecule of the invention comprises at least one masking moiety.
Others have tried to mask binding of an antibody by capping the
binding moiety with a fragment of the antigen recognized by the
binding moiety (e.g., WO2013128194). This approach has several
limitations. For example, using the antigen allows for less
flexibility in reducing the affinity of the binding moiety. This is
so because the affinity has to be high enough to be reliably masked
by the antigen mask. Also, dissociated antigen could potentially
bind to and interact with its cognate receptor(s) in vivo and cause
undesirable signals to the cell expressing such receptor. In
contrast, the approach described herein uses an anti-idiotype
antibody or fragment thereof as a mask. Two countervailing
considerations for designing an effective masking moiety are 1.
effectiveness of the masking and 2. reversibility of the masking.
If the affinity is too low, masking would be inefficient. However,
if the affinity is too high, the masking process might not be
readily reversible. It was not predictable whether a high affinity
anti-idiotype mask or a low affinity anti-idiotype mask would work
better. As described herein, higher affinity masking moieties
performed overall better in masking the antigen binding side and,
at the same time, could be effectively removed for activation of
the molecule. In one embodiment, the anti-idiotype mask has a KD of
1-8 nM. In one embodiment, anti-idiotype mask has a KD of 2 nM at
37.degree. C. In one specific embodiment, the masking moiety
recognizes the idiotype of the first antigen binding moiety capable
of specific binding to a CD3, e.g., a human CD3. In one specific
embodiment, the masking moiety recognizes the idiotype of the
second antigen binding moiety capable of binding to a target cell
antigen.
[0431] In one embodiment, the masking moiety masks a CD3-binding
moiety and comprises at least one of the heavy chain CDR1 of SEQ ID
NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the heavy chain CDR3
of SEQ ID NO: 22, the light chain CDR1 of SEQ ID NO: 23, the light
chain CDR2 of SEQ ID NO: 24, and the light chain CDR3 of SEQ ID NO:
25. In one embodiment, the masking moiety comprises the heavy chain
CDR1 of SEQ ID NO: 20, the heavy chain CDR2 of SEQ ID NO: 21, the
heavy chain CDR3 of SEQ ID NO: 22, the light chain CDR1 of SEQ ID
NO: 23, the light chain CDR2 of SEQ ID NO: 24, and the light chain
CDR3 of SEQ ID NO: 25.
[0432] In one embodiment, the masking moiety masks a CD3-binding
moiety and comprises a polypeptide sequence that is at least about
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 41. In one
embodiment, the masking moiety masks a CD3-binding moiety and
comprises the polypeptide sequence of SEQ ID NO: 41.
[0433] In one preferred embodiment, the masking moiety masks a
CD3-binding moiety and comprises at least one of the heavy chain
CDR1 of SEQ ID NO: 26, the heavy chain CDR2 of SEQ ID NO: 27, the
heavy chain CDR3 of SEQ ID NO: 28, the light chain CDR1 of SEQ ID
NO: 29, the light chain CDR2 of SEQ ID NO: 30, and the light chain
CDR3 of SEQ ID NO: 31. In one embodiment, the masking moiety
comprises the heavy chain CDR1 of SEQ ID NO: 26, the heavy chain
CDR2 of SEQ ID NO: 27, the heavy chain CDR3 of SEQ ID NO: 28, the
light chain CDR1 of SEQ ID NO: 29, the light chain CDR2 of SEQ ID
NO: 30, and the light chain CDR3 of SEQ ID NO: 31. In one
embodiment, the masking moiety masks a CD3-binding moiety and
comprises a polypeptide sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to SEQ ID NO: 42. In one preferred
embodiment, the masking moiety masks a CD3-binding moiety and
comprises the polypeptide sequence of SEQ ID NO: 42.
[0434] In one embodiment, the masking moiety masks a HER1-binding
moiety and comprises at least one of the heavy chain CDR1 of SEQ ID
NO: 48, the heavy chain CDR2 of SEQ ID NO: 49, the heavy chain CDR3
of SEQ ID NO: 50, the light chain CDR1 of SEQ ID NO: 51, the light
chain CDR2 of SEQ ID NO: 52, and the light chain CDR3 of SEQ ID NO:
53. In one embodiment, the anti-idiotypic scFv comprises the heavy
chain CDR1 of SEQ ID NO: 48, the heavy chain CDR2 of SEQ ID NO: 49,
the heavy chain CDR3 of SEQ ID NO: 50, the light chain CDR1 of SEQ
ID NO: 51, the light chain CDR2 of SEQ ID NO: 52, and the light
chain CDR3 of SEQ ID NO: 53.
[0435] In one aspect, the invention relates to an idiotype-specific
polypeptide for reversibly concealing antigen binding of an
antigen-binding of a molecule. In one embodiment, the invention
relates to an idiotype-specific polypeptide for reversibly
concealing an anti-CD3 antigen binding site of a molecule. Such
idiotype-specific polypeptide for reversibly concealing an anti-CD3
antigen binding site must be capable of specific binding to the
anti-CD3 antigen binding site's idiotype and thereby reducing or
abrogating binding of the anti-CD3 antigen binding site to CD3. In
one embodiment, the invention relates to an idiotype-specific
polypeptide for reversibly concealing an anti-HER1 antigen binding
site of a molecule. In one embodiment the idiotype-specific
polypeptide is an anti-idiotype scFv. In one embodiment the
idiotype-specific polypeptide is covalently attached to the
molecule through a linker. In one embodiment the idiotype-specific
polypeptide is covalently attached to the molecule through more
than one linker. In one embodiment the idiotype-specific
polypeptide is covalently attached to the molecule through two
linkers. In one embodiment the linker is a peptide linker. In one
embodiment the linker is a protease-cleavable linker. In one
embodiment, the linker comprises the sequence of SEQ ID NO: 7, 8,
9, or 10. In one embodiment, the linker comprises the sequence of
SEQ ID NO: 7, 8, 9, 10, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or
96. In one embodiment, the linker comprises the polypeptide
sequence of SEQ ID NO: 7. In one embodiment, the linker comprises
the polypeptide sequence of SEQ ID NO: 86. In one embodiment the
peptide linker comprises at least one protease recognition site. In
one embodiment, the protease recognition site comprises the
polypeptide sequence of SEQ ID NO: 36, 37, 38, 39, 40, 97, 98, 99,
100, 101, 102, 103, 104, 105 or 106. In one preferred embodiment,
the protease recognition site comprises the protease recognition
sequence RQARVVNG (SEQ ID NO: 36). In further embodiment, the
linker comprises more than one protease recognition site. In one
preferred embodiment, the protease recognition site comprises the
protease recognition sequence VHMPLGFLGPRQARVVNG (SEQ ID NO:97). In
one embodiment the protease is selected from the group consisting
of metalloproteinase, e.g., matrix metalloproteinase (MMP) 1-28 and
A Disintegrin And Metalloproteinase (ADAM) 2, 7-12, 15, 17-23,
28-30 and 33, serine protease, e.g., urokinase-type plasminogen
activator and Matriptase, cysteine protease, aspartic protease, and
cathepsin protease. In one specific embodiment the protease is MMP9
or MMP2. In a further specific embodiment, the protease is
Matriptase.
[0436] In one embodiment the molecule which comprises the anti-CD3
antigen binding site is a T-cell activating bispecific molecule. In
one particular embodiment the idiotype-specific polypeptide
comprises a heavy chain variable region comprising at least one of
a heavy chain complementarity determining region (CDR H) 1 amino
acid sequence of DYSIH (SEQ ID NO:20); CDR H2 amino acid sequence
of WINTETGEPAYADDFKG (SEQ ID NO:21); and a CDR H3 amino acid
sequence of PYDYDVLDY (SEQ ID NO:22). In one particular embodiment
the idiotype-specific polypeptide comprises a light chain variable
region comprising at least one of: a light chain (CDR L)1 amino
acid sequence of RASKSVSTSNYSYIH (SEQ ID NO:23); a CDR L2 amino
acid sequence of YVSYLES (SEQ ID NO:24); and a CDR L3 amino acid
sequence of QHSREFPWT (SEQ ID NO:25). In one particular embodiment
the idiotype-specific polypeptide comprises: a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
DYSIH (SEQ ID NO:20); a CDR H2 amino acid sequence of
WINTETGEPAYADDFKG (SEQ ID NO:21); a CDR H3 amino acid sequence of
PYDYDVLDY (SEQ ID NO:22); and a light chain variable region
comprising: a light chain (CDR L)1 amino acid sequence of
RASKSVSTSNYSYIH (SEQ ID NO:23); a CDR L2 amino acid sequence of
YVSYLES (SEQ ID NO:24); and a CDR L3 amino acid sequence of
QHSREFPWT (SEQ ID NO:25). In one particular embodiment the
idiotype-specific polypeptide comprises a heavy chain variable
region comprising at least one of: a heavy chain complementarity
determining region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID
NO:26); a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID
NO:27); and a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID
NO:28). In one particular embodiment the idiotype-specific
polypeptide comprises a light chain variable region comprising at
least one of: a light chain (CDR L)1 amino acid sequence of
RASENIDSYLA (SEQ ID NO:29); a CDR L2 amino acid sequence of AATFLAD
(SEQ ID NO:30); and a CDR L3 amino acid sequence of QHYYSTPYT (SEQ
ID NO:31). In one particular embodiment the idiotype-specific
polypeptide comprises a heavy chain variable region comprising: a
heavy chain complementarity determining region (CDR H) 1 amino acid
sequence of SYGVS (SEQ ID NO:26); a CDR H2 amino acid sequence of
IIWGDGSTNYHSALIS (SEQ ID NO:27); a CDR H3 amino acid sequence of
GITTVVDDYYAMDY (SEQ ID NO:28); and a light chain variable region
comprising: a light chain (CDR L)1 amino acid sequence of
RASENIDSYLA (SEQ ID NO:29); a CDR L2 amino acid sequence of AATFLAD
(SEQ ID NO:30); and a CDR L3 amino acid sequence of QHYYSTPYT (SEQ
ID NO:31). In one embodiment, the idiotype-specific polypeptide
comprises a heavy chain variable region comprising at least one of:
a heavy chain complementarity determining region (CDR H) 1 amino
acid sequence of SEQ ID NO:48; a CDR H2 amino acid sequence of SEQ
ID NO:49; and a CDR H3 amino acid sequence of SEQ ID NO:50. In one
embodiment, the idiotype-specific polypeptide comprises a light
chain variable region comprising at least one of: a light chain
complementarity determining region (CDR L) 1 amino acid sequence of
SEQ ID NO:51; a CDR L2 amino acid sequence of SEQ ID NO:52; and a
CDR L3 amino acid sequence of SEQ ID NO:53. In one embodiment, the
idiotype-specific polypeptide comprises a heavy chain variable
region comprising a heavy chain complementarity determining region
(CDR H) 1 amino acid sequence of SEQ ID NO:48; a CDR H2 amino acid
sequence of SEQ ID NO:49; and a CDR H3 amino acid sequence of SEQ
ID NO:50, and a light chain variable region comprising a light
chain complementarity determining region (CDR L) 1 amino acid
sequence of SEQ ID NO:51; a CDR L2 amino acid sequence of SEQ ID
NO:52; and a CDR L3 amino acid sequence of SEQ ID NO:53.
Polynucleotides
[0437] The invention further provides isolated polynucleotides
encoding a protease-activatable T cell activating bispecific
molecule as described herein or a fragment thereof. In some
embodiments, said fragment is an antigen binding fragment.
[0438] Polynucleotides of the invention include those that are at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequences set forth in SEQ ID NOs 62-71 or SEQ ID
NOs including functional fragments or variants thereof.
Polynucleotides of the invention further include those that are at
least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequences set forth in SEQ ID NOs 117-131
including functional fragments or variants thereof. Polynucleotides
of the invention further include those that are at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
sequences set forth in SEQ ID NOs 162-170 including functional
fragments or variants thereof.
[0439] The polynucleotides encoding protease-activatable T cell
activating bispecific molecules of the invention may be expressed
as a single polynucleotide that encodes the entire
protease-activatable T cell activating bispecific molecule or as
multiple (e.g., two or more) polynucleotides that are co-expressed.
Polypeptides encoded by polynucleotides that are co-expressed may
associate through, e.g., disulfide bonds or other means to form a
functional protease-activatable T cell activating bispecific
molecule. For example, the light chain portion of an antigen
binding moiety may be encoded by a separate polynucleotide from the
portion of the protease-activatable T cell activating bispecific
molecule comprising the heavy chain portion of the antigen binding
moiety, an Fc domain subunit and optionally (part of) another
antigen binding moiety. When co-expressed, the heavy chain
polypeptides will associate with the light chain polypeptides to
form the antigen binding moiety. In another example, the portion of
the protease-activatable T cell activating bispecific molecule
comprising one of the two Fc domain subunits and optionally (part
of) one or more antigen binding moieties could be encoded by a
separate polynucleotide from the portion of the
protease-activatable T cell activating bispecific molecule
comprising the the other of the two Fc domain subunits and
optionally (part of) an antigen binding moiety. When co-expressed,
the Fc domain subunits will associate to form the Fc domain.
[0440] In some embodiments, the isolated polynucleotide encodes the
entire protease-activatable T cell activating bispecific molecule
according to the invention as described herein. In other
embodiments, the isolated polynucleotide encodes a polypeptides
comprised in the protease-activatable T cell activating bispecific
molecule according to the invention as described herein.
[0441] In another embodiment, the present invention is directed to
an isolated polynucleotide encoding a protease-activatable T cell
activating bispecific molecule of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes a variable region sequence. In another embodiment, the
present invention is directed to an isolated polynucleotide
encoding a protease-activatable T cell activating bispecific
molecule or fragment thereof, wherein the polynucleotide comprises
a sequence that encodes a polypeptide sequence as shown in SEQ ID
NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 47, or 55. In another embodiment, the present
invention is directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule or
fragment thereof, wherein the polynucleotide comprises a sequence
that encodes a polypeptide sequence as shown in SEQ ID NOs 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 47, 55, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85.
In another embodiment, the present invention is directed to an
isolated polynucleotide encoding a protease-activatable T cell
activating bispecific molecule or fragment thereof, wherein the
polynucleotide comprises a sequence that encodes a polypeptide
sequence as shown in SEQ ID NOs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 47, 55, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 132, 133, 134, 135, 136,
137, 138, 139, 140 or 141. In another embodiment, the invention is
further directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in
SEQ ID NOs 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71. In another
embodiment, the invention is further directed to an isolated
polynucleotide encoding a protease-activatable T cell activating
bispecific molecule of the invention or a fragment thereof, wherein
the polynucleotide comprises a sequence that is at least about 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide
sequence shown in SEQ ID NOs 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,
129, 130 or 131. In another embodiment, the invention is further
directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that is at least about 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in
SEQ ID NOs 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 117, 118, 119,
120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 162,
163, 164, 165, 166, 167, 168, 169 or 170. In another embodiment,
the invention is directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises the nucleic acid sequence shown in SEQ ID NOs 62, 63, 64,
65, 66, 67, 68, 69, 70, or 71. In another embodiment, the invention
is directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises the nucleic acid sequence shown in SEQ ID NOs 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130 or 131. In another embodiment, the
invention is directed to an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises the nucleic acid sequence shown in SEQ ID NOs 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 162, 163, 164, 165, 166, 167,
168, 169 or 170. In another embodiment, the invention is directed
to an isolated polynucleotide encoding a protease-activatable T
cell activating bispecific molecule of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes a variable region sequence that is at least about 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence in SEQ ID NOs 43, 47, or 55. The invention encompasses an
isolated polynucleotide encoding a protease-activatable T cell
activating bispecific molecule of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes the variable region sequence of SEQ ID NOs SEQ ID NOs 43,
47, or 55 with conservative amino acid substitutions.
[0442] In another embodiment, the invention is directed to an
isolated polynucleotide encoding a protease-activatable T cell
activating bispecific molecule of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes a variable region sequence that is at least about 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence in SEQ ID NOs 43, 47, 55, 113, 114, 115 or 116. The
invention encompasses an isolated polynucleotide encoding a
protease-activatable T cell activating bispecific molecule of the
invention or a fragment thereof, wherein the polynucleotide
comprises a sequence that encodes the variable region sequence of
SEQ ID NOs SEQ ID NOs 43, 47, 55, 113, 114, 115 or 116 with
conservative amino acid substitutions.
[0443] In another embodiment, the invention is directed to an
isolated polynucleotide encoding a protease-activatable T cell
activating bispecific molecule of the invention or a fragment
thereof, wherein the polynucleotide comprises a sequence that
encodes a variable region sequence that is at least about 80%, 85%,
90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid
sequence in SEQ ID NOs 43, 47, 55, 113, 114, 115, 116, 157, 158,
159, 160 or 161. The invention encompasses an isolated
polynucleotide encoding a protease-activatable T cell activating
bispecific molecule of the invention or a fragment thereof, wherein
the polynucleotide comprises a sequence that encodes the variable
region sequence of SEQ ID NOs SEQ ID NOs 43, 47, 55, 113, 114, 115,
116, 157, 158, 159, 160 or 161 with conservative amino acid
substitutions.
[0444] In certain embodiments the polynucleotide or nucleic acid is
DNA. In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA (mRNA).
RNA of the present invention may be single stranded or double
stranded.
[0445] The invention further provides isolated polynucleotides
encoding an idiotype-specific polypeptide as described herein or a
fragment thereof. In some embodiments, said fragment is an idiotype
binding, i.e., anti-idiotype specific antibody or fragment thereof.
In one embodiment the idiotype-specific polypeptide is an
anti-idiotypic scFv.
[0446] The invention also encompasses an isolated polynucleotide
encoding an idiotype-specific polypeptide of the invention or a
fragment thereof, wherein the polynucleotide comprises a sequence
that encodes the polypeptide sequence of one or more of SEQ ID NOs
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 48, 49, 50, 51, 52,
and 53. The invention also encompasses an isolated polynucleotide
encoding an idiotype-specific polypeptide of the invention or a
fragment thereof, wherein the polynucleotide comprises a sequence
that encodes the polypeptide sequence of one or more of SEQ ID NOs
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 48, 49, 50, 51, 52,
and 53 with conservative amino acid substitutions.
[0447] The polynucleotides encoding idiotype-specific polypeptides
of the invention may be expressed as a single polynucleotide that
encodes the entire idiotype-specific polypeptide or as multiple
(e.g., two or more) polynucleotides that are co-expressed.
Polypeptides encoded by polynucleotides that are co-expressed may
associate through, e.g., disulfide bonds or other means to form a
functional idiotype-specific polypeptide, e.g., a masking moiety.
For example, in one embodiment the idiotype-specific polypeptide is
an anti-idiotypic scFv (single chain variable fragment) wherein the
light chain variable portion of the anti-idiotypic scFv may be
encoded by a separate polynucleotide from the portion of the
anti-idiotypic scFv comprising the heavy chain variable portion of
the anti-idiotypic scFv. When co-expressed, the heavy chain
polypeptides will associate with the light chain polypeptides to
form the anti-idiotypic scFv. In some embodiments, the isolated
polynucleotide encodes the idiotype-specific polypeptide according
to the invention as described herein. In certain embodiments the
polynucleotide or nucleic acid is DNA. In other embodiments, a
polynucleotide of the present invention is RNA, for example, in the
form of messenger RNA (mRNA). RNA of the present invention may be
single stranded or double stranded.
Recombinant Methods
[0448] protease-activatable T cell activating bispecific molecules
of the invention may be obtained, for example, by solid-state
peptide synthesis (e.g., Merrifield solid phase synthesis) or
recombinant production. For recombinant production one or more
polynucleotide encoding the protease-activatable T cell activating
bispecific molecule (fragment), e.g., as described above, is
isolated and inserted into one or more vectors for further cloning
and/or expression in a host cell. Such polynucleotide may be
readily isolated and sequenced using conventional procedures. In
one embodiment a vector, preferably an expression vector,
comprising one or more of the polynucleotides of the invention is
provided. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing the coding
sequence of a protease-activatable T cell activating bispecific
molecule (fragment) along with appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Maniatis et al., MOLECULAR CLONING: A
LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene
Publishing Associates and Wiley Interscience, N.Y (1989). The
expression vector can be part of a plasmid, virus, or may be a
nucleic acid fragment. The expression vector includes an expression
cassette into which the polynucleotide encoding the
protease-activatable T cell activating bispecific molecule
(fragment) (i.e. the coding region) is cloned in operable
association with a promoter and/or other transcription or
translation control elements. As used herein, a "coding region" is
a portion of nucleic acid which consists of codons translated into
amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not
translated into an amino acid, it may be considered to be part of a
coding region, if present, but any flanking sequences, for example
promoters, ribosome binding sites, transcriptional terminators,
introns, 5' and 3' untranslated regions, and the like, are not part
of a coding region. Two or more coding regions can be present in a
single polynucleotide construct, e.g., on a single vector, or in
separate polynucleotide constructs, e.g., on separate (different)
vectors. Furthermore, any vector may contain a single coding
region, or may comprise two or more coding regions, e.g., a vector
of the present invention may encode one or more polypeptides, which
are post- or co-translationally separated into the final proteins
via proteolytic cleavage. In addition, a vector, polynucleotide, or
nucleic acid of the invention may encode heterologous coding
regions, either fused or unfused to a polynucleotide encoding the
protease-activatable T cell activating bispecific molecule
(fragment) of the invention, or variant or derivative thereof.
Heterologous coding regions include without limitation specialized
elements or motifs, such as a secretory signal peptide or a
heterologous functional domain. An operable association is when a
coding region for a gene product, e.g., a polypeptide, is
associated with one or more regulatory sequences in such a way as
to place expression of the gene product under the influence or
control of the regulatory sequence(s). Two DNA fragments (such as a
polypeptide coding region and a promoter associated therewith) are
"operably associated" if induction of promoter function results in
the transcription of mRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not
interfere with the ability of the expression regulatory sequences
to direct the expression of the gene product or interfere with the
ability of the DNA template to be transcribed. Thus, a promoter
region would be operably associated with a nucleic acid encoding a
polypeptide if the promoter was capable of effecting transcription
of that nucleic acid. The promoter may be a cell-specific promoter
that directs substantial transcription of the DNA only in
predetermined cells. Other transcription control elements, besides
a promoter, for example enhancers, operators, repressors, and
transcription termination signals, can be operably associated with
the polynucleotide to direct cell-specific transcription. Suitable
promoters and other transcription control regions are disclosed
herein. A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions, which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (e.g., the immediate early promoter, in
conjunction with intron-A), simian virus 40 (e.g., the early
promoter), and retroviruses (such as, e.g., Rous sarcoma virus).
Other transcription control regions include those derived from
vertebrate genes such as actin, heat shock protein, bovine growth
hormone and rabbit a-globin, as well as other sequences capable of
controlling gene expression in eukaryotic cells. Additional
suitable transcription control regions include tissue-specific
promoters and enhancers as well as inducible promoters (e.g.,
promoters inducible tetracyclins). Similarly, a variety of
translation control elements are known to those of ordinary skill
in the art. These include, but are not limited to ribosome binding
sites, translation initiation and termination codons, and elements
derived from viral systems (particularly an internal ribosome entry
site, or IRES, also referred to as a CITE sequence). The expression
cassette may also include other features such as an origin of
replication, and/or chromosome integration elements such as
retroviral long terminal repeats (LTRs), or adeno-associated viral
(AAV) inverted terminal repeats (ITRs).
[0449] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. For example, if secretion of the
protease-activatable T cell activating bispecific molecule is
desired, DNA encoding a signal sequence may be placed upstream of
the nucleic acid encoding a protease-activatable T cell activating
bispecific molecule of the invention or a fragment thereof.
According to the signal hypothesis, proteins secreted by mammalian
cells have a signal peptide or secretory leader sequence which is
cleaved from the mature protein once export of the growing protein
chain across the rough endoplasmic reticulum has been initiated.
Those of ordinary skill in the art are aware that polypeptides
secreted by vertebrate cells generally have a signal peptide fused
to the N-terminus of the polypeptide, which is cleaved from the
translated polypeptide to produce a secreted or "mature" form of
the polypeptide. In certain embodiments, the native signal peptide,
e.g., an immunoglobulin heavy chain or light chain signal peptide
is used, or a functional derivative of that sequence that retains
the ability to direct the secretion of the polypeptide that is
operably associated with it. Alternatively, a heterologous
mammalian signal peptide, or a functional derivative thereof, may
be used. For example, the wild-type leader sequence may be
substituted with the leader sequence of human tissue plasminogen
activator (TPA) or mouse .beta.-glucuronidase.
[0450] DNA encoding a short protein sequence that could be used to
facilitate later purification (e.g., a histidine tag) or assist in
labeling the protease-activatable T cell activating bispecific
molecule may be included within or at the ends of the
protease-activatable T cell activating bispecific molecule
(fragment) encoding polynucleotide.
[0451] In a further embodiment, a host cell comprising one or more
polynucleotides of the invention is provided. In certain
embodiments a host cell comprising one or more vectors of the
invention is provided. The polynucleotides and vectors may
incorporate any of the features, singly or in combination,
described herein in relation to polynucleotides and vectors,
respectively. In one such embodiment a host cell comprises (e.g.,
has been transformed or transfected with) a vector comprising a
polynucleotide that encodes (part of) a protease-activatable T cell
activating bispecific molecule of the invention. As used herein,
the term "host cell" refers to any kind of cellular system which
can be engineered to generate the protease-activatable T cell
activating bispecific molecules of the invention or fragments
thereof. Host cells suitable for replicating and for supporting
expression of protease-activatable T cell activating bispecific
molecules are well known in the art. Such cells may be transfected
or transduced as appropriate with the particular expression vector
and large quantities of vector containing cells can be grown for
seeding large scale fermenters to obtain sufficient quantities of
the protease-activatable T cell activating bispecific molecule for
clinical applications. Suitable host cells include prokaryotic
microorganisms, such as E. coli, or various eukaryotic cells, such
as Chinese hamster ovary cells (CHO), insect cells, or the like.
For example, polypeptides may be produced in bacteria in particular
when glycosylation is not needed. After expression, the polypeptide
may be isolated from the bacterial cell paste in a soluble fraction
and can be further purified. In addition to prokaryotes, eukaryotic
microbes such as filamentous fungi or yeast are suitable cloning or
expression hosts for polypeptide-encoding vectors, including fungi
and yeast strains whose glycosylation pathways have been
"humanized", resulting in the production of a polypeptide with a
partially or fully human glycosylation pattern. See Gerngross, Nat
Biotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24,
210-215 (2006). Suitable host cells for the expression of
(glycosylated) polypeptides are also derived from multicellular
organisms (invertebrates and vertebrates). Examples of invertebrate
cells include plant and insect cells. Numerous baculoviral strains
have been identified which may be used in conjunction with insect
cells, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures can also be utilized as hosts. See e.g.,
U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and
6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants). Vertebrate cells may also be used
as hosts. For example, mammalian cell lines that are adapted to
grow in suspension may be useful. Other examples of useful
mammalian host cell lines are monkey kidney CV1 line transformed by
SV40 (COS-7); human embryonic kidney line (293 or 293T cells as
described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)), baby
hamster kidney cells (BHK), mouse sertoli cells (TM4 cells as
described, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkey
kidney cells (CV1), African green monkey kidney cells (VERO-76),
human cervical carcinoma cells (HELA), canine kidney cells (MDCK),
buffalo rat liver cells (BRL 3A), human lung cells (W138), human
liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI
cells (as described, e.g., in Mather et al., Annals N.Y. Acad Sci
383, 44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful
mammalian host cell lines include Chinese hamster ovary (CHO)
cells, including dhfr.sup.- CHO cells (Urlaub et al., Proc Natl
Acad Sci USA 77, 4216 (1980)); and myeloma cell lines such as YO,
NS0, P3X63 and Sp2/0. For a review of certain mammalian host cell
lines suitable for protein production, see, e.g., Yazaki and Wu,
Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana
Press, Totowa, N.J.), pp. 255-268 (2003). Host cells include
cultured cells, e.g., mammalian cultured cells, yeast cells, insect
cells, bacterial cells and plant cells, to name only a few, but
also cells comprised within a transgenic animal, transgenic plant
or cultured plant or animal tissue. In one embodiment, the host
cell is a eukaryotic cell, preferably a mammalian cell, such as a
Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK)
cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).
[0452] Standard technologies are known in the art to express
foreign genes in these systems. Cells expressing a polypeptide
comprising either the heavy or the light chain of an antigen
binding domain such as an antibody, may be engineered so as to also
express the other of the antibody chains such that the expressed
product is an antibody that has both a heavy and a light chain. In
one embodiment, a method of producing a protease-activatable T cell
activating bispecific molecule according to the invention is
provided, wherein the method comprises culturing a host cell
comprising a polynucleotide encoding the protease-activatable T
cell activating bispecific molecule, as provided herein, under
conditions suitable for expression of the protease-activatable T
cell activating bispecific molecule, and recovering the
protease-activatable T cell activating bispecific molecule from the
host cell (or host cell culture medium).
[0453] The components of the protease-activatable T cell activating
bispecific molecule are genetically fused to each other.
Protease-activatable T cell activating bispecific molecules can be
designed such that its components are fused directly to each other
or indirectly through a linker sequence. The composition and length
of the linker may be determined in accordance with methods well
known in the art and may be tested for efficacy. Examples of linker
sequences between different components of protease-activatable T
cell activating bispecific molecules are found in the sequences
provided herein. Additional sequences may also be included to
incorporate a cleavage site to separate the individual components
of the fusion if desired, for example an endopeptidase recognition
sequence. In certain embodiments the one or more antigen binding
moieties of the protease-activatable T cell activating bispecific
molecules comprise at least an antibody variable region capable of
binding an antigenic determinant. Variable regions can form part of
and be derived from naturally or non-naturally occurring antibodies
and fragments thereof. Methods to produce polyclonal antibodies and
monoclonal antibodies are well known in the art (see e.g., Harlow
and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor
Laboratory, 1988). Non-naturally occurring antibodies can be
constructed using solid phase-peptide synthesis, can be produced
recombinantly (e.g., as described in U.S. Pat. No. 4,186,567) or
can be obtained, for example, by screening combinatorial libraries
comprising variable heavy chains and variable light chains (see
e.g., U.S. Pat. No. 5,969,108 to McCafferty).
[0454] Any animal species of antibody, antibody fragment, antigen
binding domain or variable region can be used in the
protease-activatable T cell activating bispecific molecules of the
invention. Non-limiting antibodies, antibody fragments, antigen
binding domains or variable regions useful in the present invention
can be of murine, primate, or human origin. If the
protease-activatable T cell activating bispecific molecule is
intended for human use, a chimeric form of antibody may be used
wherein the constant regions of the antibody are from a human. A
humanized or fully human form of the antibody can also be prepared
in accordance with methods well known in the art (see e. g. U.S.
Pat. No. 5,565,332 to Winter). Humanization may be achieved by
various methods including, but not limited to (a) grafting the
non-human (e.g., donor antibody) CDRs onto human (e.g., recipient
antibody) framework and constant regions with or without retention
of critical framework residues (e.g., those that are important for
retaining good antigen binding affinity or antibody functions), (b)
grafting only the non-human specificity-determining regions (SDRs
or a-CDRs; the residues critical for the antibody-antigen
interaction) onto human framework and constant regions, or (c)
transplanting the entire non-human variable domains, but "cloaking"
them with a human-like section by replacement of surface residues.
Humanized antibodies and methods of making them are reviewed, e.g.,
in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are
further described, e.g., in Riechmann et al., Nature 332, 323-329
(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033
(1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and
7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et
al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv
Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536
(1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al.,
Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,
Mol Immunol 28, 489-498 (1991) (describing "resurfacing");
Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR
shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and
Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the
"guided selection" approach to FR shuffling). Human antibodies and
human variable regions can be produced using various techniques
known in the art. Human antibodies are described generally in van
Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and
Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable
regions can form part of and be derived from human monoclonal
antibodies made by the hybridoma method (see e.g., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)). Human antibodies and human variable
regions may also be prepared by administering an immunogen to a
transgenic animal that has been modified to produce intact human
antibodies or intact antibodies with human variable regions in
response to antigenic challenge (see e.g., Lonberg, Nat Biotech 23,
1117-1125 (2005). Human antibodies and human variable regions may
also be generated by isolating Fv clone variable region sequences
selected from human-derived phage display libraries (see e.g.,
Hoogenboom et al. in Methods in Molecular Biology 178, 1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and
McCafferty et al., Nature 348, 552-554; Clackson et al., Nature
352, 624-628 (1991)). Phage typically display antibody fragments,
either as single-chain Fv (scFv) fragments or as Fab fragments.
[0455] In certain embodiments, the antigen binding moieties useful
in the present invention are engineered to have enhanced binding
affinity according to, for example, the methods disclosed in U.S.
Pat. Appl. Publ. No. 2004/0132066, the entire contents of which are
hereby incorporated by reference. The ability of the
protease-activatable T cell activating bispecific molecule of the
invention to bind to a specific antigenic determinant can be
measured either through an enzyme-linked immunosorbent assay
(ELISA) or other techniques familiar to one of skill in the art,
e.g., surface plasmon resonance technique (analyzed on a BIACORE
T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)), and
traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).
Competition assays may be used to identify an antibody, antibody
fragment, antigen binding domain or variable domain that competes
with a reference antibody for binding to a particular antigen,
e.g., an antibody that competes with the V9 antibody for binding to
CD3. In certain embodiments, such a competing antibody binds to the
same epitope (e.g., a linear or a conformational epitope) that is
bound by the reference antibody. Detailed exemplary methods for
mapping an epitope to which an antibody binds are provided in
Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular
Biology vol. 66 (Humana Press, Totowa, N.J.). In an exemplary
competition assay, immobilized antigen (e.g., CD3) is incubated in
a solution comprising a first labeled antibody that binds to the
antigen (e.g., V9 antibody, described in U.S. Pat. No. 6,054,297)
and a second unlabeled antibody that is being tested for its
ability to compete with the first antibody for binding to the
antigen. The second antibody may be present in a hybridoma
supernatant. As a control, immobilized antigen is incubated in a
solution comprising the first labeled antibody but not the second
unlabeled antibody. After incubation under conditions permissive
for binding of the first antibody to the antigen, excess unbound
antibody is removed, and the amount of label associated with
immobilized antigen is measured. If the amount of label associated
with immobilized antigen is substantially reduced in the test
sample relative to the control sample, then that indicates that the
second antibody is competing with the first antibody for binding to
the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory
Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y.).
[0456] Protease-activatable T cell activating bispecific molecules
prepared as described herein may be purified by art-known
techniques such as high performance liquid chromatography, ion
exchange chromatography, gel electrophoresis, affinity
chromatography, size exclusion chromatography, and the like. The
actual conditions used to purify a particular protein will depend,
in part, on factors such as net charge, hydrophobicity,
hydrophilicity etc., and will be apparent to those having skill in
the art. For affinity chromatography purification an antibody,
ligand, receptor or antigen can be used to which the
protease-activatable T cell activating bispecific molecule binds.
For example, for affinity chromatography purification of
protease-activatable T cell activating bispecific molecules of the
invention, a matrix with protein A or protein G may be used.
Sequential Protein A or G affinity chromatography and size
exclusion chromatography can be used to isolate a
protease-activatable T cell activating bispecific molecule
essentially as described in the Examples. The purity of the
protease-activatable T cell activating bispecific molecule can be
determined by any of a variety of well-known analytical methods
including gel electrophoresis, high pressure liquid chromatography,
and the like. For example, the heavy chain fusion proteins
expressed as described in the Examples were shown to be intact and
properly assembled as demonstrated by reducing SDS-PAGE (see, e.g.,
FIGS. 8-12). Three bands were resolved at approximately Mr 25,000,
Mr 50,000 and Mr 75,000, corresponding to the predicted molecular
weights of the protease-activatable T cell activating bispecific
molecule light chain, heavy chain and heavy chain/light chain
fusion protein.
Assays
[0457] protease-activatable T cell activating bispecific molecules
provided herein may be identified, screened for, or characterized
for their physical/chemical properties and/or biological activities
by various assays known in the art.
Affinity Assays
[0458] The affinity of the protease-activatable T cell activating
bispecific molecule for an Fc receptor or a target antigen can be
determined in accordance with the methods set forth in the Examples
by surface plasmon resonance (SPR), using standard instrumentation
such as a BIAcore instrument (GE Healthcare), and receptors or
target proteins such as may be obtained by recombinant expression.
Alternatively, binding of protease-activatable T cell activating
bispecific molecules for different receptors or target antigens may
be evaluated using cell lines expressing the particular receptor or
target antigen, for example by flow cytometry (FACS). A specific
illustrative and exemplary embodiment for measuring binding
affinity is described in the following and in the Examples below.
According to one embodiment, K.sub.D is measured by surface plasmon
resonance using a BIACORE.RTM. T100 machine (GE Healthcare) at
25.degree. C.
[0459] To analyze the interaction between the Fc-portion and Fc
receptors, His-tagged recombinant Fc-receptor is captured by an
anti-Penta His antibody (Qiagen) immobilized on CMS chips and the
bispecific constructs are used as analytes. Briefly,
carboxymethylated dextran biosensor chips (CMS, GE Healthcare) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Anti Penta-His antibody is diluted with 10
mM sodium acetate, pH 5.0, to 40 m/ml before injection at a flow
rate of 5 .mu.l/min to achieve approximately 6500 response units
(RU) of coupled protein. Following the injection of the ligand, 1 M
ethanolamine is injected to block unreacted groups. Subsequently
the Fc-receptor is captured for 60 s at 4 or 10 nM. For kinetic
measurements, four-fold serial dilutions of the bispecific
construct (range between 500 nM and 4000 nM) are injected in HBS-EP
(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%
Surfactant P20, pH 7.4) at 25.degree. C. at a flow rate of 30
.mu.l/min for 120 s.
[0460] To determine the affinity to the target antigen, bispecific
constructs are captured by an anti-human Fab specific antibody (GE
Healthcare) that is immobilized on an activated CMS-sensor chip
surface as described for the anti Penta-His antibody. The final
amount of coupled protein is is approximately 12000 R U. The
bispecific constructs are captured for 90 s at 300 nM. The target
antigens are passed through the flow cells for 180 s at a
concentration range from 250 to 1000 nM with a flowrate of 30
.mu.l/min. The dissociation is monitored for 180 s.
[0461] Bulk refractive index differences are corrected for by
subtracting the response obtained on reference flow cell. The
steady state response was used to derive the dissociation constant
K.sub.D by non-linear curve fitting of the Langmuir binding
isotherm. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIACORE.RTM. T100 Evaluation Software version 1.1.1)
by simultaneously fitting the association and dissociation
sensorgrams. The equilibrium dissociation constant (K.sub.D) is
calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen et al.,
J Mol Biol 293, 865-881 (1999).
Activity Assays
[0462] Biological activity of the protease-activatable T cell
activating bispecific molecules of the invention can be measured by
various assays as described in the Examples. Biological activities
may for example include the induction of proliferation of T cells,
the induction of signaling in T cells, the induction of expression
of activation markers in T cells, the induction of cytokine
secretion by T cells, the induction of lysis of target cells such
as tumor cells, and the induction of tumor regression and/or the
improvement of survival.
Compositions, Formulations, and Routes of Administration
[0463] In a further aspect, the invention provides pharmaceutical
compositions comprising any of the protease-activatable T cell
activating bispecific molecules provided herein, e.g., for use in
any of the below therapeutic methods. In one embodiment, a
pharmaceutical composition comprises any of the
protease-activatable T cell activating bispecific molecules
provided herein and a pharmaceutically acceptable carrier. In
another embodiment, a pharmaceutical composition comprises any of
the protease-activatable T cell activating bispecific molecules
provided herein and at least one additional therapeutic agent,
e.g., as described below.
[0464] Further provided is a method of producing a
protease-activatable T cell activating bispecific molecule of the
invention in a form suitable for administration in vivo, the method
comprising (a) obtaining a protease-activatable T cell activating
bispecific molecule according to the invention, and (b) formulating
the protease-activatable T cell activating bispecific molecule with
at least one pharmaceutically acceptable carrier, whereby a
preparation of protease-activatable T cell activating bispecific
molecule is formulated for administration in vivo.
[0465] Pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of one or more
protease-activatable T cell activating bispecific molecule
dissolved or dispersed in a pharmaceutically acceptable carrier.
The phrases "pharmaceutical or pharmacologically acceptable" refers
to molecular entities and compositions that are generally non-toxic
to recipients at the dosages and concentrations employed, i.e. do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, such as, for example, a human, as
appropriate. The preparation of a pharmaceutical composition that
contains at least one protease-activatable T cell activating
bispecific molecule and optionally an additional active ingredient
will be known to those of skill in the art in light of the present
disclosure, as exemplified by Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, incorporated herein by
reference. Moreover, for animal (e.g., human) administration, it
will be understood that preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biological Standards or corresponding authorities in
other countries. Preferred compositions are lyophilized
formulations or aqueous solutions. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, buffers, dispersion media, coatings, surfactants,
antioxidants, preservatives (e.g., antibacterial agents, antifungal
agents), isotonic agents, absorption delaying agents, salts,
preservatives, antioxidants, proteins, drugs, drug stabilizers,
polymers, gels, binders, excipients, disintegration agents,
lubricants, sweetening agents, flavoring agents, dyes, such like
materials and combinations thereof, as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.
1289-1329, incorporated herein by reference). Except insofar as any
conventional carrier is incompatible with the active ingredient,
its use in the therapeutic or pharmaceutical compositions is
contemplated. The composition may comprise different types of
carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection. Protease-activatable T cell
activating bispecific molecules of the present invention (and any
additional therapeutic agent) can be administered intravenously,
intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostatically,
intrasplenically, intrarenally, intrapleurally, intratracheally,
intranasally, intravitreally, intravaginally, intrarectally,
intratumorally, intramuscularly, intraperitoneally, subcutaneously,
subconjunctivally, intravesicularlly, mucosally,
intrapericardially, intraumbilically, intraocularally, orally,
topically, locally, by inhalation (e.g., aerosol inhalation),
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), or by other method
or any combination of the forgoing as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference). Parenteral administration, in
particular intravenous injection, is most commonly used for
administering polypeptide molecules such as the
protease-activatable T cell activating bispecific molecules of the
invention.
[0466] Parenteral compositions include those designed for
administration by injection, e.g., subcutaneous, intradermal,
intralesional, intravenous, intraarterial intramuscular,
intrathecal or intraperitoneal injection. For injection, the
protease-activatable T cell activating bispecific molecules of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. The solution may
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the protease-activatable T cell
activating bispecific molecules may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use. Sterile injectable solutions are prepared by
incorporating the protease-activatable T cell activating bispecific
molecules of the invention in the required amount in the
appropriate solvent with various of the other ingredients
enumerated below, as required. Sterility may be readily
accomplished, e.g., by filtration through sterile filtration
membranes. Generally, dispersions are prepared by incorporating the
various sterilized active ingredients into a sterile vehicle which
contains the basic dispersion medium and/or the other ingredients.
In the case of sterile powders for the preparation of sterile
injectable solutions, suspensions or emulsion, the preferred
methods of preparation are vacuum-drying or freeze-drying
techniques which yield a powder of the active ingredient plus any
additional desired ingredient from a previously sterile-filtered
liquid medium thereof. The liquid medium should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic prior to injection with sufficient saline or glucose. The
composition must be stable under the conditions of manufacture and
storage, and preserved against the contaminating action of
microorganisms, such as bacteria and fungi. It will be appreciated
that endotoxin contamination should be kept minimally at a safe
level, for example, less that 0.5 ng/mg protein. Suitable
pharmaceutically acceptable carriers include, but are not limited
to: buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride; benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugars such as sucrose, mannitol, trehalose or
sorbitol; salt-forming counter-ions such as sodium; metal complexes
(e.g., Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene glycol (PEG). Aqueous injection suspensions may
contain compounds which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, dextran, or the
like. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl cleats or
triglycerides, or liposomes.
[0467] Active ingredients may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing
Company, 1990). Sustained-release preparations may be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
e.g., films, or microcapsules. In particular embodiments, prolonged
absorption of an injectable composition can be brought about by the
use in the compositions of agents delaying absorption, such as, for
example, aluminum monostearate, gelatin or combinations
thereof.
[0468] In addition to the compositions described previously, the
protease-activatable T cell activating bispecific molecules may
also be formulated as a depot preparation. Such long acting
formulations may be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the protease-activatable T cell activating
bispecific molecules may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt. Pharmaceutical
compositions comprising the protease-activatable T cell activating
bispecific molecules of the invention may be manufactured by means
of conventional mixing, dissolving, emulsifying, encapsulating,
entrapping or lyophilizing processes. Pharmaceutical compositions
may be formulated in conventional manner using one or more
physiologically acceptable carriers, diluents, excipients or
auxiliaries which facilitate processing of the proteins into
preparations that can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0469] The protease-activatable T cell activating bispecific
molecules may be formulated into a composition in a free acid or
base, neutral or salt form. Pharmaceutically acceptable salts are
salts that substantially retain the biological activity of the free
acid or base. These include the acid addition salts, e.g., those
formed with the free amino groups of a proteinaceous composition,
or which are formed with inorganic acids such as for example,
hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic, tartaric or mandelic acid. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides; or such organic bases as isopropylamine,
trimethylamine, histidine or procaine. Pharmaceutical salts tend to
be more soluble in aqueous and other protic solvents than are the
corresponding free base forms.
Therapeutic Methods and Compositions
[0470] Any of the protease-activatable T cell activating bispecific
molecules provided herein may be used in therapeutic methods.
Protease-activatable T cell activating bispecific molecules of the
invention can be used as immunotherapeutic agents, for example in
the treatment of cancers.
[0471] For use in therapeutic methods, protease-activatable T cell
activating bispecific molecules of the invention would be
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical
practitioners.
[0472] In one aspect, protease-activatable T cell activating
bispecific molecules of the invention for use as a medicament are
provided. In further aspects, protease-activatable T cell
activating bispecific molecules of the invention for use in
treating a disease are provided. In certain embodiments,
protease-activatable T cell activating bispecific molecules of the
invention for use in a method of treatment are provided. In one
embodiment, the invention provides a protease-activatable T cell
activating bispecific molecule as described herein for use in the
treatment of a disease in an individual in need thereof. In certain
embodiments, the invention provides a protease-activatable T cell
activating bispecific molecule for use in a method of treating an
individual having a disease comprising administering to the
individual a therapeutically effective amount of the
protease-activatable T cell activating bispecific molecule. In
certain embodiments the disease to be treated is a proliferative
disorder. In a particular embodiment the disease is cancer. In
certain embodiments the method further comprises administering to
the individual a therapeutically effective amount of at least one
additional therapeutic agent, e.g., an anti-cancer agent if the
disease to be treated is cancer.
[0473] In further embodiments, the invention provides a
protease-activatable T cell activating bispecific molecule as
described herein for use in inducing lysis of a target cell,
particularly a tumor cell. In certain embodiments, the invention
provides a protease-activatable T cell activating bispecific
molecule for use in a method of inducing lysis of a target cell,
particularly a tumor cell, in an individual comprising
administering to the individual an effective amount of the
protease-activatable T cell activating bispecific molecule to
induce lysis of a target cell. An "individual" according to any of
the above embodiments is a mammal, preferably a human.
[0474] In a further aspect, the invention provides for the use of a
protease-activatable T cell activating bispecific molecule of the
invention in the manufacture or preparation of a medicament. In one
embodiment the medicament is for the treatment of a disease in an
individual in need thereof. In a further embodiment, the medicament
is for use in a method of treating a disease comprising
administering to an individual having the disease a therapeutically
effective amount of the medicament. In certain embodiments the
disease to be treated is a proliferative disorder. In a particular
embodiment the disease is cancer. In one embodiment, the method
further comprises administering to the individual a therapeutically
effective amount of at least one additional therapeutic agent,
e.g., an anti-cancer agent if the disease to be treated is cancer.
In a further embodiment, the medicament is for inducing lysis of a
target cell, particularly a tumor cell. In still a further
embodiment, the medicament is for use in a method of inducing lysis
of a target cell, particularly a tumor cell, in an individual
comprising administering to the individual an effective amount of
the medicament to induce lysis of a target cell. An "individual"
according to any of the above embodiments may be a mammal,
preferably a human.
[0475] In a further aspect, the invention provides a method for
treating a disease. In one embodiment, the method comprises
administering to an individual having such disease a
therapeutically effective amount of a protease-activatable T cell
activating bispecific molecule of the invention. In one embodiment
a composition is administered to said individual, comprising the
protease-activatable T cell activating bispecific molecule of the
invention in a pharmaceutically acceptable form. In certain
embodiments the disease to be treated is a proliferative disorder.
In a particular embodiment the disease is cancer. In certain
embodiments the method further comprises administering to the
individual a therapeutically effective amount of at least one
additional therapeutic agent, e.g., an anti-cancer agent if the
disease to be treated is cancer. An "individual" according to any
of the above embodiments may be a mammal, preferably a human.
[0476] In a further aspect, the invention provides a method for
inducing lysis of a target cell, particularly a tumor cell. In one
embodiment the method comprises contacting a target cell with a
protease-activatable T cell activating bispecific molecule of the
invention in the presence of a T cell, particularly a cytotoxic T
cell. In a further aspect, a method for inducing lysis of a target
cell, particularly a tumor cell, in an individual is provided. In
one such embodiment, the method comprises administering to the
individual an effective amount of a protease-activatable T cell
activating bispecific molecule to induce lysis of a target cell. In
one embodiment, an "individual" is a human.
[0477] In certain embodiments the disease to be treated is a
proliferative disorder, particularly cancer. Non-limiting examples
of cancers include bladder cancer, brain cancer, head and neck
cancer, pancreatic cancer, lung cancer, breast cancer, ovarian
cancer, uterine cancer, cervical cancer, endometrial cancer,
esophageal cancer, colon cancer, colorectal cancer, rectal cancer,
gastric cancer, prostate cancer, blood cancer, skin cancer,
squamous cell carcinoma, bone cancer, and kidney cancer. Other cell
proliferation disorders that can be treated using a
protease-activatable T cell activating bispecific molecule of the
present invention include, but are not limited to neoplasms located
in the: abdomen, bone, breast, digestive system, liver, pancreas,
peritoneum, endocrine glands (adrenal, parathyroid, pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous
system (central and peripheral), lymphatic system, pelvic, skin,
soft tissue, spleen, thoracic region, and urogenital system. Also
included are pre-cancerous conditions or lesions and cancer
metastases. In certain embodiments the cancer is chosen from the
group consisting of renal cell cancer, skin cancer, lung cancer,
colorectal cancer, breast cancer, brain cancer, head and neck
cancer. A skilled artisan readily recognizes that in many cases the
protease-activatable T cell activating bispecific molecule may not
provide a cure but may only provide partial benefit. In some
embodiments, a physiological change having some benefit is also
considered therapeutically beneficial. Thus, in some embodiments,
an amount of protease-activatable T cell activating bispecific
molecule that provides a physiological change is considered an
"effective amount" or a "therapeutically effective amount". The
subject, patient, or individual in need of treatment is typically a
mammal, more specifically a human. In some embodiments, an
effective amount of a protease-activatable T cell activating
bispecific molecule of the invention is administered to a cell. In
other embodiments, a therapeutically effective amount of a
protease-activatable T cell activating bispecific molecule of the
invention is administered to an individual for the treatment of
disease.
[0478] For the prevention or treatment of disease, the appropriate
dosage of a protease-activatable T cell activating bispecific
molecule of the invention (when used alone or in combination with
one or more other additional therapeutic agents) will depend on the
type of disease to be treated, the route of administration, the
body weight of the patient, the type of T cell activating
bispecific antigen binding molecule, the severity and course of the
disease, whether the T cell activating bispecific antigen binding
molecule is administered for preventive or therapeutic purposes,
previous or concurrent therapeutic interventions, the patient's
clinical history and response to the protease-activatable T cell
activating bispecific molecule, and the discretion of the attending
physician. The practitioner responsible for administration will, in
any event, determine the concentration of active ingredient(s) in a
composition and appropriate dose(s) for the individual subject.
Various dosing schedules including but not limited to single or
multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein.
[0479] The protease-activatable T cell activating bispecific
molecule is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 15 mg/kg (e.g., 0.1 mg/kg-10
mg/kg) of protease-activatable T cell activating bispecific
molecule can be an initial candidate dosage for administration to
the patient, whether, for example, by one or more separate
administrations, or by continuous infusion. One typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment would generally be sustained until a
desired suppression of disease symptoms occurs. One exemplary
dosage of the T cell activating bispecific antigen binding molecule
would be in the range from about 0.005 mg/kg to about 10 mg/kg. In
other non-limiting examples, a dose may also comprise from about 1
microgram/kg body weight, about 5 microgram/kg body weight, about
10 microgram/kg body weight, about 50 microgram/kg body weight,
about 100 microgram/kg body weight, about 200 microgram/kg body
weight, about 350 microgram/kg body weight, about 500 microgram/kg
body weight, about 1 milligram/kg body weight, about 5 milligram/kg
body weight, about 10 milligram/kg body weight, about 50
milligram/kg body weight, about 100 milligram/kg body weight, about
200 milligram/kg body weight, about 350 milligram/kg body weight,
about 500 milligram/kg body weight, to about 1000 mg/kg body weight
or more per administration, and any range derivable therein. In
non-limiting examples of a derivable range from the numbers listed
herein, a range of about 5 mg/kg body weight to about 100 mg/kg
body weight, about 5 microgram/kg body weight to about 500
milligram/kg body weight, etc., can be administered, based on the
numbers described above. Thus, one or more doses of about 0.5
mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combination
thereof) may be administered to the patient. Such doses may be
administered intermittently, e.g., every week or every three weeks
(e.g., such that the patient receives from about two to about
twenty, or e.g., about six doses of the protease-activatable T cell
activating bispecific molecule). An initial higher loading dose,
followed by one or more lower doses may be administered. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0480] The protease-activatable T cell activating bispecific
molecule of the invention will generally be used in an amount
effective to achieve the intended purpose. For use to treat or
prevent a disease condition, the protease-activatable T cell
activating bispecific molecules of the invention, or pharmaceutical
compositions thereof, are administered or applied in a
therapeutically effective amount. Determination of a
therapeutically effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0481] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays, such as cell
culture assays. A dose can then be formulated in animal models to
achieve a circulating concentration range that includes the
IC.sub.50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans.
[0482] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0483] Dosage amount and interval may be adjusted individually to
provide plasma levels of the protease-activatable T cell activating
bispecific molecules which are sufficient to maintain therapeutic
effect. Usual patient dosages for administration by injection range
from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1
mg/kg/day. Therapeutically effective plasma levels may be achieved
by administering multiple doses each day. Levels in plasma may be
measured, for example, by HPLC. In cases of local administration or
selective uptake, the effective local concentration of the
protease-activatable T cell activating bispecific molecules may not
be related to plasma concentration. One having skill in the art
will be able to optimize therapeutically effective local dosages
without undue experimentation.
[0484] A therapeutically effective dose of the protease-activatable
T cell activating bispecific molecules described herein will
generally provide therapeutic benefit without causing substantial
toxicity. Toxicity and therapeutic efficacy of a
protease-activatable T cell activating bispecific molecule can be
determined by standard pharmaceutical procedures in cell culture or
experimental animals. Cell culture assays and animal studies can be
used to determine the LD.sub.50 (the dose lethal to 50% of a
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of a population). The dose ratio between toxic and
therapeutic effects is the therapeutic index, which can be
expressed as the ratio LD.sub.50/ED.sub.50. Protease-activatable T
cell activating bispecific molecule that exhibit large therapeutic
indices are preferred. In one embodiment, the protease-activatable
T cell activating bispecific molecule according to the present
invention exhibits a high therapeutic index. The data obtained from
cell culture assays and animal studies can be used in formulating a
range of dosages suitable for use in humans. The dosage lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon a variety of factors, e.g.,
the dosage form employed, the route of administration utilized, the
condition of the subject, and the like. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition (see, e.g., Fingl et
al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p.
1, incorporated herein by reference in its entirety). The attending
physician for patients treated with protease-activatable T cell
activating bispecific molecules of the invention would know how and
when to terminate, interrupt, or adjust administration due to
toxicity, organ dysfunction, and the like. Conversely, the
attending physician would also know to adjust treatment to higher
levels if the clinical response were not adequate (precluding
toxicity). The magnitude of an administered dose in the management
of the disorder of interest will vary with the severity of the
condition to be treated, with the route of administration, and the
like. The severity of the condition may, for example, be evaluated,
in part, by standard prognostic evaluation methods. Further, the
dose and perhaps dose frequency will also vary according to the
age, body weight, and response of the individual patient.
Other Agents and Treatments
[0485] The protease-activatable T cell activating bispecific
molecules of the invention may be administered in combination with
one or more other agents in therapy. For instance, a
protease-activatable T cell activating bispecific molecule of the
invention may be co-administered with at least one additional
therapeutic agent. The term "therapeutic agent" encompasses any
agent administered to treat a symptom or disease in an individual
in need of such treatment. Such additional therapeutic agent may
comprise any active ingredients suitable for the particular
indication being treated, preferably those with complementary
activities that do not adversely affect each other. In certain
embodiments, an additional therapeutic agent is an immunomodulatory
agent, a cytostatic agent, an inhibitor of cell adhesion, a
cytotoxic agent, an activator of cell apoptosis, or an agent that
increases the sensitivity of cells to apoptotic inducers. In a
particular embodiment, the additional therapeutic agent is an
anti-cancer agent, for example a microtubule disruptor, an
antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an
alkylating agent, a hormonal therapy, a kinase inhibitor, a
receptor antagonist, an activator of tumor cell apoptosis, or an
antiangiogenic agent.
[0486] Such other agents are suitably present in combination in
amounts that are effective for the purpose intended. The effective
amount of such other agents depends on the amount of
protease-activatable T cell activating bispecific molecule used,
the type of disorder or treatment, and other factors discussed
above. The protease-activatable T cell activating bispecific
molecule are generally used in the same dosages and with
administration routes as described herein, or about from 1 to 99%
of the dosages described herein, or in any dosage and by any route
that is empirically/clinically determined to be appropriate.
[0487] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate compositions), and separate administration,
in which case, administration of the protease-activatable T cell
activating bispecific molecule of the invention can occur prior to,
simultaneously, and/or following, administration of the additional
therapeutic agent and/or adjuvant. Protease-activatable T cell
activating bispecific molecules of the invention can also be used
in combination with radiation therapy.
Articles of Manufacture
[0488] In another aspect of the invention, an article of
manufacture containing materials useful for the treatment,
prevention and/or diagnosis of the disorders described above is
provided. The article of manufacture comprises a container and a
label or package insert on or associated with the container.
Suitable containers include, for example, bottles, vials, syringes,
IV solution bags, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is by itself or combined with another composition
effective for treating, preventing and/or diagnosing the condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is a protease-activatable T cell
activating bispecific molecule of the invention. The label or
package insert indicates that the composition is used for treating
the condition of choice. Moreover, the article of manufacture may
comprise (a) a first container with a composition contained
therein, wherein the composition comprises a protease-activatable T
cell activating bispecific molecule of the invention; and (b) a
second container with a composition contained therein, wherein the
composition comprises a further cytotoxic or otherwise therapeutic
agent. The article of manufacture in this embodiment of the
invention may further comprise a package insert indicating that the
compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
EXEMPLARY EMBODIMENTS
[0489] 1. A protease-activatable T cell activating bispecific
molecule comprising [0490] (a) a first antigen binding moiety
capable of specific binding to CD3; [0491] (b) a second antigen
binding moiety capable of specific binding to a target cell
antigen; and [0492] (c) a masking moiety covalently attached to the
T cell bispecific binding molecule through a protease-cleavable
linker, wherein the masking moiety is capable of specific binding
to the idiotype of the first or the second antigen binding moiety
thereby reversibly concealing the first or the second antigen
binding moiety. [0493] 2. The protease-activatable T cell
activating bispecific molecule of embodiment 1, wherein the masking
moiety is covalently attached to the first antigen binding moiety
and reversibly conceals the first antigen binding moiety. [0494] 3.
The protease-activatable T cell activating bispecific molecule of
embodiment 1 or 2, wherein the masking moiety is covalently
attached to the heavy chain variable region of the first antigen
binding moiety. [0495] 4. The protease-activatable T cell
activating bispecific molecule of embodiment 1 or 2, wherein the
masking moiety is covalently attached to the light chain variable
region of the first antigen binding moiety. [0496] 5. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 4, wherein the masking moiety is an
anti-idiotypic scFv. [0497] 6. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 2 to 5,
wherein the protease-activatable T cell activating bispecific
molecule comprises a second masking moiety reversibly concealing
the second antigen binding moiety. [0498] 7. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 6, wherein the protease is expressed by the
target cell. [0499] 8. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 7, wherein the
second antigen binding moiety is a crossover Fab molecule wherein
either the variable or the constant regions of the Fab light chain
and the Fab heavy chain are exchanged. [0500] 9. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 8, wherein the second antigen binding
moiety is a crossover Fab molecule wherein the constant regions of
the Fab light chain and the Fab heavy chain are exchanged. [0501]
10. The protease-activatable T cell activating bispecific molecule
of any one of embodiments 1 to 9, wherein the first antigen binding
moiety is a conventional Fab molecule. [0502] 11. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 10, comprising not more than one antigen
binding moiety capable of specific binding to CD3. [0503] 12. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 11, comprising a third antigen binding
moiety which is a Fab molecule capable of specific binding to a
target cell antigen. [0504] 13. The protease-activatable T cell
activating bispecific molecule of embodiment 12, wherein the third
antigen binding moiety is identical to the second antigen binding
moiety. [0505] 14. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 13, wherein the
second antigen binding moiety is capable of specific binding to
FolR1 or HER. [0506] 15. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 13, wherein the
second antigen binding moiety is capable of specific binding to a
target cell antigen selected from the group consisting of FolR1,
HER1 and Mesothelin. [0507] 16. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 13,
wherein the second antigen binding moiety is capable of specific
binding to a target cell antigen selected from the group consisting
of FolR1, HER1, HER2 and Mesothelin. [0508] 17. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 16, wherein the first and the second
antigen binding moiety are fused to each other, optionally via a
peptide linker. [0509] 18. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 17,
wherein the second antigen binding moiety is fused at the
C-terminus of the Fab heavy chain to the N-terminus of the Fab
heavy chain of the first antigen binding moiety. [0510] 19. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 17, wherein the first antigen binding
moiety is fused at the C-terminus of the Fab heavy chain to the
N-terminus of the Fab heavy chain of the second antigen binding
moiety. [0511] 20. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 19, wherein the
Fab light chain of the first antigen binding moiety and the Fab
light chain of the second antigen binding moiety are fused to each
other, optionally via a peptide linker. [0512] 21. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 20, additionally comprising an Fc domain
composed of a first and a second subunit capable of stable
association. [0513] 22. The protease-activatable T cell activating
bispecific molecule of embodiment 21, wherein the Fc domain is an
IgG, specifically an IgG.sub.1 or IgG.sub.4, Fc domain. [0514] 23.
The protease-activatable T cell activating bispecific molecule of
embodiment 21 or 22, wherein the Fc domain is a human Fc domain.
[0515] 24. The protease-activatable T cell activating bispecific
molecule of any one of embodiments 21 to 23, wherein the Fc domain
exhibits reduced binding affinity to an Fc receptor and/or reduced
effector function, as compared to a native IgG.sub.1 Fc domain.
[0516] 25. The protease-activatable T cell activating bispecific
molecule of embodiment 24, wherein the Fc domain comprises one or
more amino acid substitution that reduces binding to an Fc receptor
and/or effector function. [0517] 26. The protease-activatable T
cell activating bispecific molecule of embodiment 25, wherein said
one or more amino acid substitution is at one or more position
selected from the group of L234, L235, and P329 (Kabat numbering).
[0518] 27. The protease-activatable T cell activating bispecific
molecule of embodiment 26, wherein each subunit of the Fc domain
comprises three amino acid substitutions that reduce binding to an
activating Fc receptor and/or effector function wherein said amino
acid substitutions are L234A, L235A and P329G. [0519] 28. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 24 to 27, wherein the Fc receptor is an
Fc.gamma. receptor. [0520] 29. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 24 to 28,
wherein the effector function is antibody-dependent cell-mediated
cytotoxicity (ADCC). [0521] 30. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 29,
wherein the masking moiety comprises a heavy chain variable region
comprising at least one of: [0522] (a) a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
DYSIH (SEQ ID NO:20); [0523] (b) a CDR H2 amino acid sequence of
WINTETGEPAYADDFKG (SEQ ID NO:21); and [0524] (c) a CDR H3 amino
acid sequence of PYDYDVLDY (SEQ ID NO:22). [0525] 31. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 30, wherein the masking moiety comprises a
light chain variable region comprising at least one of: [0526] (d)
a light chain (CDR L)1 amino acid sequence of RASKSVSTSNYSYIH (SEQ
ID NO:23); [0527] (e) a CDR L2 amino acid sequence of YVSYLES (SEQ
ID NO:24); and [0528] (f) a CDR L3 amino acid sequence of QHSREFPWT
(SEQ ID NO:25). [0529] 32. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 31,
wherein the masking moiety comprises a heavy chain variable region
comprising: [0530] (a) a heavy chain complementarity determining
region (CDR H) 1 amino acid sequence of DYSIH (SEQ ID NO:20);
[0531] (b) a CDR H2 amino acid sequence of WINTETGEPAYADDFKG (SEQ
ID NO:21); [0532] (c) a CDR H3 amino acid sequence of PYDYDVLDY
(SEQ ID NO:22); and a light chain variable region comprising:
[0533] (d) a light chain (CDR L)1 amino acid sequence of
RASKSVSTSNYSYIH (SEQ ID NO:23); [0534] (e) a CDR L2 amino acid
sequence of YVSYLES (SEQ ID NO:24); and [0535] (f) a CDR L3 amino
acid sequence of QHSREFPWT (SEQ ID NO:25). [0536] 33. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 29, wherein the masking moiety comprises a
heavy chain variable region comprising at least one of: [0537] (a)
a heavy chain complementarity determining region (CDR H) 1 amino
acid sequence of SYGVS (SEQ ID NO:26); [0538] (b) a CDR H2 amino
acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); and [0539] (c) a
CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO:28). [0540]
34. The protease-activatable T cell activating bispecific molecule
of any one of embodiments 1 to 29 and 33, wherein the masking
moiety comprises a light chain variable region comprising at least
one of: [0541] (d) a light chain (CDR L)1 amino acid sequence of
RASENIDSYLA (SEQ ID NO:29); [0542] (e) a CDR L2 amino acid sequence
of AATFLAD (SEQ ID NO:30); and [0543] (f) a CDR L3 amino acid
sequence of QHYYSTPYT (SEQ ID NO:31). [0544] 35. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 29 and 33 to 34, wherein the masking moiety
comprises a heavy chain variable region comprising: [0545] (a) a
heavy chain complementarity determining region (CDR H) 1 amino acid
sequence of SYGVS (SEQ ID NO:26); [0546] (b) a CDR H2 amino acid
sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); [0547] (c) a CDR H3
amino acid sequence of GITTVVDDYYAMDY (SEQ ID NO:28); and a light
chain variable region comprising: [0548] (d) a light chain (CDR L)1
amino acid sequence of RASENIDSYLA (SEQ ID NO:29); [0549] (e) a CDR
L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and [0550] (f) a
CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31). [0551] 36.
The protease-activatable T cell activating bispecific molecule of
any one of embodiments 1 to 35, wherein the masking moiety
comprises a heavy chain variable region comprising at least one of:
[0552] (a) a heavy chain complementarity determining region (CDR H)
1 amino acid sequence of DYYIN (SEQ ID NO:48); [0553] (b) a CDR H2
amino acid sequence of VINPDSGGTDYNQNFKG (SEQ ID NO:49); and [0554]
(c) a CDR H3 amino acid sequence of RDSYGFDY (SEQ ID NO:50). [0555]
37. The protease-activatable T cell activating bispecific molecule
of any one of embodiments 1 to 36, wherein the masking moiety
comprises a light chain variable region comprising at least one of:
[0556] (a) a light chain (CDR L)1 amino acid sequence of
KASLSVTNDVA (SEQ ID NO:51); [0557] (b) a CDR L2 amino acid sequence
of YASNRNA (SEQ ID NO:52); and [0558] (c) a CDR L3 amino acid
sequence of QQDYTSPPT (SEQ ID NO:53). [0559] 38. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 37, wherein the masking moiety comprises a
heavy chain variable region comprising: [0560] a) a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
DYYIN (SEQ ID NO:48); [0561] b) a CDR H2 amino acid sequence of
VINPDSGGTDYNQNFKG (SEQ ID NO:49); and [0562] c) a CDR H3 amino acid
sequence of RDSYGFDY (SEQ ID NO:50); and a light chain variable
region comprising: [0563] d) a light chain (CDR L)1 amino acid
sequence of KASLSVTNDVA (SEQ ID NO:51); [0564] e) a CDR L2 amino
acid sequence of YASNRNA (SEQ ID NO:52); and [0565] f) a CDR L3
amino acid sequence of QQDYTSPPT (SEQ ID NO:53). [0566] 39. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 38, wherein the protease cleavable linker
comprises at least one protease recognition sequence. [0567] 40.
The protease-activatable T cell activating bispecific molecule of
embodiment 39, wherein the protease cleavable linker comprises a
protease recognition sequence. [0568] 41. The protease-activatable
T cell activating bispecific molecule of embodiment 40, wherein the
protease recognition sequence is selected from the group consisting
of:
TABLE-US-00004 [0568] (a) (SEQ ID NO: 36) RQARVVNG; (b) (SEQ ID NO:
37) VHMPLGFLGPGRSRGSFP; (c) (SEQ ID NO: 38) RQARVVNGXXXXXVPLSLYSG;
and (d) (SEQ ID NO: 39) RQARVVNGVPLSLYSG (e) (SEQ ID NO: 40)
PLGLWSQ, wherein X is any amino acid.
[0569] 42. The protease-activatable T cell activating bispecific
molecule of embodiment 40, wherein the protease recognition
sequence is selected from the group consisting of:
TABLE-US-00005 [0569] (a) (SEQ ID NO: 36) RQARVVNG; (b) (SEQ ID NO:
37) VHMPLGFLGPGRSRGSFP; (c) (SEQ ID NO: 38) RQARVVNGXXXXXVPLSLYSG;
(d) (SEQ ID NO: 39) RQARVVNGVPLSLYSG; (e) (SEQ ID NO: 40) PLGLWSQ;
(f) (SEQ ID NO: 97) VHMPLGFLGPRQARVVNG; (g) (SEQ ID NO: 98) FVGGTG;
(h) (SEQ ID NO: 99) KKAAPVNG; (i) (SEQ ID NO: 100) PMAKKVNG; (j)
(SEQ ID NO: 101) QARAKVNG; (k) (SEQ ID NO: 102) VHMPLGFLGP; (l)
(SEQ ID NO: 103) QARAK; (m) (SEQ ID NO: 104) VHMPLGFLGPPMAKK; (n)
(SEQ ID NO: 105) KKAAP; and (o) (SEQ ID NO: 106) PMAKK, wherein X
is any amino acid.
[0570] 43. The protease-activatable T cell activating bispecific
molecule of embodiment 39 or 40, wherein the protease cleavable
linker comprises the protease recognition sequence RQARVVNG (SEQ ID
NO:36). [0571] 44. The protease-activatable T cell activating
bispecific molecule of embodiment 39 or 40, wherein the protease
cleavable linker comprises the protease recognition sequence
VHMPLGFLGPRQARVVNG (SEQ ID NO:97). [0572] 45. The
protease-activatable T cell activating bispecific molecule of
embodiment 39 or 40, wherein the protease cleavable linker
comprises the protease recognition sequence RQARVVNG (SEQ ID NO:36)
or the protease recognition sequence VHMPLGFLGPRQARVVNG (SEQ ID
NO:97). [0573] 46. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 45, wherein the
protease is selected from the group consisting of
metalloproteinase, serine protease, cysteine protease, aspartic
proteases, and cathepsin protease. [0574] 47. The
protease-activatable T cell activating bispecific molecule of
embodiment 46, wherein the metalloproteinase is a matrix
metalloproteinase (MMP), preferably MMP9 or MMP2. [0575] 48. The
protease-activatable T cell activating bispecific molecule of
embodiment 46, wherein the serine protease is Matriptase. [0576]
49. The protease-activatable T cell activating bispecific molecule
of any one of embodiments 1 to 48, wherein the first antigen
binding moiety comprises at least one heavy chain complementarity
determining region (CDR) selected from the group consisting of SEQ
ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46 and at least one light
chain CDR selected from the group of SEQ ID NO: 17, SEQ ID NO: 18
and SEQ ID NO: 19. [0577] 50. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 49,
wherein the first antigen binding moiety is capable of specific
binding to CD3 and comprises a heavy chain variable region
comprising: [0578] a) a heavy chain complementarity determining
region (CDR H) 1 amino acid sequence of TYAMN (SEQ ID NO:44);
[0579] b) a CDR H2 amino acid sequence of RIRSKYNNYATYYADSVKG (SEQ
ID NO:45); and [0580] c) a CDR H3 amino acid sequence of
HGNFGNSYVSWFAY (SEQ ID NO:46); and a light chain variable region
comprising: [0581] d) a light chain (CDR L)1 amino acid sequence of
GSSTGAVTTSNYAN (SEQ ID NO:17); [0582] e) a CDR L2 amino acid
sequence of GTNKRAP (SEQ ID NO:18); and [0583] f) a CDR L3 amino
acid sequence of ALWYSNLWV (SEQ ID NO:19). [0584] 51. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 50, wherein the first antigen binding
moiety comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 43 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 55. [0585] 52. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 50, wherein the first antigen binding
moiety is capable of specific binding to CD3 and comprises a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO: 43 and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 55. [0586] 53. The protease-activatable T
cell activating bispecific molecule of any one of embodiments 1 to
52, wherein the second antigen binding moiety is capable of
specific binding to FolR1 and comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 and at
least one light chain CDR selected from the group of SEQ ID NO: 17,
SEQ ID NO: 18 and SEQ ID NO: 19. [0587] 54. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 53, wherein the second antigen binding
moiety is capable of specific binding to FolR1 and comprises a
heavy chain variable region comprising: [0588] a) a heavy chain
complementarity determining region (CDR H) 1 amino acid sequence of
NAWMS (SEQ ID NO:14); [0589] b) a CDR H2 amino acid sequence of
RIKSKTDGGTTDYAAPVKG (SEQ ID NO:15); and [0590] c) a CDR H3 amino
acid sequence of PWEWSWYDY (SEQ ID NO:16); and a light chain
variable region comprising: [0591] d) a light chain (CDR L)1 amino
acid sequence of GSSTGAVTTSNYAN (SEQ ID NO:17); [0592] e) a CDR L2
amino acid sequence of GTNKRAP (SEQ ID NO:18); and [0593] f) a CDR
L3 amino acid sequence of ALWYSNLWV (SEQ ID NO:19). [0594] 55. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 54, wherein the second antigen binding
moiety comprises a heavy chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 47 and a
light chain variable region comprising an amino acid sequence that
is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the
amino acid sequence of SEQ ID NO: 55. [0595] 56. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 54, wherein the second antigen binding
moiety is capable of specific binding to FolR1 and comprises a
heavy chain variable region comprising the amino acid sequence of
SEQ ID NO: 47 and a light chain variable region comprising the
amino acid sequence of SEQ ID NO: 55. [0596] 57. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 52, wherein the second antigen binding
moiety is capable of specific binding to FolR1 and comprises at
least one heavy chain complementarity determining region (CDR)
selected from the group consisting of SEQ ID NO: 151, SEQ ID NO:
152 and SEQ ID NO: 153 and at least one light chain CDR selected
from the group of SEQ ID NO: 154, SEQ ID NO: 155 and SEQ ID NO:
156. [0597] 58. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 52 or 57,
wherein the second antigen binding moiety is capable of specific
binding to FolR1 and comprises a heavy chain variable region
comprising: [0598] a) a heavy chain complementarity determining
region (CDR H) 1 amino acid sequence of SYYMH (SEQ ID NO:151);
[0599] b) a CDR H2 amino acid sequence of IINPSGGSTSYAQKFQG (SEQ ID
NO:152); and [0600] c) a CDR H3 amino acid sequence of SFFTGFHLDY
(SEQ ID NO:153); and a light chain variable region comprising:
[0601] d) a light chain (CDR L)1 amino acid sequence of
RASQSVSSSYLA (SEQ ID NO:154); [0602] e) a CDR L2 amino acid
sequence of GASSRAT (SEQ ID NO:155); and [0603] f) a CDR L3 amino
acid sequence of QQYTNEHYYT (SEQ ID NO:156). [0604] 59. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 52, 57 or 58, wherein the second antigen
binding moiety comprises a heavy chain variable region comprising
an amino acid sequence that is at least about 95%, 96%, 97%, 98%,
99% or 100% identical to the amino acid sequence of SEQ ID NO: 157
and a light chain variable region comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 158. [0605] 60. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 52 or 57 to 59, wherein the second antigen
binding moiety wherein the second antigen binding moiety is capable
of specific binding to ForR1 and comprises a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 157 and a
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 158. [0606] 61. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 52,
wherein the second antigen binding moiety is capable of specific
binding to Mesothelin and comprises at least one heavy chain
complementarity determining region (CDR) selected from the group
consisting of SEQ ID NO: 107, SEQ ID NO: 108 and SEQ ID NO: 109 and
at least one light chain CDR selected from the group of SEQ ID NO:
110, SEQ ID NO: 111 and SEQ ID NO: 112. [0607] 62. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 52 or 61, wherein the second antigen
binding moiety is capable of specific binding to Mesothelin and
comprises a heavy chain variable region comprising: [0608] a) a
heavy chain complementarity determining region (CDR H) 1 amino acid
sequence of GYTMN (SEQ ID NO:107); [0609] b) a CDR H2 amino acid
sequence of LITPYNGASSYNQKFRG (SEQ ID NO:108); and [0610] c) a CDR
H3 amino acid sequence of GGYDGRGFDY (SEQ ID NO:109); and a light
chain variable region comprising: [0611] d) a light chain (CDR L)1
amino acid sequence of SASSSVSYMH (SEQ ID NO:110); [0612] e) a CDR
L2 amino acid sequence of DTSKLAS (SEQ ID NO:111); and [0613] f) a
CDR L3 amino acid sequence of QQWSKHPLT (SEQ ID NO:112). [0614] 63.
The protease-activatable T cell activating bispecific molecule of
any one of embodiments 1 to 52, 61 or 62, wherein the second
antigen binding moiety comprises a heavy chain variable region
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 113 and a light chain variable region comprising an amino
acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or
100% identical to the amino acid sequence of SEQ ID NO: 114. [0615]
64. The protease-activatable T cell activating bispecific molecule
of any one of embodiments 1 to 52 or 61 to 63, wherein the second
antigen binding moiety is capable of specific binding to Mesothelin
and comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 113 and a light chain variable region
comprising to the amino acid sequence of SEQ ID NO: 114. [0616] 65.
The protease-activatable T cell activating bispecific molecule of
any one of embodiments 1 to 52, wherein the second antigen binding
moiety comprises a heavy chain comprising an amino acid sequence
that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to
the amino acid sequence of SEQ ID NO: 32 and a light chain
comprising an amino acid sequence that is at least about 95%, 96%,
97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ
ID NO: 33. [0617] 66. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 52 or 65,
wherein the second antigen binding moiety is capable of specific
binding to HER1 and comprises a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 115 and a light
chain variable region comprising the amino acid sequence of SEQ ID
NO: 116. [0618] 67. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 48, comprising
[0619] (a) a first heavy chain comprising the amino acid sequence
of SEQ ID NO:2; [0620] (b) a second heavy chain comprising the
amino acid sequence of SEQ ID NO:3; and [0621] (c) a light chain
comprising an amino acid sequence of SEQ ID NO:1. [0622] 68. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 48, comprising [0623] (a) a first heavy
chain comprising the amino acid sequence of SEQ ID NO:4; [0624] (b)
a second heavy chain comprising the amino acid sequence of SEQ ID
NO:3; and [0625] (c) a light chain comprising an amino acid
sequence of SEQ ID NO:1. [0626] 69. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 48,
comprising [0627] a) at least one heavy chain comprising the amino
acid sequence of SEQ ID NO:32; [0628] b) at least one light chain
comprising the amino acid sequence of SEQ ID NO:34. [0629] 70. The
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 48, comprising [0630] (a) a first heavy
chain comprising the amino acid sequence of SEQ ID NO:72; [0631]
(b) a second heavy chain comprising the amino acid sequence of SEQ
ID NO:3; and [0632] (c) a light chain comprising an amino acid
sequence of SEQ ID NO:1. [0633] 71. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 48,
comprising [0634] (a) a first heavy chain comprising the amino acid
sequence of SEQ ID NO:85; [0635] (b) a second heavy chain
comprising the amino acid sequence of SEQ ID NO:3; and [0636] (c) a
light chain comprising an amino acid sequence of SEQ ID NO:1.
[0637] 72. The protease-activatable T cell activating bispecific
molecule of any one of embodiments 1 to 48, comprising [0638] (a) a
first heavy chain comprising the amino acid sequence of SEQ ID
NO:73; [0639] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:3; [0640] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:1; and [0641] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO: 74.
[0642] 73. The protease-activatable T cell activating bispecific
molecule of any one of embodiments 1 to 48, comprising [0643] (a) a
first heavy chain comprising the amino acid sequence of SEQ ID
NO:77; [0644] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:82; [0645] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:78; and [0646] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO:81.
[0647] 74. The protease-activatable T cell activating bispecific
molecule of any one of embodiments 1 to 48, comprising [0648] (a) a
first heavy chain comprising the amino acid sequence of SEQ ID
NO:76; [0649] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:77; [0650] (c) a first light chain comprising
an amino acid sequence of SEQ ID NO:78; and [0651] (d) a second
light chain comprising an amino acid sequence of SEQ ID NO:79.
[0652] 75. The protease-activatable T cell activating bispecific
molecule of any one of embodiments 1 to 48, comprising [0653] (a) a
first heavy chain comprising the amino acid sequence of SEQ ID
NO:132; [0654] (b) a second heavy chain comprising the amino acid
sequence of SEQ ID NO:136; [0655] (c) a first light chain
comprising an amino acid sequence of SEQ ID NO:81; and [0656] (d) a
second light chain comprising an amino acid sequence of SEQ ID
NO:133. [0657] 76. The protease-activatable T cell activating
bispecific molecule of any one of embodiments 1 to 48, comprising
[0658] (a) a first heavy chain comprising the amino acid sequence
of SEQ ID NO:137; [0659] (b) a second heavy chain comprising the
amino acid sequence of SEQ ID NO:139; [0660] (c) a first light
chain comprising an amino acid sequence of SEQ ID NO:81; and [0661]
(d) a second light chain comprising an amino acid sequence of SEQ
ID NO:138. [0662] 77. An idiotype-specific polypeptide for
reversibly concealing an anti-CD3 antigen binding site of a
molecule. [0663] 78. The idiotype-specific polypeptide of
embodiment 77, wherein the idiotype-specific polypeptide is an
anti-idiotype scFv.
[0664] 79. The idiotype-specific polypeptide of embodiment 77 or
78, wherein the idiotype-specific polypeptide is covalently
attached to the molecule through a linker. [0665] 80. The
idiotype-specific polypeptide of embodiment 79, wherein the linker
is a peptide linker. [0666] 81. The idiotype-specific polypeptide
of embodiment 79 or 80, wherein the linker is a protease-cleavable
linker. [0667] 82. The idiotype-specific polypeptide of any one of
embodiments 79 to 81, wherein the peptide linker comprises at least
one protease recognition site. [0668] 83. The idiotype-specific
polypeptide of embodiment 82, wherein the protease is selected from
the group consisting of metalloproteinase, serine protease,
cysteine protease, aspartic proteases, and cathepsin protease.
[0669] 84. The idiotype-specific polypeptide of embodiment 83,
wherein the metalloproteinase is a matrix metalloproteinase (MMP),
preferably MMP9 or MMP2. [0670] 85. The idiotype-specific
polypeptide of embodiment 83, wherein the serine protease is
Matriptase. [0671] 86. The idiotype-specific polypeptide of
embodiment 82, wherein the protease cleavable linker comprises the
protease recognition sequence RQARVVNG (SEQ ID NO:36) or the
protease recognition sequence VHMPLGFLGPRQARVVNG (SEQ ID NO:97).
[0672] 87. The idiotype-specific polypeptide of any one of
embodiments 77 to 86, wherein the molecule is a T-cell activating
bispecific molecule. [0673] 88. The idiotype-specific polypeptide
of any one of embodiments 77 to 87, comprising a heavy chain
variable region comprising at least one of: [0674] (a) a heavy
chain complementarity determining region (CDR H) 1 amino acid
sequence of DYSIH (SEQ ID NO:20); [0675] (b) a CDR H2 amino acid
sequence of WINTETGEPAYADDFKG (SEQ ID NO:21); and [0676] (c) a CDR
H3 amino acid sequence of PYDYDVLDY (SEQ ID NO:22). [0677] 89. The
idiotype-specific polypeptide of any one of embodiments 77 to 88,
comprising a light chain variable region comprising at least one
of: [0678] (d) a light chain (CDR L)1 amino acid sequence of
RASKSVSTSNYSYIH (SEQ ID NO:23); [0679] (e) a CDR L2 amino acid
sequence of YVSYLES (SEQ ID NO:24); and [0680] (f) a CDR L3 amino
acid sequence of QHSREFPWT (SEQ ID NO:25). [0681] 90. The
idiotype-specific polypeptide of any one of embodiments 77 to 87,
comprising a heavy chain variable region comprising: [0682] (a) a
heavy chain complementarity determining region (CDR H) 1 amino acid
sequence of DYSIH (SEQ ID NO:20); [0683] (b) a CDR H2 amino acid
sequence of WINTETGEPAYADDFKG (SEQ ID NO:21); [0684] (c) a CDR H3
amino acid sequence of PYDYDVLDY (SEQ ID NO:22); and a light chain
variable region comprising: [0685] (d) a light chain (CDR L)1 amino
acid sequence of RASKSVSTSNYSYIH (SEQ ID NO:23); [0686] (e) a CDR
L2 amino acid sequence of YVSYLES (SEQ ID NO:24); and [0687] (f) a
CDR L3 amino acid sequence of QHSREFPWT (SEQ ID NO:25). [0688] 91.
The idiotype-specific polypeptide of any one of embodiments 77 to
87, comprising a heavy chain variable region comprising at least
one of: [0689] (a) a heavy chain complementarity determining region
(CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO:26); [0690] (b) a
CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID NO:27); and
[0691] (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY (SEQ ID
NO:28). [0692] 92. The idiotype-specific polypeptide of any one of
embodiments 77 to 87 and 91, comprising a light chain variable
region comprising at least one of: [0693] (d) a light chain (CDR
L)1 amino acid sequence of RASENIDSYLA (SEQ ID NO:29); [0694] (e) a
CDR L2 amino acid sequence of AATFLAD (SEQ ID NO:30); and [0695]
(f) a CDR L3 amino acid sequence of QHYYSTPYT (SEQ ID NO:31).
[0696] 93. The idiotype-specific polypeptide of any one of
embodiments 77 to 87, comprising a heavy chain variable region
comprising: [0697] (a) a heavy chain complementarity determining
region (CDR H) 1 amino acid sequence of SYGVS (SEQ ID NO:26);
[0698] (b) a CDR H2 amino acid sequence of IIWGDGSTNYHSALIS (SEQ ID
NO:27); [0699] (c) a CDR H3 amino acid sequence of GITTVVDDYYAMDY
(SEQ ID NO:28); and a light chain variable region comprising:
[0700] (d) a light chain (CDR L)1 amino acid sequence of
RASENIDSYLA (SEQ ID NO:29); [0701] (e) a CDR L2 amino acid sequence
of AATFLAD (SEQ ID NO:30); and [0702] (f) a CDR L3 amino acid
sequence of QHYYSTPYT (SEQ ID NO:31). [0703] 94. The
idiotype-specific polypeptide of embodiments 77 to 93, wherein the
anti-CD3 antigen binding site comprises a heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 43 and a
light chain variable region comprising the amino acid sequence of
SEQ ID NO: 55. [0704] 95. An isolated polynucleotide encoding the
protease-activatable T cell activating bispecific antigen binding
molecule of any one of embodiments 1 to 76 or the idiotype-specific
polypeptide of any one of embodiments 77 to 94. [0705] 96. A
polypeptide encoded by the polynucleotide of embodiment 95. [0706]
97. A vector, particularly an expression vector, comprising the
polynucleotide of embodiment 95. [0707] 98. A host cell comprising
the polynucleotide of embodiment 95 or the vector of embodiment 97.
[0708] 99. A method of producing a protease-activatable T cell
activating bispecific molecule, comprising the steps of a)
culturing the host cell of embodiment 98 under conditions suitable
for the expression of the protease-activatable T cell activating
bispecific molecule and b) recovering the protease-activatable T
cell activating bispecific molecule. [0709] 100. A
protease-activatable T cell activating bispecific molecule produced
by the method of embodiment 99. [0710] 101. A method of producing
an idiotype-specific polypeptide, comprising the steps of a)
culturing the host cell of embodiment 98 under conditions suitable
for the expression of the idiotype-specific polypeptide and b)
recovering the an idiotype-specific polypeptide. [0711] 102. An
idiotype-specific polypeptide produced by the method of embodiment
101. [0712] 103. A pharmaceutical composition comprising the
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 76 and a pharmaceutically acceptable
carrier. [0713] 104. A pharmaceutical composition comprising the
idiotype-specific polypeptide of any one of embodiments 77 to 94
and a pharmaceutically acceptable carrier. [0714] 105. A
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 76, the idiotype-specific polypeptide of
any one of embodiments 77 to 94 or the composition of embodiment
103 for use as a medicament. [0715] 106. The protease-activatable T
cell activating bispecific molecule for use according to embodiment
105, wherein the medicament is for treating or delaying progression
of cancer, treating or delaying progression of an immune related
disease, or enhancing or stimulating an immune response or function
in an individual. [0716] 107. The protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 76 or
the idiotype-specific polypeptide of any one of embodiments 77 to
94 for use in the treatment of a disease in an individual in need
thereof. [0717] 108. The protease-activatable T cell activating
bispecific molecule or the idiotype-specific polypeptide for use in
the treatment of a disease in an individual in need thereof of
embodiment 107, wherein the disease is a cancer. [0718] 109. Use of
the protease-activatable T cell activating bispecific molecule of
any one of embodiments 1 to 76 or the idiotype-specific polypeptide
of any one of embodiments 77 to 94 for the manufacture of a
medicament. [0719] 110. The use of embodiment 109, wherein the
disease is a cancer. [0720] 111. A method of treating a disease in
an individual, comprising administering to said individual a
therapeutically effective amount of a composition comprising the
protease-activatable T cell activating bispecific molecule of any
one of embodiments 1 to 76 or composition of embodiment 103. [0721]
112. A method for inducing lysis of a target cell, comprising
contacting a target cell with the protease-activatable T cell
activating bispecific molecule of any one of embodiments 1 to 76 or
composition of embodiment 103 in the presence of a T cell. [0722]
113. The method of embodiment 112 wherein the target cell is a
cancer cell. [0723] 114. The method of embodiment 112 or 113,
wherein the target cell expresses a protease capable of activating
the protease-activatable T cell activating bispecific molecule.
[0724] 115. An anti-idiotype CD3 antibody or antigen-binding
fragment thereof specific for an idiotype of an anti-CD3
antigen-binding molecule, wherein the anti-idiotype CD3 antibody or
fragment thereof when bound to the anti-CD3 antigen-binding
molecule specifically blocks binding of the anti-CD3
antigen-binding molecule to CD3. [0725] 116. The anti-idiotype CD3
antibody or antigen-binding fragment thereof of embodiment 115,
wherein the anti-idiotype CD3 antibody or fragment thereof is
reversibly associated with the anti-CD3 antigen-binding molecule
through a peptide linker comprising a protease recognition site.
[0726] 117. The anti-idiotype CD3 antibody or antigen-binding
fragment thereof of embodiment 115 or 116, wherein the CD3 is a
mouse, monkey or human CD3. [0727] 118. A method of reducing in
vivo toxicity of a T cell activating bispecific molecule comprising
attaching an idiotype-specific polypeptide of any one of
embodiments 77 to 94 to the T cell activating bispecific molecule
with a protease-cleavable linker to form a protease-activatable T
cell activating bispecific molecule, wherein the in vivo toxicity
of the protease-activatable T cell activating bispecific molecule
is reduced compared to toxicity of the T cell activating bispecific
molecule. [0728] 119. The invention as described hereinbefore.
EXAMPLES
[0729] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above.
Example 1
Synthesis of Monovalent Anti-CD3 IgG Molecules with Anti-Idiotypic
scFv
[0730] Described herein are CD3 binders that are masked with an
N-terminally linked anti-idiotypic CD3 scFv. These constructs
include a protease recognition site which is recognized by a tumor
specific protease. In the presence of protease-expressing tumor
cells, the linker connecting the masking moiety will be cleaved
and, thereby, CD3 binding by the CD3 binder is recovered. Several
monovalent anti-CD3 IgG molecules with various anti-idiotypic scFv
were produced and are schematically depicted in FIGS. 1A-1E with
their respective ID number. The following molecules were prepared:
[0731] Identification No. 7859: monovalent CD3 IgG, (anti-idiotypic
scFv 4.15.64--MK062 Matriptase site--CD3--N-terminal fused to CD3
Fab--inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and
protease-cleavable linker. [0732] Identification No. 7860:
monovalent CD3 IgG, (anti-idiotypic scFv 4.32.63--MK062 Matriptase
site--CD3--N-terminal fused to CD3 Fab--inert Fc) with N-terminal
fused anti CD3 scFv 4.32.63 and protease-cleavable linker. [0733]
Identification No. 7857: monovalent CD3 IgG, (anti-idiotypic scFv
4.15.64--non-cleavable linker--CD3--N-terminal fused to CD3
Fab--inert Fc) with N-terminal fused anti CD3 scFv 4.15.64 and
protease-cleavable linker. [0734] Identification No. 7858:
monovalent CD3 IgG, (anti-idiotypic scFv 4.32.63--non-cleavable
linker--CD3--N-terminal fused to CD3 Fab--inert Fc) with N-terminal
fused anti CD3 scFv 4.32.63 and protease-cleavable linker. [0735]
Identification No. 7861: monovalent CD3 IgG, (CD3 Fab--inert Fc)
with N-terminal fused anti CD3 scFv 4.15.64/4.32.63 and protease
linker.
[0736] Anti-idiotypic (ID) binder sequences were obtained by
RACE-PCR (rapid amplification of cDNA ends) from RNA of Hybridoma
cells. Hybridoma cells were obtained by immunization of mice with
CH2527 (VL_7-46(13)/VH_3-23(12)) Fab-fragment. Single chain Fv
(ScFv) sequence synthesis was ordered from Invitrogen including the
necessary restriction sites for cloning. Six different
anti-idiotypic CH2527 binders were compared for their affinities
(FIG. 2, result Biacore-Analytics (AG M. Schraml) at 25.degree.
C./37.degree. C. (Analyt: MAK<CEA/CD3>rH)) and two of them
were cloned as N-terminal fusions at the heavy chain of CD3
Fab-Fc.
[0737] The anti-ID single chain Fv DNA sequences were subcloned in
frame with the CD3 VH chain pre-inserted into the respective
recipient mammalian expression vector. Protein expression is driven
by an MPSV promoter and a synthetic polyA signal sequence is
present at the 3' end of the CDS. In addition each vector contains
an EBV OriP sequence.
[0738] The molecules were produced by co-transfecting HEK293-EBNA
cells growing in suspension with the mammalian expression vectors
using polyethylenimine (PEI). The cells were transfected with the
corresponding expression vectors in a 1:1:2 ratio ("Fc hole
(CH2-CH3)":"common light chain (CLC)":"vector heavy chain knob
(scFv-VH-CH1-CH2-CH3)").
[0739] For transfection, HEK293 EBNA cells were cultivated in serum
free ExCell culture medium containing 6 mM L-glutamine and 250 mg/l
G418. For the production in 600 ml tubespin flasks (max. working
volume 400 mL) 800 million HEK293 EBNA cells were seeded 24 hours
before transfection without G418. For transfection 800 mio cells
were centrifuged for 5 min at 210.times.g and supernatant was
replaced by 40 ml pre-warmed CD CHO medium containing 6 mM
L-Glutamine. Expression vectors were mixed with 40 ml CD CHO medium
containing 6 mM L-Glutamine to a total amount of 400 .mu.g DNA.
After addition of 1080 .mu.l PEI solution (2.7 .mu.g/ml) the
mixture was vortexed for 15 s and subsequently incubated for 10 min
at room temperature. Afterwards cells were mixed with the DNA/PEI
solution, transferred to a 600 ml tubespin flask and incubated for
3 hours at 37.degree. C. in an incubator with a 5% CO2 atmosphere.
After incubation, 320 ml ExCell+6 mM L-glutamine+5 g/L Pepsoy+1.0
mM VPA+3 g/l glucose medium was added and cells were cultivated for
24 hours prior to feeding with 7% Feed 7. After 6-7 days
cultivation supernatant was collected for purification by
centrifugation for 20-30 min at 210.times.g (Sigma 8K centrifuge).
The solution was sterile filtered (0.22 .mu.m filter) and sodium
azide in a final concentration of 0.01% w/v was added. The solution
was kept at 4.degree. C. until purification. The secreted protein
was purified from cell culture supernatants by affinity
chromatography using ProteinA affinity chromatography, followed by
one to two size exclusion chromatographic steps.
[0740] For affinity chromatography supernatant was loaded on a
HiTrap Protein A FF column (CV=5 mL, GE Healthcare) equilibrated
with 25 ml 20 mM sodium phosphate, 20 mM sodium citrate, 0.5M
sodium chloride, 0.01% Tween-20 pH 7.5. Unbound protein was removed
by washing with at least 10 column volumes 20 mM sodium phosphate,
20 mM sodium citrate, 0.5M sodium chloride, 0.01% Tween-20 pH 7.5
and target protein was eluted in 20 column volumes (gradient from
0%-100%) 20 mM sodium citrate, 0.5M sodium chloride, 0.01% Tween-20
pH 2.5. Protein solution was neutralized by adding 1/10 of 2 M Tris
pH 10.5. Target protein was concentrated with Amicon.RTM. Ultra-15
Ultracel 30K (Merck Millipore Ltd.) to a volume of 4 ml maximum
prior loading on a HiLoad Superdex 200 column (GE Healthcare)
equilibrated with 20 mM histidine, 140 mM sodium chloride, pH 6.0,
0.01% Tween20.
[0741] For analytics after size exclusion chromatography the purity
and molecular weight of the molecules in the single fractions were
analyzed by SDS-PAGE in the absence of a reducing agent and
staining with Coomassie (InstantBlue.TM., Expedeon). The
NuPAGE.RTM. Pre-Cast gel system (4-12% Bis-Tris, Invitrogen or 3-8%
Tris-Acetate, Invitrogen) was used according to the manufacturer's
instruction. The protein concentration of purified protein samples
was determined by measuring the optical density (OD) at 280 nm
divided by the molar extinction coefficient calculated on the basis
of the amino acid sequence.
[0742] Purity and molecular weight of the molecules after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction. The aggregate content of the molecules was analyzed
using a TSKgel G3000 SW XL analytical size-exclusion column (Tosoh)
in 25 mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrocloride,
0.02% (w/v) NaN3, pH 6.7 running buffer at 25.degree. C. The final
quality of all molecules was good, with .gtoreq.92% monomer
content.
TABLE-US-00006 TABLE 2 Summary of production and purification of
protease activated monovalent CD3 IgG molecules. Analytical SEC
Titer Yield (HMW/Monomer/LMW) Molecule [mg/l] [mg/l] [%] 1 12 3.38
2.21/95.5/2.29 2 9 1.75 4.86/95.14/0 3 15 4.8 6.93/93.07/0 4 4.5
0.26 4.88/95.12/0 5 105.3 26.3 0/100/0
Example 2
Cleavage and Stability of Protease Activated IgGs
[0743] Capillary Electrophoresis of protease activated IgG
molecules. Comparison of untreated sample and treated sample showed
that the anti-ID scFv was completely cleaved off after treatment
with rhMatriptase/ST14 (R&D Systems) indicated by the size
shift in the SDS page analysis (FIG. 3). Analysis of samples
incubated for 48 h at 37.degree. C. confirmed stability of the
molecules in formulation buffer (FIG. 3A-D).
Example 3
Masking Effect of Anti-Idiotypic scFv for CD3 IgG
[0744] The efficiency of masking the CD3 binder by N-terminal
fusion of an anti-idiotypic CD3 scFv was shown by a Jurkat-NFAT
reporter assay. Jurkat-NFAT reporter cells (a human acute lymphatic
leukemia reporter cell line with a NFAT promoter-regulated
luciferase expression, GloResponse Jurkat NFAT-RE-luc2P, Promega
#CS176501) express active firefly luciferase if the NFAT promoter
is activated by binding of CD3.epsilon.. The intensity of the
luminescence signal upon addition of luciferase substrate is
proportional to the intensity of CD3 activation and signaling.
Completely unmasked monovalent CD3 molecules served as a positive
control. The treatment was done with rhMatriptase/ST14 (R&D
Systems) for 48 h at 37.degree. C. In parallel Bug/ml Anti human Fc
Antibody (BioLegends) were coated in 0.025 ul/well PBS for 48 h at
4.degree. C. in white-walled, clear bottom 96-well (flat)-plate
(Greiner Bio-One). PBS was removed by pipetting before monovalent
IgGs were added at the indicated concentration range of 200 nM-2.56
pM. Plates were incubated for about 30 min at 4.degree. C.
Subsequently, Jurkat-NFAT reporter cells were harvested and
viability assessed using ViCell. Cells were resuspended in Jurkat
medium (RPMI1640, 2 g/l Glucose, 2 g/l NaHCO.sub.3, 10% FCS, 25 mM
HEPES, 2 mM L-Glutamin, 1.times.NEAA, 1.times.Sodium-pyruvate)
without Hygromycine and 100 .mu.l per well (25.000 cells/well) were
added to the crosslinked monovalent CD3 IgGs. Cells were incubated
for 3 h at 37.degree. C. in a humidified incubator. Plates were
taken out of the incubator for about 10 min to adapt to room
temperature prior to Luminescence read out. 100 .mu.l/well of
ONE-Glo solution (1:1 ONE-Glo and assay medium volume per well)
were added to wells and incubated for 10 min at room temperature in
the dark. Luminescence was detected using WALLAC Victor3 ELISA
reader (PerkinElmer2030), 1 sec/well as detection time. 7857
(4.15.64 mask with non-cleavable linker) and 7859 (untreated) show
significantly reduced CD3.epsilon. binding compared to unmasked
(7861) and pretreated molecule (7859 treated) (FIG. 4A). 7760 was
included as a control to show that N-terminal linkage does not
block CD3 binding itself. 7858 (4.32.63 mask with non-cleavable
linker) and 7860 (untreated) show significantly reduced
CD3.epsilon. binding compared to unmasked (7861) and pretreated
molecule (7860 treated) (FIG. 4B). In line with the affinities of
the anti-idiotypic CD3 binders the 4.32.63 mask is much more
efficient than the 4.15.64. In terms of EC50 values (FIG. 4C) the
4.32.63 masked CD3 binder binds 54 fold less than the unmasked CD3
binder 7861. For the 4.15.64 mask it is only 16 fold less binding
than for 7861. Depending on the tumor target and the target binder
the best mask can be evaluated.
Example 4
Preparation of Anti FolR1/Anti-CD3 T Cell Bispecific (TCB)
Molecules with Anti CD3 scFv
[0745] Several T cell bispecific (TCB) molecules with various
anti-idiotypic scFv were produced and are schematically depicted in
FIGS. 5A-5H with their respective ID number. The following
molecules were prepared: [0746] ID7344: FolR1 16D5 2+1 IgG, classic
format (anti-idiotypic scFv 4.15.64--MK062 Matriptase
site--CD3--N-terminal fused to FolR1 VH--inert Fc) with N-terminal
fused anti CD3 scFv 4.15.64 and protease linker (FIG. 5A, SEQ ID
NOs 1, 2 and 3). [0747] ID7496: FolR1 16D5 2+1 IgG, classic format
(anti-idiotypic scFv 4.32.63--MK062 Matriptase
site--CD3--N-terminal fused to FolR1 VH--inert Fc) with N-terminal
fused anti CD3 scFv 4.32.63 and protease linker (FIG. 5C, SEQ ID
NOs 1, 3 and 4). [0748] ID7676: FolR1 16D5 2+1 IgG, classic format
(anti-idiotypic scFv 4.15.64--non-cleavable GS
linker--CD3--N-terminal fused to FolR1 VH--inert Fc) with
N-terminal fused anti CD3 scFv 4.15.64 and protease linker (FIG.
5B, SEQ ID NOs 1, 3 and 6). [0749] ID7611: FolR1 16D5 2+1 IgG,
classic format (anti-idiotypic scFv 4.32.63--non-cleavable GS
linker--CD3--N-terminal fused to FolR1 VH--inert Fc) with
N-terminal fused anti CD3 scFv 4.32.63 and protease linker (FIG.
5D, SEQ ID NOs 1, 3 and 5).
[0750] Anti-idiotypic (ID) binder sequences were obtained by
RACE-PCR (rapid amplification of cDNA ends) from RNA of Hybridoma
cells. Hybridoma cells were obtained by immunization of mice.
Single chain Fv (ScFv) sequence synthesis was ordered at Invitrogen
including the necessary restriction sites for cloning. Six
different anti-idiotypic CH2527 binders were compared for their
affinities (FIG. 2, result Biacore-Analytics (AG M. Schraml) at
25.degree. C./37.degree. C. (Analyt: MAK<CEA/CD3>rH)) and
four of them were cloned as N-terminal fusions at the HC of
CD3-FolR1 16D5 TCB.
[0751] The anti-ID single chain Fv DNA sequences were subcloned in
frame with the CD3 VH chain pre-inserted into the respective
recipient mammalian expression vector. Protein expression is driven
by an MPSV promoter and a synthetic polyA signal sequence is
present at the 3' end of the coding sequence (CDS). In addition
each vector contains an EBV OriP sequence.
[0752] The molecules were produced by co-transfecting HEK293-EBNA
cells growing in suspension with the mammalian expression vectors
using polyethylenimine (PEI). The cells were transfected with the
corresponding expression vectors in a 1:3:2 ratio ("vector heavy
chain hole (VH-CH1-CH2-CH3)": "common light chain (CLC)": "vector
heavy chain knob (scFv-VH-CH1-VH-CH1-CH2-CH3)").
[0753] For transfection HEK293 EBNA cells were cultivated in serum
free ExCell culture medium containing 6 mM L-glutamine and 250 mg/l
G418. For the production in 600 ml tubespin flasks (max. working
volume 400 mL) 800 million HEK293 EBNA cells were seeded 24 hours
before transfection without G418. For transfection 800 mio cells
were centrifuged for 5 min at 210.times.g and supernatant was
replaced by 40 ml pre-warmed CD CHO medium containing 6 mM
L-Glutamine. Expression vectors were mixed with 40 ml CD CHO medium
containing 6 mM L-Glutamine to a total amount of 400 .mu.g DNA.
After addition of 1080 .mu.l PEI solution (2.7 .mu.g/ml) the
mixture was vortexed for 15 s and subsequently incubated for 10 min
at room temperature. Afterwards cells were mixed with the DNA/PEI
solution, transferred to a 600 ml tubespin flask and incubated for
3 hours at 37.degree. C. in an incubator with a 5% CO.sub.2
atmosphere. After incubation, 320 ml ExCell+6 mM L-glutamine+5 g/L
Pepsoy+1.0 mM VPA+3 g/l glucose medium was added and cells were
cultivated for 24 hours prior to feeding with 7% Feed 7. After 6-7
days cultivation supernatant was collected for purification by
centrifugation for 20-30 min at 210.times.g (Sigma 8K centrifuge).
The solution was sterile filtered (0.22 .mu.m filter) and sodium
azide in a final concentration of 0.01% w/v was added. The solution
was kept at 4.degree. C. until purification.
[0754] The secreted protein was purified from cell culture
supernatants by affinity chromatography using ProteinA affinity
chromatography, followed by one to two size exclusion
chromatographic steps. For affinity chromatography supernatant was
loaded on a HiTrap Protein A FF column (CV=5 mL, GE Healthcare)
equilibrated with 25 ml 20 mM sodium phosphate, 20 mM sodium
citrate, 0.5M sodium chloride, 0.01% Tween-20 pH 7.5. Unbound
protein was removed by washing with at least 10 column volumes 20
mM sodium phosphate, 20 mM sodium citrate, 0.5M sodium chloride,
0.01% Tween-20 pH 7.5 and target protein was eluted in 20 column
volumes (gradient from 0%-100%) 20 mM sodium citrate, 0.5M sodium
chloride, 0.01% Tween-20 pH 2.5. Protein solution was neutralized
by adding 1/10 of 2 M Tris pH 10.5. Target protein was concentrated
with Amicon.RTM. Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a
volume of 4 ml maximum prior loading on a HiLoad Superdex 200
column (GE Healthcare) equilibrated with 20 mM histidine, 140 mM
sodium chloride, pH 6.0, 0.01% Tween20.
[0755] For analytics after size exclusion chromatography the purity
and molecular weight of the molecules in the single fractions were
analyzed by SDS-PAGE in the absence of a reducing agent and
staining with Coomassie (InstantBlue.TM., Expedeon). The
NuPAGE.RTM. Pre-Cast gel system (4-12% Bis-Tris, Invitrogen or 3-8%
Tris-Acetate, Invitrogen) was used according to the manufacturer's
instruction. The protein concentration of purified protein samples
was determined by measuring the optical density (OD) at 280 nm
divided by the molar extinction coefficient calculated on the basis
of the amino acid sequence.
[0756] Purity and molecular weight of the molecules after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction.
[0757] The aggregate content of the molecules was analyzed using a
TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrocloride, 0.02%
(w/v) NaN3, pH 6.7 running buffer at 25.degree. C. The final
quality of all molecules was good, with .gtoreq.92% monomer
content.
TABLE-US-00007 TABLE 2 Summary of production and purification of
protease activated TCB molecules. Analytical SEC Titer Yield
(HMW/Monomer/LMW) Molecule [mg/l] [mg/l] [%] 1 33 3.7
0.98/92.7/6.32 2 11 0.55 3.76/96.24/0 3 12.9 0.89 2.9/93.82/2.19 4
6.7 0.35 4.59/95.41/0
Example 5
Transient Expression of Protease Activated TCBs
[0758] Different plasmid ratios used for transfection were compared
by size exclusion chromatograpy as the knob chain was suspected to
be expressed in lower levels compared to the hole chain and the
light chain. As shown in FIGS. 6 and 7, using a plasmid ratio of
1(hole):2 (knob):3 (CLC) (FIG. 7) instead of 1(hole):1 (knob):3
(CLC) (FIG. 6) increased the yield of correct molecule (left peak)
and decreased the amount of hole hole homodimers (right peak).
Example 6
Cleavage and Stability of Protease Activated TCB
[0759] Protease activated TCBs were analyzed by capillary
electrophoresis. Comparison of untreated sample and treated sample
showed that the anti-idiotype scFc moiety was completely cleaved
off after treatment with rhMatriptase/ST14. Analysis of samples
incubated for 48 h at 37.degree. C. confirmed stability of the
molecules in formulation buffer (FIGS. 12A-12D).
Example 7
Cell Killing Using Target Cell Lines that Express Different Levels
of FolR1
[0760] T-cell-mediated cell killing induced by protease activated
TCB molecules was assessed using target cell lines expressing
different levels of FolR1 (FIG. 13). Human PBMCs were used as
effector cells and cell killing was detected at 48 h of incubation
with the protease activated TCB molecules. Human Peripheral blood
mononuclear cells (PBMCs) were isolated from fresh taken blood or
from buffy coats obtained from healthy human donors. For fresh
blood 50 ml Leucosep tubes (GreinerBioOne) were used for
preparation. For enriched lymphocyte preparations (buffy coats)
Histopaque-1077 density preparation was used. Blood/buffy coat was
diluted 1:1 with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, w/o
break, room temperature), the plasma above the PBMC-containing
interphase was discarded and PBMCs transferred in a new falcon tube
subsequently filled with 50 ml of PBS. The mixture was centrifuged
(400.times.g, 10 minutes, room temperature), the supernatant
discarded and the PBMC pellet resuspended in 2 ml ACK buffer for
Erythrocytes lysis. After incubation at 37.degree. C. for about 2-3
minutes the tubes were filled with sterile PBS to 50 ml and
centrifuged at 350.times.g for 10 minutes. This washing step was
repeated once prior to resuspension of PBMCs in RPMI1640 medium
containing 2% FCS and 1.times. GlutaMax at 37.degree. C., 5%
CO.sub.2 in cell incubator until further use. Briefly, adherent
target cells were harvested with Trypsin/EDTA, counted, checked for
viability and resuspended at 0.4.times.10.sup.6 cells/ml in assay
medium (RPMI1640, 2% FCS, 1.times. GlutaMax). Target cells were
plated at a density of 20 000 cells/well using round-bottom 96-well
plates. For the killing assay, the molecules were added at the
indicated concentrations in triplicates. FolR1 16D5 TCB was
included as positive control and an untargeted TCB molecule
(binding to CD3 but not to a target cell antigen) was included as
negative control. PBMCs were added to target cells at final E:T
ratio of 10:1. Target cell killing was assessed after 48 h of
incubation at 37.degree. C., 5% CO.sub.2 by quantification of LDH
release into cell supernatants by apoptotic/necrotic cells (LDH
detection kit, Roche Applied Science, #11 644 793 001). Maximal
lysis of the target cells (=100%) was achieved by incubation of
target cells with 1% Triton X-100 1 h before LDH readout. Minimal
lysis (=0%) refers to target cells co-incubated with effector cells
without any TCB.
[0761] The results (FIGS. 14A, 15A, 16, 17, 18A, 19A and 20A) show
that the protease activated TCB with anti-idiotypic CD3 scFv moiety
N-terminally linked by a non-cleavable linker (#7676 and #7611,
FIGS. 5B and D, respectively) were able to significantly reduce
cell lysis on Skov3 and HT29 cells. #7611 (FIG. 5D) led to reduced
killing on Hela cells while anti-idiotypic CD3 scFv 4.15.64 in
#7676 (FIG. 5B) was less efficient in reduction of cell lysis. This
is in line with affinities of the anti-idiotypic CD3 scFv N moiety.
The higher affinity scFv moiety masks more efficiently. Comparable
potency of treated and untreated TCBs suggests Matriptase
expression of Hela and Skov3 cells. Expression of Matriptase seems
to be lower in HT29 cells. Treatment of Mkn-45, a FolR1 negative
cell line, shows only weak killing with all molecules used herein
(FIG. 15A).
Example 8
T-Cell Activation after Co-Incubation of Tumor Cell Lines with
Human PBMCs
[0762] T-cell activation mediated by protease activated TCB
molecules was assessed on Hela, Skov3 and HT29 cells. Human PBMCs
were used as effector cells and the T cell activation was detected
at 48 h of incubation with target cells and the antibodies. Target
cells were plated at a density of 20 000 cells/well using
round-bottom 96-well plates. Molecules were added at the indicated
concentrations in triplicates. FolR1 16D5 TCB was included as
positive control and an untargeted TCB molecule (binding to CD3 but
not to a target cell antigen) was included as negative control.
PBMCs were added to target cells at final E:T ratio of 10:1. T-cell
activation was assessed after 48 h of incubation at 37.degree. C.,
5% CO.sub.2 by quantification of CD25 and CD69 on CD4 positive and
CD8 positive T cells. T cell activation results are consistent with
the results observed in the previous example assessing target cell
killing (Example 7).
Example 9
T-Cell Activation Mediated by Protease-Activated TCBs and Target
Cell Lines Expressing Low Antigen Levels
[0763] T-cell activation mediated by protease activated TCB
molecules was assessed on HT29 cells expressing only low levels of
FolR1 (FIG. 13). Human PBMCs isolated from buffy coat were used as
effector cells. For enriched lymphocyte preparations (buffy coats)
Histopaque-1077 density preparation was used. Buffy coat was
diluted 1:1 with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, w/o
break, room temperature), the plasma above the PBMC-containing
interphase was discarded and PBMCs transferred in a new falcon tube
subsequently filled with 50 ml of PBS. The mixture was centrifuged
(400.times.g, 10 minutes, room temperature), the supernatant
discarded and the PBMC pellet resuspended in 2 ml ACK buffer for
Erythrocytes lysis. After incubation at 37.degree. C. for about 2-3
minutes the tubes were filled with sterile PBS to 50 ml and
centrifuged at 350.times.g for 10 minutes. This washing step was
repeated once prior to resuspension of PBMCs in RPMI1640 medium
containing 2% FCS and 1.times. GlutaMax at 37.degree. C., 5%
CO.sub.2 in cell incubator until further use. Briefly, adherent
target cells were harvested with Trypsin/EDTA, counted, assessed
for viability and resuspended at 0.4.times.10.sup.6 cells/ml in
assay medium (RPMI1640, 2% FCS, 1.times. GlutaMax). Target cells
were plated at a density of 20 000 cells/well using round-bottom
96-well plates. Molecules were added at the indicated
concentrations in triplicates. FolR1 16D5 TCB was included as
positive control and an untargeted TCB molecule (binding to CD3 but
not to a target cell antigen) was included as negative control.
PBMCs were added to target cells at final E:T ratio of 10:1. T-cell
activation was assessed after 48 h of incubation at 37.degree. C.,
5% CO.sub.2 by quantification of CD25 and CD69 on CD4 positive and
CD8-positive T cells. The potency of treated protease activated TCB
is comparable to 16D5 TCB (6298). The 16D5 TCB (inverted format)
show higher potency than the classic format. Masked TCBs with
non-cleavable linker or without Matriptase pre-treatment do not
induce T cell activation on this cell line. For cell lines with low
or medium FolR1 expression levels both anti-idiotypic scFvs are
sufficient in masking the CD3 Fab (FIGS. 22A and B).
Example 10
T-Cell Activation Mediated by Protease Activated TCB with Primary
Cell Line HRCEpiC
[0764] T-cell activation mediated by protease activated TCB
molecules was assessed on primary Human Renal Cortical Epithelial
Cell (ScienceCell) cells expressing only very little amounts of
FolR1 (FIG. 13). Human PBMCs isolated from buffy coat were used as
effector cells. For enriched lymphocyte preparations (buffy coats)
Histopaque-1077 density preparation was used. Buffy coat was
diluted 1:1 with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes,
without break at room temperature), the plasma above the
PBMC-containing interphase was discarded and PBMCs transferred in a
new falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant discarded and the PBMC pellet resuspended in 2 ml ACK
buffer for Erythrocytes lysis. After incubation at 37.degree. C.
for about 2-3 minutes the tubes were filled with sterile PBS to 50
ml and centrifuged at 350.times.g for 10 minutes. This washing step
was repeated once prior to resuspension of PBMCs in RPMI1640 medium
containing 2% FCS and 1.times.GlutaMax at 37.degree. C., 5%
CO.sub.2 in cell incubator until further use. Briefly, adherent
target cells were harvested with Trypsin/EDTA, counted, checked for
viability and resuspended at 0.4.times.10.sup.6 cells/ml in assay
medium (RPMI1640, 2% FCS, 1.times.GlutaMax). Target cells were
plated at a density of 20 000 cells/well using round-bottom 96-well
plates. Protease activatable TCB molecules were added at the
indicated concentrations in triplicates. FolR1 16D5 TCB was
included as positive control and an untargeted TCB molecule
(binding to CD3 but not to a target cell antigen) was included as
negative control. PBMCs were added to target cells at final E:T
ratio of 10:1. T-cell activation was assessed after 48 h of
incubation at 37.degree. C., 5% CO.sub.2 by quantification of CD25
and CD69 on CD4 positive and CD8 positive T cells. Masked 16D5 TCB
does not induce T cell activation upon incubation with primary
human renal cortical epithelial cells despite low level FolR1
expression at the highest concentration of 10.000 pM of TCB,
demonstrating the effectiveness of the anti-idiotype masking
moiety. Little T cell activation can be observed for the 16D5 TCBs
(inverted and classic format) (FIG. 23).
Example 11
Anti-ID CD3 Fab Masking CD3 Binder of 16D5 TCB. Killing on Ovcar3
Cells
[0765] T-cell-mediated target cell killing mediated by protease
activated TCB molecules was assessed on OVCAR3 cells (FIG. 24).
Human PBMCs were used as effector cells and cell killing was
detected at 48 h of incubation with the molecules. Human Peripheral
blood mononuclear cells (PBMCs) were isolated from fresh taken
blood of a healthy donor. 50 ml Leucosep tubes (GreinerBioOne) were
used for preparation. Blood was diluted 1:1 with sterile PBS and
layered over Histopaque gradient (Sigma, #H8889). After
centrifugation (450.times.g, 30 minutes, w/o break, room
temperature), the plasma above the PBMC-containing interphase was
discarded and PBMCs transferred in a new falcon tube subsequently
filled with 50 ml of PBS. The mixture was centrifuged (400.times.g,
10 minutes, room temperature), the supernatant discarded and the
PBMC pellet resuspended in 2 ml ACK buffer for Erythrocytes lysis.
After incubation at 37.degree. C. for about 2-3 minutes the tubes
were filled with sterile PBS to 50 ml and centrifuged at
350.times.g for 10 minutes. This washing step was repeated once
prior to resuspension of PBMCs in RPMI1640 medium containing 2% FCS
and 1.times.GlutaMax at 37.degree. C., 5% CO.sub.2 in cell
incubator until further use. Briefly, adherent target cells were
harvested with Trypsin/EDTA, counted, checked for viability and
resuspended at 0.4.times.10.sup.6 cells/ml in assay medium
(RPMI1640, 2% FCS, 1.times.GlutaMax). Target cells were plated at a
density of 20 000 cells/well using round-bottom 96-well plates. For
the killing assay, the molecules were added at the indicated
concentrations in triplicates. FolR1 16D5 TCB was included as
positive control and an untargeted TCB molecule (binding to CD3 but
not to a target cell antigen) was included as negative control.
PBMCs were added to target cells at final E:T ratio of 10:1. Target
cell killing was assessed after 48 h of incubation at 37.degree.
C., 5% CO.sub.2 by quantification of LDH release into cell
supernatants by apoptotic/necrotic cells (LDH detection kit, Roche
Applied Science, #11 644 793 001). Maximal lysis of the target
cells (=100%) was achieved by incubation of target cells and PBMCs
with 1% Triton X-100 1 h before LDH readout. Minimal lysis (=0%)
refers to target cells co-incubated with effector cells without any
TCB. The result (FIG. 24) shows that protease activated TCB with
anti-idiotypic CD3 4.15.64 crossed Fab N--terminally linked by a
non-cleavable linker is not significantly masking the CD3 binder.
Further, Ovcar3 cells appear to express Matriptase because
untreated molecule also induces killing of these cells.
Example 12
Killing on Skov3 and HeLa Cells with Three Different Human PBMC
Donors
[0766] T-cell killing mediated by protease activated TCB molecules
was assessed on two different cell lines expressing different
levels of FolR1 (FIGS. 25-27). Human PBMCs were used as effector
cells and cell killing was detected at 48 h of incubation with the
molecules. Human Peripheral blood mononuclear cells (PBMCs) were
isolated from buffy coats obtained from healthy human donors. For
enriched lymphocyte preparations (buffy coats) Histopaque-1077
density preparation was used. Blood/buffy coat was diluted 1:1 with
sterile PBS and layered over Histopaque gradient (Sigma, #H8889).
After centrifugation (450.times.g, 30 minutes, w/o break, room
temperature), the plasma above the PBMC-containing interphase was
discarded and PBMCs transferred in a new falcon tube subsequently
filled with 50 ml of PBS. The mixture was centrifuged (400.times.g,
10 minutes, room temperature), the supernatant discarded and the
PBMC pellet resuspended in 2 ml ACK buffer for Erythrocytes lysis.
After incubation at 37.degree. C. for about 2-3 minutes the tubes
were filled with sterile PBS to 50 ml and centrifuged at
350.times.g for 10 minutes. This washing step was repeated once
prior to resuspension of PBMCs in RPMI1640 medium containing 10%
FCS and 1.times.GlutaMax. PBMCs were resuspended in RPMI1640 medium
containing 10% FCS, 1.times.GlutaMax and 10% DMSO. PBMCs were
frozen overnight at -80.degree. C. in Cool Cell boxes before they
were transferred to liquid nitrogen. 24 h before assay start, PBMCs
were thawed and kept in RPMI1640 medium containing 10% FCS and
1.times.GlutaMax at 37.degree. C., 5% CO2 in cell incubator. The
day before assay start adherent target cells were harvested with
Trypsin/EDTA, counted, checked for viability and resuspended at
0.4.times.10.sup.6 cells/ml in appropriate medium. Target cells
were plated at a density of 20 000 cells/well using flat-bottom
96-well plates. On the day of assay start PBMCs were counted and
checked for viability. PBMCs were centrifuged at 350 g for 5 min
and resupsended in assay medium (RPMI1640, 2% FCS,
1.times.GlutaMax). The medium of target cells was removed and PBMCs
were added to the target cells before diluted antibodies were added
at the indicated concentrations in triplicates. FolR1 16D5 TCB was
included as positive control and an untargeted TCB molecule
(binding to CD3 but not to a target cell antigen) was included as
negative control. PBMCs were added to target cells at E:T ratio of
10:1. Target cell killing was assessed after 48 h of incubation at
37.degree. C., 5% CO2 by quantification of LDH release into cell
supernatants by apoptotic/necrotic cells (LDH detection kit, Roche
Applied Science, #11 644 793 001). Maximal lysis of the target
cells (=100%) was achieved by incubation of target cells with 1%
Triton X-100 2 h before LDH readout. Minimal lysis (=0%) refers to
target cells co-incubated with effector cells without any TCB.
[0767] The results (FIGS. 25-27) show that FolR1 TCB with scFv
4.32.63 N-terminally linked by a non-cleavable linker (FIG. 5D)
induced reduced killing on Hela cells at concentration of 100 pM
and on Skov3 cells at a concentration of 10 nM. FolR1 TCB with scFv
4.15.64 N-terminally linked by a non-cleavable linker (FIG. 5B) was
less efficient in reducing killing on Skov3 cells at a
concentration of 10 nM. The stronger mask, meaning the
anti-idiotypic scFv 4.32.63 with the higher affinity, is more
efficient in masking the CD3 binder than the weak anti-idiotypic
scFv 4.15.64. Comparable potency of treated and untreated TCBs
suggests protease, e.g. Matriptase, expression by Hela and Skov3
cells.
Example 13
Preparation of the HER1 Binding Antibody GA201 Masked with an
Anti-Idiotype GA201 scFv
[0768] The following molecules were prepared in this example:
[0769] 1: GA201 IgG1 antibody with N-terminal fusion of an
anti-idiotypic GA201 scFv and Matrix Metalloprotease site in
glycine serine linker (SEQ ID NOs 32 and 34); and [0770] 2:
HER1-binding IgG1 antibody GA201 (SEQ ID NOs 32 and 33).
[0771] Schematic illustrations thereof are shown in FIGS. 28 and
29. The GA201 anti-idiotypic (ID) binder sequence was obtained by
RT-PCR (reverse transcription) from RNA of Hybridoma cells using
degenerated primers binding to the ends of the variable light and
heavy chain, respectively. Hybridoma cells were obtained by
immunization of mice. Single chain Fv (scFv) DNA sequence synthesis
with flanking singular restriction endonuclease sites was ordered
at Geneart and cloned as N-terminal fusion at the GA201 light
chain.
[0772] A Roche expression vector was used for the construction of
all heavy and light chain scFv fusion protein encoding expression
plasmids. The vector is composed of the following elements: [0773]
a hygromycin resistance gene as a selection marker, [0774] an
origin of replication, oriP, of Epstein-Barr virus (EBV), [0775] an
origin of replication from the vector pUC18 which allows
replication of this plasmid in E. coli [0776] a beta-lactamase gene
which confers ampicillin resistance in E. coli, [0777] the
immediate early enhancer and promoter from the human
cytomegalovirus (HCMV), [0778] the human 1-immunoglobulin
polyadenylation ("poly A") signal sequence, and [0779] unique BamHI
and XbaI restriction sites.
[0780] The molecules were produced by co-transfecting human
embryonic kidney 293-F cells growing in suspension with the
mammalian expression vectors using the FreeStyle.TM. 293 Expression
System according to the manufacturer's instruction (Invitrogen,
USA). Briefly, suspension FreeStyle.TM. 293-F cells were cultivated
in FreeStyle.TM. 293 Expression medium at 37.degree. C./8% CO.sub.2
and the cells were seeded in fresh medium at a density of
1-2.times.10.sup.6 viable cells/ml on the day of transfection.
DNA-293fectin.TM. complexes were prepared in Opti-MEM I medium
(Invitrogen, USA) using 325 .mu.l of 293fectin.TM. (Invitrogen,
Germany) and 250 .mu.g of heavy ("GA201 heavy chain") and light
chain ("anti-GA201 VH-VL scFv MMP cleavable linker G4S GA201 light
chain" or "GA201 light chain") plasmid DNA in a 1:1 molar ratio for
a 250 ml final transfection volume. Antibody containing cell
culture supernatants were harvested 7 days after transfection by
centrifugation at 14000 g for 30 minutes and filtered through a
sterile filter (0.22 .mu.m). Supernatants were stored at
-20.degree. C. until purification.
[0781] The secreted protein was purified from cell culture
supernatants by affinity chromatography using ProteinA affinity
chromatography, followed by size exclusion chromatography. Briefly,
sterile filtered cell culture supernatants were applied to a HiTrap
ProteinA HP (5 ml) column equilibrated with PBS buffer (10 mM
Na2HPO4, 1 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4). Unbound
proteins were washed out with equilibration buffer. Antibody and
antibody variants were eluted with 0.1 M citrate buffer, pH 2.8,
and the protein containing fractions were neutralized with 0.1 ml 1
M Tris, pH 8.5. Then, the eluted protein fractions were pooled,
concentrated with an Amicon Ultra centrifugal filter device (MWCO:
30 K, Millipore) to a volume of 3 ml and loaded on a Superdex200
HiLoad 120 ml 16/60 gel filtration column (GE Healthcare, Sweden)
equilibrated with 20 mM Histidin, 140 mM NaCl, pH 6.0. Fractions
containing purified GA201-anti-GA201-scFv or GA201 with less than
5% high molecular weight aggregates were pooled and stored as 1.0
mg/ml aliquots at -80.degree. C.
[0782] For Protein analytics after size exclusion chromatography,
the purity and molecular weight of the molecules in the single
fractions were analyzed by SDS-PAGE in the absence of a reducing
agent and staining with Coomassie (InstantBlue.TM., Expedeon). The
NuPAGE.RTM. Pre-Cast gel system (4-12% Bis-Tris, Invitrogen or 3-8%
Tris-Acetate, Invitrogen) was used according to the manufacturer's
instruction.
[0783] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm divided
by the molar extinction coefficient calculated on the basis of the
amino acid sequence. Purity and molecular weight of the molecules
after the final purification step were analyzed by CE-SDS analyses
in the presence and absence of a reducing agent. The Caliper
LabChip GXII system (Caliper Lifescience) was used according to the
manufacturer's instruction. The aggregate content of the molecules
was analyzed by high-performance SEC using a Superdex 200
analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM
KH2PO4, 250 mM KCl, pH 7.0 running buffer at 25.degree. C. 25 .mu.g
protein were injected on the column at a flow rate of 0.5 ml/min
and eluted isocratic over 50 minutes.
[0784] The final purity of all molecules was .gtoreq.95% monomer
content as detected by high performance SEC. The molecular weight
of the anti-idiotypic scFv masked GA201 was determined by CE-SDS
analysis as 216.3 kDa under non reducing conditions (FIG. 1A) and
under reducing conditions as 58.3 kDa for the GA201 heavy chain and
60.3 kDa for the scFv linked GA201 light chain (FIG. 30B),
respectively. The molecular weight based on the amino acid sequence
was calculated as 49.2 kDa for the heavy chain and 51.9 kDa for the
scFv fused GA201 light chain, which indicates glycosylation of both
chains in HEK293 cells.
TABLE-US-00008 TABLE 3 Summary of production and purification of
protease-activated GA201 IgG (FIG. 28) and GA201 (FIG. 29) control
molecules. Molecule Supernatant Protein A-Yield SEC-Yield 1 1.0 L
1.3 mg 0.4 mg 2 1.0 L 26.4 mg 24 mg
Example 14
Masking Effect of an Anti-Idiotypic scFv for GA201 IgG
[0785] The efficiency of masking the HER1 binding of GA201 by
N-terminal fusion of an anti-idiotypic GA201 scFv was shown by FACS
analysis on HER1 expressing H322M cells and Surface Plasmon
Resonance (SPR) analysis on a HER1 coated chip surface. For
proteolytic cleavage of GA201-anti-GA201-scFv recombinant active
human MMP2 (Calbiochem) was used. 1 mg of GA201 anti-idiotypic scFv
fused to GA201 by a glycine serine linker containing a MMP cleavage
site was incubated with 1.2 .mu.g MMP2 overnight at 37.degree. C.
in PBS.
[0786] For FACS analysis of HER1 binding of cleaved and uncleaved
GA201-anti-GA201-scFv, the non-small cell lung cancer line H322M
was used. Cells were adjusted to 1.times.10.sup.6/ml and
distributed to a 96-well round-bottom plate. The molecules were
added and incubated on ice for 30 minutes. Cells were washed once
with FACS buffer (PBS+2% FCS+0.1% sodium azide) and re-suspended
with a F(ab')2-goat anti-human IgG Fc secondary antibody FITC
conjugate (ThermoFisher Scientific). After another 20 minutes on
ice, cells were washed twice and re-suspended in FACS buffer and
analyzed in a BD FACS Canto II. 10000 cells were measured and the
median of the fluorescence signal was used for analysis. Before
MMP-2 cleavage of GA201-anti-GA201-scFv no binding to HER1 on H322M
cells was measurable, indicating complete masking of the GA201
binding domains by the anti-idiotypic scFv (FIG. 31). Binding of
uncleaved GA201-anti-GA201-scFv was comparable to an unspecific
isotype IgG control antibody (FIG. 31). In contrast, MMP cleavage
of the anti-idiotypic scFv leads to activation of GA201 and binding
to HER1 on H322M cells was restored to similar levels as the
unmasked parental antibody GA201 (FIG. 31)
[0787] To confirm the FACS binding data of masked GA201 binding
after MMP cleavage, we also performed a SPR experiment as second
analytical method using a Biacore T100 instrument (GE Healthcare
Biosciences AB, Uppsala, Sweden). HER1 was immobilized on the
surface of a CMS biosensorchip using standard amine-coupling
chemistry. The HER1 extracellular domain was injected in sodium
acetate, pH 5.0 at 1 .mu.g/ml. Reference control flow cells were
treated in the same way but with vehicle buffer only.
GA201-anti-GA201-scFv, before and after an overnight MMP cleavage,
and GA201 were diluted in 1.times.PBS pH 7.4, 0.05% Tween20 Roche
Diagnostics GmbH) and injected at increasing concentrations between
3.125 and 50 nM with a flow rate of 30 .mu.l/min. The association
phase was 3 minutes and the dissociation time was 10 minutes. HER1
binding was regenerated with an inject of 0.85% phosphoric acid for
30 s at a flow rate of 5 .mu.l/min. Kinetic rate constants and
equilibrium dissociation constants were calculated by using the 1:1
Langmuir binding model within the Biaevaluation software. A K.sub.D
value of 1 nM for binding of HER1 was determined for the GA201
parental unmasked antibody (FIG. 32). After an overnight MMP-2
incubation of GA201-anti-GA201-scFv, a K.sub.D value of 2 nM was
measured with similar k.sub.a and k.sub.d rate constants for
association and dissociation as the unmasked control antibody,
indicating complete restoration of HER1 binding by protease
cleavage (FIG. 32). Uncleaved GA201-anti-GA201-scFv did not show
any binding to HER1 in SPR analysis (FIG. 32). In summary, we have
demonstrated a complete loss of binding to HER1 by fusion of an
anti-idiotypic scFv to the N-terminus of the IgG.sub.1 antibody
GA201 with two independent analytical methods. Furthermore, binding
to HER1 was fully restored by removal of the scFv through protease
cleavage in the MMP cleavage site in the glycine serine linker.
Example 15
Preparation of Anti FolR1/Anti-CD3 and antiMesothelin/Anti-CD3 T
Cell Bispecific (TCB) Molecules with Anti CD3 scFv
[0788] Several T cell bispecific (TCB) molecules with various
anti-idiotypic scFv were produced and are schematically depicted in
FIGS. 33A-33J with their respective ID number. The following
molecules were prepared: [0789] ID 8364: "FolR1 16D5 2+1 IgG,
classic format (anti idiotypic scFv 4.32.63--MMP9--MK062 Matriptase
site--CD3--N-terminal fused to FolR1 VH--inert Fc) with N-terminal
fused anti CD3 scFv 4.32.63 and MMP9--MK062 protease linker" (FIG.
33A, SEQ ID NOs 1, 3 and 72). [0790] ID 8363: "FolR1 16D5 2+1 IgG,
classic format (anti idiotypic scFv 4.32.63--Cathepsin S/B
site--CD3--N-terminal fused to FolR1 VH--inert Fc) with N-terminal
fused anti CD3 scFv 4.32.63 and Cathepsin S/B protease linker"
(FIG. 33B, SEQ ID NOs 1, 3 and 85). [0791] ID 8365: "FolR1 16D5 2+1
IgG, inverted format, (anti idiotypic scFv 4.32.63--MK062
Matriptase linker--CD3--N-terminal fused to CD3 VL--inert Fc) with
N-terminal fused anti CD3 scFv 4.32.63 and MK062 Matriptase linker"
(FIG. 33C, SEQ ID NOs 1, 3, 73 and 74). [0792] ID 8366: "FolR1 16D5
2+1 IgG, inverted format, (anti idiotypic scFv
4.32.63--non-cleavable GS linker--CD3--N-terminal fused to CD3
VL--inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and
non-cleavable GS linker" (FIG. 33D). [0793] ID 8672: "aMesothelin
2+1 IgG, classic format, MSLN charged variants, CD3 crossed (anti
idiotypic scFv 4.32.63--MMP9--MK062 Matriptase--CD3--N-terminal
fused to aMesothelin VH--inert Fc) with N-terminal fused anti CD3
scFv 4.32.63 and MMP9--MK062 Matriptase" (FIG. 33E, SEQ ID NOs 77,
78, 81, 82). [0794] ID 8673: "aMesothelin 2+1 IgG, classic format,
MSLN charged variants, CD3 crossed (anti idiotypic scFv
4.32.63--non-cleavable GS linker--CD3--N-terminal fused to
aMesothelin VH--inert Fc) with N-terminal fused anti CD3 scFv
4.32.63 non-cleavable GS linker" (FIG. 33F). [0795] ID 8674:
"aMesothelin 2+1 IgG, inverted format, MSLN charged variants, CD3
crossed (anti idiotypic scFv 4.32.63--MMP9--MK062
Matriptase--CD3--N-terminal fused to CD3 VH--inert Fc) with
N-terminal fused anti CD3 scFv 4.32.63 and MMP9--MK062 Matriptase"
(FIG. 33G, SEQ ID NOs 76, 77, 78, 79). [0796] ID 8675: "aMesothelin
2+1 IgG, inverted format, MSLN charged variants, CD3 crossed (anti
idiotypic scFv 4.32.63--non-cleavable GS linker--CD3--N-terminal
fused to CD3 VH--inert Fc) with N-terminal fused anti CD3 scFv
4.32.63 and non-cleavable GS linker" (FIG. 33H). [0797] ID 8505:
"aMesothelin 2+1 IgG, inverted format, MSLN charged variants, CD3
(aMesothelin HC N-terminally fused to CD3 VL--inert Fc)" (FIG.
33I). [0798] ID 8676: "aMesothelin 2+1 IgG, classic format, MSLN
charged variants, CD3 crossed (aMesothelin IgG with CD3--N-terminal
fused to aMesothelin VH--inert Fc)" (FIG. 33J)
[0799] The variable domains were subcloned in frame with the
pre-inserted domains into the respective recipient mammalian
expression vector. Protein expression is driven by an MPSV promoter
and a synthetic polyA signal sequence is present at the 3' end of
the CDS. In addition each vector contains an EBV OriP sequence.
[0800] The molecules (except 8505, this molecule was produced by
co-transfecting CHO cells growing in suspension with the mammalian
expression vectors. Transient transfection was done at Evitria AG
(Switzerland).) were produced by co-transfecting HEK293-EBNA cells
growing in suspension with the mammalian expression vectors using
polyethylenimine (PEI). For transfection HEK293 EBNA cells were
cultivated in serum free ExCell culture medium containing 6 mM
L-glutamine and 250 mg/l G418. For the production in 600 ml
tubespin flasks (max. working volume 400 ml) 800 million HEK293
EBNA cells were seeded 24 hours before transfection without G418.
For transfection 800 mio cells were centrifuged for 5 min at
210.times.g and supernatant was replaced by 40 ml pre-warmed CD CHO
medium containing 6 mM L-Glutamine. Expression vectors were mixed
with 40 ml CD CHO medium containing 6 mM L-Glutamine to a total
amount of 400 .mu.g DNA. After addition of 1080 .mu.l PEI solution
(2.7 .mu.g/ml) the mixture was vortexed for 15 s and subsequently
incubated for 10 min at room temperature. Afterwards cells were
mixed with the DNA/PEI solution, transferred to a 600 ml tubespin
flask and incubated for 3 hours at 37.degree. C. in an incubator
with a 5% CO.sub.2 atmosphere. After incubation, 320 ml ExCell+6 mM
L-glutamine+5 g/L Pepsoy+1.0 mM VPA+3 g/l glucose medium was added
and cells were cultivated for 24 hours prior to feeding with 7%
Feed 7. After 6-7 days the cultivation supernatant was collected
for purification by centrifugation for 20-30 min at 210.times.g
(Sigma 8K centrifuge). The solution was sterile filtered (0.22
.mu.m filter) and sodium azide in a final concentration of 0.01%
w/v was added. The solution was kept at 4.degree. C. until
purification.
[0801] The secreted protein was purified from cell culture
supernatants by affinity chromatography using ProteinA affinity
chromatography, followed by one to two size exclusion
chromatographic steps. For affinity chromatography supernatant was
loaded on a Protein A MabSelectSure (CV=5 mL, GE Healthcare)
equilibrated with 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH
7.5. Unbound protein was removed by washing with at least 10 column
volumes 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5 and
target protein was eluted in 20 column volumes (gradient from
0%-100%) 20 mM Sodium Citrate, 100 mM Sodium Chloride, 100 mM
Glycine, pH 3.0. Protein solution was neutralized by adding 1/10 of
0.5 M Na2HPO4 pH 8.0. Target protein was concentrated with
Amicon.RTM. Ultra-15 Ultracel 30K (Merck Millipore Ltd.) to a
volume of 4 ml maximum prior loading on a HiLoad Superdex 200
column (GE Healthcare) equilibrated with 20 mM Histidine, 140 mM
NaCl, 0.01% Tween pH 6.0.
[0802] The protein concentration of purified protein samples was
determined by measuring the optical density (OD) at 280 nm divided
by the molar extinction coefficient calculated on the basis of the
amino acid sequence.
[0803] Purity and molecular weight of the molecules after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction.
[0804] The aggregate content of the molecules was analyzed using a
TSKgel G3000 SW XL analytical size-exclusion column (Tosoh) in 25
mM K2HPO4, 125 mM NaCl, 200 mM L-arginine monohydrocloride, 0.02%
(w/v) NaN3, pH 6.7 running buffer at 25.degree. C.
[0805] The final quality of all molecules was good, with
.gtoreq.95% monomer content.
TABLE-US-00009 TABLE 4 Summary of production and purification of
protease activated TCB molecules. Analytical SEC Titer Yield
(HMW/Monomer/LMW) Molecule [mg/l] [mg/l] [%] 1 (8364) 34.55 1.72
0.68/99.32/0 2 (8363) 33.75 1.59 4.02/95.98/0 3 (8365) 5.35 0.24
2.71/96.46/0.83 4 (8366) 4.2 0.43 4.908/96.02/0 5 (8672) 13.8 1.59
3.96/96.04/0 6 (8673) 14 1.99 2.15/97.85/0 7 (8674) 3.6 0.96
6.27/93.73/0 8 (8675) 5.2 0.59 5.81/90.63/3.57 9 (8505) 120 20.46
0.47/99.32/0.22 10 (8676) 22.5 3.84 1.98/96.21/1.81
Example 16
Quality Control and Stability--Capillary Electrophoresis SDS
Analysis of Different TCB Molecules
[0806] Purity and molecular weight of the molecules after the final
purification step were analyzed by CE-SDS analyses in the presence
and absence of a reducing agent. The Caliper LabChip GXII system
(Caliper Lifescience) was used according to the manufacturer's
instruction. Comparison of untreated molecules (stored at 4.degree.
C.), treated molecules (treated with appropriate recombinant
protease (R&D Systems) for 24 h at 37.degree. C. and molecule
incubated for 72 h at 37.degree. C. (FIGS. 34, 35A, and 35B).
Comparison of the untreated and treated molecule shows complete
cleavage of the anti ID scFv after rhMatriptase/ST14 treatment for
the inverted format containing MK062 Matriptase linker but
incomplete cleavage of MMP9-MK062 Matriptase linker. rhCathepsin B
and rhCathepsin S treatment is incomplete as well. The conditions
for the purified enzymes have not been optimal. Molecules incubated
at 37.degree. C. for 72 h are running on the same height than pure
molecules suggesting that the molecules are stable at 37.degree. C.
for the time of in vitro assay duration. Pre-stained protein Marker
Mark 12 (Invitrogen) was used for estimation of correct molecule
weight.
Example 17
Comparison of Different Linkers and Formats of Protease Activated
FolR1 TCBs
[0807] Jurkat NFAT activation assay. Jurkat NFAT activation assay
for comparison of different formats and linkers of protease
activated TCB. Jurkat-NFAT reporter cell line (Promega) is a human
acute lymphatic leukemia reporter cell line with a NFAT promoter,
expressing human CD3c. If the TCB binds the tumor target and the
CD3 binder (crosslinkage) binds the CD3.epsilon. Luciferase
expression can be measured in Luminescence after addition of
One-Glo substrate (Promega). 20.000 target cells were seeded in
96-well white walled clear bottom plate (Greiner BioOne) in 50
ul/well Jurkat medium (RPMI1640, 2 g/l Glucose, 2 g/l NaHCO.sub.3,
10% FCS, 25 mM HEPES, 2 mM L-Glutamin, 1.times.NEAA,
1.times.Sodium-pyruvate) without Hygromycine. Plates were incubated
for about 20 hours at 37.degree. C. Jurkat-NFAT reporter cells were
harvested and viability was assessed using ViCell. Cells were
resuspended in Jurkat medium without Hygromycine and 50 .mu.l per
well (50.000 cells/well) were added. The E:T ratio was 2.5:1 (based
on cell number seeded). Antibodies were diluted in Jurkat medium
without Hygromycine and 50 ul/well were added. Cells were incubated
at 37.degree. C. for 6 h in a humidified incubator before they were
taken out of the incubator for about 10 min to adapt to room
temperature prior to Luminescence read out. 50 .mu.l/well of
ONE-Glo solution were added to wells and incubated for 10 min at
room temperature in the dark. Luminescence was detected using
WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/well as
detection time. Comparison of the pretreated protease activated TCB
(8364, grey filled squares) and FolR1 TCB (black triangles pointing
down) showed that potency after cleavage is recovered completely.
No Luminescence was detectable for cells incubated with the masked
TCB (containing a GS non-cleavable linker, grey triangles pointing
up) and the non-targeted TCB control (empty triangle pointing down)
for both cell lines in this concentration range. The dotted line
shows the Luminescence of target cells and effector cells without
any TCB (FIGS. 36A and 36B).
Example 18
Tumor Cell Cytotoxicity Mediated by Different Formats of Protease
Activated TCB
[0808] T-cell killing mediated by protease activated TCB molecules
was assessed on cell lines expressing different levels of FolR1.
Human Peripheral blood mononuclear cells (PBMCs) were isolated from
buffy coats obtained from healthy human donors. Buffy coat was
diluted 1:1 with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, w/o
break, room temperature) the PBMC-containing interphase was
transferred in a new falcon tube that was subsequently filled with
50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes,
room temperature), the supernatant was discarded and the PBMC
pellet was resuspended in 2 ml ACK buffer for Erythrocytes lysis.
After incubation for about 2-3 minutes at 37.degree. C. the tubes
were filled with sterile PBS to 50 ml and centrifuged for 10
minutes at 350.times.g. This washing step was repeated once prior
to resuspension of PBMCs in RPMI1640 medium containing 10% FCS,
1.times.GlutaMax and 10% DMSO. PBMCs were slowly frozen in
CoolCell.RTM. Cell Freezing Containers (BioCision) at -80.degree.
C. and then transferred to liquid nitrogen. One day before assay
start adherent target cells were harvested with Trypsin/EDTA,
counted, checked for viability and resuspended in assay medium
(RPMI1640, 2% FCS, 1.times.GlutaMax). Target cells were plated at a
density of 20 000 cells/well using 96-well flat-bottom plates and
incubated for about 20 h at 37.degree. C. in a humidified
incubator. About 20 h before assay start PBMCs were thawed in
RPMI1640 medium (10% FCS, 1.times.GlutaMax). PBMCs were centrifuged
at 350 g for 7 min. The pellet was resuspended in fresh medium
(RPMI1640, 10% FCS, 1.times.GlutaMax) and incubated for max 24 h at
37.degree. C. in a humidified incubator. On the day of the assay
start PBMCs were harvested and centrifuged at 350 g for 7 min. The
pellet was resuspended in assay medium and 0.2 mio PBMCs in 100
ul/well (E:T 10:1, based on the number of seeded target cells) were
added to the target cells. The molecules were diluted in assay
medium (RPMI1640, 2% FCS, 1.times.GlutaMax) and 50 ul/well were
added at the indicated concentrations in triplicates before the
plates were incubated for about 48 h at 37.degree. C. in a
humidified incubator. Target cell killing was assessed after 48 h
of incubation at 37.degree. C., 5% CO2 by quantification of LDH
release into cell supernatants by apoptotic/necrotic cells (LDH
detection kit, Roche Applied Science, #11 644 793 001). Maximal
lysis of the target cells (=100%) was achieved by incubation of
target cells with 1% Triton X-100 20 h before LDH readout. Minimal
lysis (=0%) refers to target cells co-incubated with effector cells
without any TCB.
[0809] The results (FIGS. 37A and 37B) show the comparison of two
different formats of the Protease activated TCBs both containing
the anti idiotypic CD3 scFv 4.32.63 linked with a MK062 Matriptase
linker. The inverted format of the protease activated TCB (8365,
grey circles) seems to be more potent in killing (HeLa and Skov-3
target cells) than the classic format of the protease activated TCB
(8408, dark grey triangles pointing up). However the inverted
molecule containing the non-cleavable linker (8366, light grey
squares) is less efficient in masking than the classic molecule
(8409, dark grey triangles pointing down).
[0810] FIG. 37C HeLa target cell cytotoxicity. Comparison of
classic Protease activated TCB containing the anti idiotypic CD3
scFv 4.32.63 and GS linkers with different protease sites. Protease
activated TCB containing the MMP9-Matriptase MK062 linker (8364,
grey squares) reaches the potency of FolR1 TCB (light grey
triangles pointing down) whereas the protease activated TCB
containing only Matriptase MK062 (light grey rhomb) is less potent
in killing HeLa cells. Molecules containing Cathepsin site (grey
circles) or non-cleavable linker (black triangles pointing down)
are comparable. FIG. 37D Skov-3 target cell cytotoxicity.
Comparison of classic Protease activated TCB containing the anti
idiotypic CD3 scFv 4.32.63 and GS linkers with different protease
sites. Protease activated TCB containing the MMP9-Matriptase MK062
linker (8364, grey squares) nearly reaches the potency of FolR1 TCB
(light grey triangles pointing down) whereas the protease activated
TCB containing only Matriptase MK062 (light grey rhomb) is less
potent in killing Skov-3 cells. The molecule containing Cathepsin
site (grey circles) is less potent than the molecule containing
only the Matriptase MK062 site and the molecule containing the
non-cleavable linker (black triangles pointing down) only induces
killing below 10% in the indicated concentration range for Skov-3
cells.
Example 19
T-Cell Activation after Co-Incubation of Human Renal Epithelial
Cortical Cells or Human Bronchial Epithelial Cells with TCBs and
Human PBMCs
[0811] T-cell activation mediated by protease activated TCB
molecules was assessed for HRCEpi (Human renal cortical epithelial
cells) and HBEpiC (human bronchial epithelial cells expressing only
little amounts of FolR1. Human PBMCs were used as effector cells
and T cell activation markers were stained after 48 h of incubation
with the molecules and cells. Human Peripheral blood mononuclear
cells (PBMCs) were isolated from buffy coats obtained from healthy
human donors. Buffy coat was diluted 1:1 with sterile PBS and
layered over Histopaque gradient (Sigma, #H8889). After
centrifugation (450.times.g, 30 minutes, w/o break, room
temperature) the PBMC-containing interphase was transferred in a
new falcon tube subsequently filled with 50 ml of PBS. The mixture
was centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant was discarded and the PBMC pellet was resuspended in 2
ml ACK buffer for Erythrocytes lysis. After incubation for about
two minutes at 37.degree. C. the tubes were filled with sterile PBS
to 50 ml and centrifuged for 10 minutes at 350.times.g. This
washing step was repeated once prior to resuspension of PBMCs in
RPMI1640 medium containing 10% FCS, 1.times.GlutaMax and 10% DMSO.
PBMCs were slowly frozen in CoolCell.RTM. Cell Freezing Containers
(BioCision) at -80.degree. C. and then transferred to liquid
nitrogen. One day before the assay was started adherent target
cells were harvested with Trypsin/EDTA, counted, checked for
viability and resuspended in assay medium (RPMI1640, 2% FCS,
1.times.GlutaMax). Target cells were plated at a density of 20 000
cells/well using 96-well flat-bottom plates and incubated for about
20 h at 37.degree. C. in a humidified incubator. About 20 h before
assay start PBMCs were thawed in RPMI1640 medium (10% FCS,
1.times.GlutaMax). PBMCs were centrifuged for 7 min at 350 g. The
pellet was resuspended in fresh medium (RPMI1640, 10% FCS,
1.times.GlutaMax) and incubated for max 24 h at 37.degree. C. in a
humidified incubator. On the day of the assay start PBMCs were
harvested and centrifuged for 7 min at 350 g. The pellet was
resuspended in assay medium and 0.2 mio PBMCs in 100 ul/well (E:T
10:1, based on the number of seeded target cells) were added to the
target cells. The molecules were diluted in assay medium (RPMI1640,
2% FCS, 1.times.GlutaMax) and added at the indicated concentrations
in triplicates before the plates were incubated for about 48 h at
37.degree. C. in a humidified incubator.
[0812] T-cell activation was assessed after 48 h of incubation at
37.degree. C., 5% CO2 by quantification of CD25 and CD69 on CD4
positive and CD8 positive T cells. FolR1 16D5 TCB (6298) and an
untargeted TCB (binding to CD3 but not to a target cell antigen,
7235) were included as controls. Each point represents the mean
value of triplicates of three different human PBMC donors. Standard
deviation is indicated in error bars. Unpaired t test was used for
statistical analysis. The results show an increase in CD69 for CD8
positive cells for the FolR1 TCB that is significantly higher than
the median fluorescence intensity for the protease activated TCBs
(FIGS. 38A and 38B).
Example 20
Tumor Cell Cytotoxicity Mediated by Different Formats of Protease
Activated Mesothelin (MSLN) TCB
[0813] T-cell killing mediated by protease activated TCB molecules
was assessed on cell lines expressing different levels of
Mesothelin (MSLN). Human Peripheral blood mononuclear cells (PBMCs)
were isolated from buffy coats obtained from healthy human donors.
Buffy coat was diluted 1:1 with sterile PBS and layered over
Histopaque gradient (Sigma, #H8889). After centrifugation
(450.times.g, 30 minutes, w/o break, room temperature) the
PBMC-containing interphase was transferred in a new falcon tube
subsequently filled with 50 ml of PBS. The mixture was centrifuged
(400.times.g, 10 minutes, room temperature), the supernatant was
discarded and the PBMC pellet was resuspended in 2 ml ACK buffer
for Erythrocytes lysis. After incubation for about two minutes at
37.degree. C. the tubes were filled with sterile PBS to 50 ml and
centrifuged for 10 minutes at 350.times.g. This washing step was
repeated once prior to resuspension of PBMCs in RPMI1640 medium
containing 10% FCS, 1.times.GlutaMax and 10% DMSO. PBMCs were
slowly frozen in CoolCell.RTM. Cell Freezing Containers (BioCision)
at -80.degree. C. and then transferred to liquid nitrogen. Adherent
target cells were harvested with Trypsin/EDTA, counted, checked for
viability and resuspended in assay medium (RPMI1640, 2% FCS,
1.times.GlutaMax) one day before the assay was started. Target
cells were plated at a density of 20 000 cells/well using 96-well
flat-bottom plates and incubated for about 20 h at 37.degree. C. in
a humidified incubator. PBMCs were thawed in RPMI1640 medium (10%
FCS, 1.times.GlutaMax) about 20 h before assay start. PBMCs were
centrifuged for 7 min at 350 g. The pellet was resuspended in fresh
medium (RPMI1640, 10% FCS, 1.times.GlutaMax) and incubated for max
24 h at 37.degree. C. in a humidified incubator. On the day of the
assay start PBMCs were harvested and centrifuged for 7 min at 350
g. The pellet was resuspended in assay medium and 0.2 mio PBMCs in
100 ul/well (E:T 10:1, based on the number of seeded target cells)
were added to the target cells. The molecules were diluted in assay
medium (RPMI1640, 2% FCS, 1.times.GlutaMax) and added at the
indicated concentrations in triplicates before the plates were
incubated for about 48 h at 37.degree. C. in a humidified
incubator. Target cell killing was assessed after 48 h of
incubation at 37.degree. C., 5% CO2 by quantification of LDH
release into cell supernatants by apoptotic/necrotic cells (LDH
detection kit, Roche Applied Science, #11 644 793 001). Maximal
lysis of the target cells (=100%) was achieved by incubation of
target cells with 1% Triton X-100 20 h before LDH readout. Minimal
lysis (=0%) refers to target cells co-incubated with effector cells
without any TCB.
[0814] The results (FIGS. 39A and 39B) show target cell killing
mediated by Protease activated MSLN TCB (8672) for NCI H596 and
AsPC-1 cell lines. The protease activated TCBs nearly reaches the
potency of MSLN TCB (8676) for NCI H596 and AsPC-1. The molecule
containing the non-cleavable GS linker (8673) does not induce
killing in the indicated concentration range for both cell
lines.
Example 21
Jurkat-NFAT Reporter Assay to Monitor Target Expression (FOLR1 TCB)
and Protease Activity (Protease Activated FOLR1 TCB) in Primary
Tumor Samples
[0815] The intention of this assay was to show tumor target antigen
(FolR1) expression and activity of tumor specific proteases like
MMP9, Matriptase or Cathepsin in human tumor samples.
[0816] Jurkat-NFAT reporter cell line (Promega) is a human acute
lymphatic leukemia reporter cell line with a NFAT promoter,
expressing human CD3c. Luciferase expression can be measured, if
the T cell bispecific molecule binds the tumor target and the
CD3.epsilon. (crosslinkage). Luminescence is measured after
addition of One-Glo substrate (Promega).
[0817] Primary tumor samples were received from Indivumed GmbH,
Germany. Samples were shipped over night in transport medium. About
24 h after surgery the sample was cut in small pieces. 96-well
white walled, flat (clear) bottom plate was prepared by adding 18
ul cold Matrigel (Matrigel (734-1101, Corning/VWR). Plate was
incubated for 2 min at 37.degree. C. before tumor pieces were added
(triplicates). 33 ul of cold Matrigel were added per well and plate
was incubated again for 2 min at 37.degree. C. 50 ul of antibody
dilution (in Jurkat medium without Hygromycine but containing
2.times.Penicillin/Streptomycine) was added per well and plate was
incubated for about 48 hours at 37.degree. C., 5% CO.sub.2.
[0818] Jurkat-NFAT reporter cells were harvested and viability was
assessed using ViCell. Cells were centrifuged at 350.times.g, 7 min
before they were resuspended in Jurkat medium without Hygromycine
and 50 .mu.l per well (50.000 cells/well) were added. Plate was
incubated for 5 h at 37.degree. C. in a humidified incubator before
it was taken out for Luminescence read out. 80 ul of each well were
transferred into a white walled 96-well plate. 27 .mu.l/well of
ONE-Glo solution were added to each well and incubated for 10 min
at room temperature in the dark. Luminescence was detected using
WALLAC Victor3 ELISA reader (PerkinElmer2030), 1 sec/well as
detection time.
[0819] Jurkat NFAT reporter cells are activated after co-incubation
with FolR1 TCB (6298) and Protease activated FolR1 TCB containing
MMP9-Matriptase cleavage site (8364). Protease activated FolR1 TCBs
(8363, 8408) and control TCBs (8409, 7235) do not induce Luciferase
expression. The dotted line indicates the baseline Luminescence for
Jurkat NFAT cells co-incubated with tumor (FIG. 40).
Example 22
Serum Stability of Protease Activated TCBs
[0820] Capillary electrophoresis of protease activated TCBs after
incubation in human serum. Molecules were incubated for 0 or 14
days in human IgG depleted serum at 37.degree. C. in a humidified
incubator (5% CO2). All molecules were purified by affinity
chromatography (ProteinA) and then analyzed by Capillary
electrophoresis.
[0821] 100 ug of each molecule was added either in buffer
(Histidine buffer (Bichsel) with 0.01% Tween-20) or in human serum
(IgG depleted, SP1839, TL-15216, 16FSP63814). The concentration of
the molecules was higher than 2 mg/ml and the final concentration
was 0.5 mg/ml. The pretreatment for one molecule (8408) was done
with rhMatriptase (R&D Systems) for 24 h at 37.degree. C., 5%
CO2 in a humidified incubator (otherwise pH of serum could change).
The samples for day 0 were directly frozen in liquid nitrogen and
stored at -80.degree. C. until analysis. Samples for day 14 were
incubated for 14 days at 37.degree. C., 5% CO2 in a humidified
incubator until they were also snap frozen.
[0822] Prior to CE-SDS analysis all samples were purified via HPLC
affinity chromatography (Agilent technologies 1200series, column:
Upchurch scientific C-130B, packaging material: Applied Biosystems
POROS 20A 60 .mu.l, buffer: 10 mM Tris, 50 mM Glycine, 500 mM NaCl
pH 8.0 and pH 2.0, injection volume: 100 .mu.l, flow rate 1 ml/min,
collection: peak based, neutralization: 0.5 M Na-phosphate pH 8.0
10% volume). Protease activated TCB is stable in human IgG depleted
serum for a minimum of 14 days (FIGS. 41A-41C).
Example 23
Design of Anti Her2/Anti-CD3 and AntiFolR1/Anti-CD3 T Cell
Bispecific (TCB) Molecules with Anti CD3 scFv
[0823] Several T cell bispecific (TCB) molecules designed and are
schematically depicted in FIGS. 42A-42F with their respective ID
number. The following molecules were designed: [0824] ID 8955:
"Herceptarg 2+1 IgG, classic format, Herceptarg charged variants,
CD3 crossed (anti idiotypic scFv 4.32.63--MMP9--MK062
Matriptase--CD3--N-terminal fused to Herceptarg VH--inert Fc) with
N-terminal fused anti CD3 scFv 4.32.63 and MMP9--MK062 Matriptase"
(FIG. 42A, SEQ ID NOs 81, 132, 133 and 136). [0825] ID 8957:
"Herceptarg 2+1 IgG, classic format, Herceptarg charged variants,
CD3 crossed (anti idiotypic scFv 4.32.63--non cleavable GS
linker--CD3--N-terminal fused to Herceptarg VH--inert Fc) with
N-terminal fused anti CD3 scFv 4.32.63 and non cleavable GS linker"
(FIG. 42B, SEQ ID NOs 81, 132, 133 and 135). [0826] ID 8959:
"Herceptarg 2+1 IgG, classic format, Herceptarg charged variants,
CD3 crossed (Herceptarg IgG with CD3--N-terminal fused to
Herceptarg VH--inert Fc)" (FIG. 42C, SEQ ID NOs 81, 132, 133 and
134). [0827] ID 8997: "FolR1 36F2 2+1 IgG, classic format, FolR1
36F2 charged variants, CD3 crossed (anti idiotypic scFv
4.32.63--MMP9--MK062 Matriptase--CD3--N-terminal fused to FolR1
36F2 VH--inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and
MMP9--MK062 Matriptase" (FIG. 42D, SEQ ID NOs 81, 137, 138 and
139). [0828] ID 8998: "FolR1 36F2 2+1 IgG, classic format, FolR1
36F2 charged variants, CD3 crossed (anti idiotypic scFv
4.32.63--non cleavable GS linker--CD3--N-terminal fused to FolR1
36F2 VH--inert Fc) with N-terminal fused anti CD3 scFv 4.32.63 and
non cleavable GS linker" (FIG. 42E, SEQ ID NOs 81, 137, 138 and
140). [0829] ID 8996: "FolR1 36F2 2+1 IgG, classic format, FolR1
36F2 charged variants, CD3 crossed (FolR1 36F2 IgG with
CD3--N-terminal fused to FolR1 36F2 VH--inert Fc)" (FIG. 42F, SEQ
ID NOs 81, 137, 138 and 141).
[0830] The variable domains were subcloned in frame with the
pre-inserted domains into the respective recipient mammalian
expression vector. Protein expression is driven by an MPSV or CMV
(for Herceptarg) promoter and a synthetic polyA signal sequence is
present at the 3' end of the CDS. In addition each vector contains
an EBV OriP sequence.
Example 24
Primary Cell Cytotoxicity Mediated by Protease Activated FolR1
TCB
[0831] T-cell killing mediated by protease activated FolR1 TCB
molecule was assessed on primary cell lines expressing low levels
of FolR1 (FIG. 43). Human Peripheral blood mononuclear cells
(PBMCs) were isolated from buffy coats obtained from healthy human
donors. Buffy coat was diluted 1:1 with sterile PBS and layered
over Histopaque gradient (Sigma, #H8889). After centrifugation
(450.times.g, 30 minutes, w/o break, room temperature) the
PBMC-containing interphase was transferred in a new falcon tube
that was subsequently filled with 50 ml of PBS. The mixture was
centrifuged (400.times.g, 10 minutes, room temperature), the
supernatant was discarded and the PBMC pellet was resuspended in 2
ml ACK buffer for Erythrocytes lysis. After incubation for about
2-3 minutes at 37.degree. C. the tubes were filled with sterile PBS
to 50 ml and centrifuged for 10 minutes at 350.times.g. This
washing step was repeated once prior to resuspension of PBMCs in
RPMI1640 medium containing 10% FCS, 1.times.GlutaMax and 10% DMSO.
PBMCs were slowly frozen in CoolCell.RTM. Cell Freezing Containers
(BioCision) at -80.degree. C. and then transferred to liquid
nitrogen. One day before assay start adherent target cells were
harvested with Trypsin/EDTA, counted, checked for viability and
resuspended in assay medium (RPMI1640, 2% FCS, 1.times.GlutaMax).
Target cells were plated at a density of 20 000 cells/well using
96-well flat-bottom plates and incubated for about 20 h at
37.degree. C. in a humidified incubator. About 20 h before assay
start PBMCs were thawed in RPMI1640 medium (10% FCS, 1.times.
GlutaMax). PBMCs were centrifuged at 350 g for 7 min. The pellet
was resuspended in fresh medium (RPMI1640, 10% FCS,
1.times.GlutaMax) and incubated for max 24 h at 37.degree. C. in a
humidified incubator. On the day of the assay start PBMCs were
harvested and centrifuged at 350 g for 7 min. The pellet was
resuspended in assay medium and 0.2 mio PBMCs in 100 ul/well (E:T
10:1, based on the number of seeded target cells) were added to the
target cells. The molecules were diluted in assay medium (RPMI1640,
2% FCS, 1.times.GlutaMax) and 50 ul/well were added at the
indicated concentrations in triplicates before the plates were
incubated for about 48 h, 72 h or 96 h at 37.degree. C. in a
humidified incubator. Target cell killing was assessed after 48 h,
72 h and 96 h of incubation at 37.degree. C., 5% CO2 by
quantification of LDH release into cell supernatants by
apoptotic/necrotic cells (LDH detection kit, Roche Applied Science,
#11 644 793 001). Maximal lysis of the target cells (=100%) was
achieved by incubation of target cells with 1% Triton X-100 20 h
before LDH readout. Minimal lysis (=0%) refers to target cells
co-incubated with effector cells without any TCB.
[0832] Human Bronchial Epithelial Cell toxicity mediated by human
PBMCs and 100 nM or 10 nM of FolR1 TCB is higher compared to
Protease activated TCB.
Example 25
FolR1 Negative Target Cell Cytotoxicity Mediated by Protease
Activated FolR1 TCB
[0833] T-cell killing mediated by protease activated FolR1 TCB
molecule was assessed on FolR1 negative Mkn-45 cell line (FIG. 44).
Human Peripheral blood mononuclear cells (PBMCs) were isolated from
buffy coats obtained from healthy human donors. Buffy coat was
diluted 1:1 with sterile PBS and layered over Histopaque gradient
(Sigma, #H8889). After centrifugation (450.times.g, 30 minutes, w/o
break, room temperature) the PBMC-containing interphase was
transferred in a new falcon tube that was subsequently filled with
50 ml of PBS. The mixture was centrifuged (400.times.g, 10 minutes,
room temperature), the supernatant was discarded and the PBMC
pellet was resuspended in 2 ml ACK buffer for Erythrocytes lysis.
After incubation for about 2-3 minutes at 37.degree. C. the tubes
were filled with sterile PBS to 50 ml and centrifuged for 10
minutes at 350.times.g. This washing step was repeated once prior
to resuspension of PBMCs in RPMI1640 medium containing 10% FCS,
1.times.GlutaMax and 10% DMSO. PBMCs were slowly frozen in
CoolCell.RTM. Cell Freezing Containers (BioCision) at -80.degree.
C. and then transferred to liquid nitrogen. One day before assay
start adherent target cells were harvested with Trypsin/EDTA,
counted, checked for viability and resuspended in assay medium
(RPMI1640, 2% FCS, 1.times.GlutaMax). Target cells were plated at a
density of 20 000 cells/well using 96-well flat-bottom plates and
incubated for about 20 h at 37.degree. C. in a humidified
incubator. About 20 h before assay start PBMCs were thawed in
RPMI1640 medium (10% FCS, 1.times.GlutaMax). PBMCs were centrifuged
at 350 g for 7 min. The pellet was resuspended in fresh medium
(RPMI1640, 10% FCS, lx GlutaMax) and incubated for max 24 h at
37.degree. C. in a humidified incubator. On the day of the assay
start PBMCs were harvested and centrifuged at 350 g for 7 min. The
pellet was resuspended in assay medium and 0.2 mio PBMCs in 100
ul/well (E:T 10:1, based on the number of seeded target cells) were
added to the target cells. The molecules were diluted in assay
medium (RPMI1640, 2% FCS, 1.times.GlutaMax) and 50 ul/well were
added at the indicated concentrations in triplicates before the
plates were incubated for about 48 h and 72 h at 37.degree. C. in a
humidified incubator. Target cell killing was assessed after 48 h,
72 h and 96 h of incubation at 37.degree. C., 5% CO2 by
quantification of LDH release into cell supernatants by
apoptotic/necrotic cells (LDH detection kit, Roche Applied Science,
#11 644 793 001). Maximal lysis of the target cells (=100%) was
achieved by incubation of target cells with 1% Triton X-100 20 h
before LDH readout. Minimal lysis (=0%) refers to target cells
co-incubated with effector cells without any TCB. Protease
activated TCB did not induce target cell killing at 100 nM.
TABLE-US-00010 EXAMPLARY SEQUENCES SEQ ID Construct Amino acid
Sequence No LC Common light
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 1 chain pETR13197
PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP
EDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPP
SSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT
TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS anti CD3
(CH2527 QIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG 2 VH_3-23(12)
VL7- KCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQI 46(13)) scFv15-
NNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGS Matriptase MK062
GGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRA CH2527 VH3_23-VH12
SKSVSTSNYSYIHWYQQKPGQPPKLLIKYVSYLESGVPARFS CH1 FolR1 16D5 VH
GSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGCGTKL CH1 hum Fc knob PG
EIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGSEV LALA, pETR15422
QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45A)
KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVES
GGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE
WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNS
LKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA
PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 16D5 VH CH1
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA 3 Fc hole P329G LALA
PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTL HRYF, pETR15214
YLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS
CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV
SKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGK anti CD3 (CH2527
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 4 VH_3-23(12) VL7-
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG Matriptase MK062
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT CH2527 VH3_23-VH12
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 FolR1 16D5 VH
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum Fc knob PG
EIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGSEV LALA, pETR15599
QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45B)
KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVES
GGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE
WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNS
LKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA
PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti CD3 (CH2527
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 5 VH_3-23(12) VL7-
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG non-cleavable linker
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT CH2527 VH3_2-VH12
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 FolR1 16D5 VH
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum Fc knob PG
EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSEV LALA, pETR15603
QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG (FIG. 45C)
KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVES
GGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE
WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNS
LKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA
PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti CD3 (CH2527
QIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG 6 VH_3-23(12) VL7-
KCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQI 46(13)) scFv15 non-
NNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGS cleavable linker
GGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRA CH2527 VH3_23-VH12
SKSVSTSNYSYIHWYQ CH1 FolR1 16D5 VH
QKPGQPPKLLIKYVSYLESGVPARFSGSGSGTDFTLNIHPVE CH1 hum Fc knob PG
EEDAATYYCQHSREFPWTFGCGTKLEIKGGGGSGGGGSGG LALA, pETR14759
GGSGGGGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGS (FIG. 45D)
LRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNN
YATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCV
RHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSC
AASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDY
AAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWE
WSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK MK062
Protease linker GGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGS 7 Combined
NF9/Mat5 GGGGSVHMPLGFLGPGRSRGSFPGGGGS 8 linker Combined MK062
GGGGSGGGGSRQARVVNGGGGGSVPLSLYSGGGGGSGG 9 MMP9 GGS Combined MK062
GGGGSGGGGSRQARVVNGVPLSLYSGGGGGSGGGGS 10 MMP9 H2527 CDR H1 Kabat
TYAMN 11 CH252 CDR H2 Kabat RIRSKYNNYATYYADSVKG 12 CH252 CDR H3
Kabat HGNFGNSYVSWFAY 13 FolR1 CDR H1 Kabat NAWMS 14 FolR1 CDR H2
Kabat RIKSKTDGGTTDYAAPVKG 15 FolR1 CDR H3 Kabat PWEWSWYDY 16 CLC
CDR1 L1 Kabat GSSTGAVTTSNYAN 17 CLC CDR L2 Kabat GTNKRAP 18 CLC CDR
L3 Kabat ALWYSNLWV 19 Anti-ID 4.15.64 CDR DYSIH 20 H1 Kabat Anti-ID
4.15.64 CDR WINTETGEPAYADDFKG 21 H2 Kabat Anti-ID 4.15.64 CDR
PYDYDVLDY 22 H3 Kabat Anti-ID 4.15.64 CDR L1 RASKSVSTSNYSYIH 23
Kabat Anti-ID 4.15.64 CDR L2 YVSYLES 24 Kabat Anti-ID 4.15.64 CDR
L3 QHSREFPWT 25 Kabat Anti-ID 4.32.63 CDR SYGVS 26 H1 Kabat Anti-ID
4.32.63 CDR IIWGDGSTNYHSALIS 27 H2 Kabat Anti-ID 4.32.63 CDR
GITTVVDDYYAMDY 28 H3 Kabat Anti-ID 4.32.63 CDR L1 RASENIDSYLA 29
Kabat Anti-ID 4.32.63 CDR L2 AATFLAD 30 Kabat Anti-ID 4.32.63 CDR
L3 QHYYSTPYT 31 Kabat anti HER1 (GA201
QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVRQAP 32 heavy chain, pUC-Exp-
GQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYM GA201-HC) (FIG. 45E)
ELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti HER1 (GA201 light
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQKPG 33 chain, pUC-Exp-GA201-
KAPKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDF LC) (FIG. 45F)
ATYYCLQHNSFPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC anti HER1
(anti-GA201 EVQLEQSGPVLVKPGTSVKMSCKASGYTFTDYYINWIIQSHG 34 VH-VL
scFv MMP KCLEWIGVINPDSGGTDYNQNFKGKATLTVDKSSTTAYMELT cleavable
linker G4S SLTSEDSAVYYCARRDSYGFDYWGQGTTLTVSSGGGGSGG GA201 light
chain, pUC- GGSGGGGSGGGGSDIVLTQTPKFLLVPAGDRITMTCKASLS I_GA201
MMP_LC) VTNDVAWYQQKPGQSPKLLLYYASNRNAGVPDRFTGSGYG (FIG. 45G)
TDFTFTITTLQAEDLAVYFCQQDYTSPPTFGCGTKLEIRGGG
GSGGGGSGPLGLWSQGGGGSGGGGSGGGGSGGDIQMTQ
SPSSLSASVGDRVTITCRASQGINNYLNWYQQKPGKAPKRLI
YNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQ
HNSFPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV
CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC MMP Protease linker
GGGGSGGGGSGPLGLWSQGGGGSGGGGSGGGGSGG 35 Protease recognition
RQARVVNG 36 site 1 Protease recognition VHMPLGFLGPGRSRGSFP 37 site
2 Protease recognition RQARVVNGXXXXXVPLSLYSG 38 site 3 Protease
recognition RQARVVNGVPLSLYSG 39 site 4 Protease recognition PLGLWSQ
40 site 5 4.15.64 Anti-idiotypic
QIQLVQSGPELKKPGETVRISCKASGYTFTDYSIHWVKQAPG 41 scFv
KCLKWMGWINTETGEPAYADDFKGRFAFSLETSASTAYLQI
NNLKNEDTATFFCAHPYDYDVLDYWGQGTSVTVSSGGGGS
GGGGSGGGGSGGGGSDTVLTQSPASLGVSLGQRATISCRA
SKSVSTSNYSYIHWYQQKPGQPPKLLIKYVSYLESGVPARFS
GSGSGTDFTLNIHPVEEEDAATYYCQHSREFPWTFGCGTKL EIK 4.32.63
Anti-idiotypic QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 42
scFv GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL EIK Anti-CD3 variable
heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 43 chain (VH)
PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY
LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT VSS CD3 heavy chain TYAMN
44 (VH)_CDR1 CD3 heavy chain RIRSKYNNYATYYADSVKG 45 (VH)_CDR2 CD3
heavy chain HGNFGNSYVSWFAY 46 (VH)_CDR3 Anti-FolR1 16D5
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA 47 variable region
PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTL
YLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSS anti-idiotypic GA201 DYYIN
48 CDR H1 Kabat anti-idiotypic GA201 VINPDSGGTDYNQNFKG 49 CDR H2
Kabat anti-idiotypic GA201 RDSYGFDY 50 CDR H3 Kabat anti-idiotypic
GA201 KASLSVTNDVA 51 CDR L1 Kabat anti-idiotypic GA201 YASNRNA 52
CDR L2 Kabat anti-idiotypic GA201 QQDYTSPPT 53 CDR L3 Kabat hu CD3E
MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSI 54
SGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL
SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC
MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRG
AGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQR RI LC Common light
QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 55 chain pETR13197
PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP V region
EDEAEYYCALWYSNLWVFGGGTKLTVL GA201 CDR H1 Kabat DYKIH 56 GA201 CDR
H2 Kabat YFNPNSGYSTYAQKFQG 57 GA201 CDR H3 Kabat LSPGGYYVMDA 58
GA201 CDR L1 Kabat RASQGINNYLN 59 GA201 CDR L2 Kabat NTNNLQT 60
GA201 CDR L3 Kabat LQHNSFPT 61 SEQ ID Construct DNA Sequence No LC
Common CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 62 light chain
GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC pETR13197
ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC
TTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC
CCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTG
ACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGC
GCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAG
CTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTG
TTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTG
GTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC
TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCAC
CACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTA
CCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAG
CTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGG CCCCCACCGAGTGCAGCTGA
anti CD3 CAGATCCAGCTGGTGCAGAGCGGCCCTGAGCTGAAGAAACCCGGC 63 (CH2527
GAGACAGTGCGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACC VH_3-23(12)
GACTACAGCATCCACTGGGTCAAGCAGGCCCCTGGCAAGTGCCTG VL7-46(13))
AAGTGGATGGGCTGGATCAACACCGAGACAGGCGAGCCCGCCTAC scFv15-
GCCGACGATTTCAAGGGCAGATTCGCCTTCAGCCTGGAAACCAGC Matriptase
GCCAGCACCGCCTACCTGCAGATCAACAACCTGAAGAACGAGGAC MK062
ACCGCCACCTTTTTCTGCGCCCACCCCTACGACTACGACGTGCTG CH2527
GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGCGGAGGC VH3_23-VH12
GGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGG CH1 FolR1
GAGGCGGATCTGATACCGTGCTGACACAGAGCCCTGCCAGCCTGG 16D5 VH CH1
GAGTGTCCCTGGGACAGAGAGCCACCATCAGCTGTCGGGCCAGCA hum Fc knob
AGAGCGTGTCCACCAGCAACTACAGCTATATCCACTGGTATCAGCA PG LALA,
GAAGCCCGGCCAGCCCCCCAAGCTGCTGATCAAATACGTGTCCTA pETR15422
CCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGG (FIG. 45H)
CACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGC
CGCCACCTACTACTGCCAGCACAGCAGAGAGTTCCCTTGGACCTTC
GGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGG
AGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGGG
GGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGT
GCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGAT
CTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCT
ACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAAT
GGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTA
CGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACA
GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACA
GCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA
CCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGG
CCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGC
TGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGG
AACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTG
CCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAAC
CACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAG
AGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGATCCGAGGT
GCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC
CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGC
GTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGT
GGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTA
CGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGC
AAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACAC
CGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGAT
TATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACC
AAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACA
TCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTC
CCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGC
GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTAC
TCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACA
CAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGT
CGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 16D5
GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGC 64 VH CH1 Fc
GGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCC hole P329G
AACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCT LALA HRYF,
CGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCAC pETR15214
GGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGAC
GATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGA
AGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGG
TACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCT
AGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAG
AGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGA
CTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCT
GACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGG
CCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCT
GGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAA
CACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAAC
TCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 65 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG Matriptase
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MK062
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC CH2527
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG VH3_23-VH12
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 FolR1
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC 16D5 VH CH1
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT hum Fc knob
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC PG LALA,
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT pETR15599
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG (FIG. 451)
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGG
GGGGGAAGTGGGGGCGGAGGCAGTGGGGGCGGAGGATCCGAGG
TGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA
TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACC
TACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA
TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT
ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGAC
AGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAG
GACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAAC
AGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTG
ACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTG
GCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGG
CTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTG
GAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGT
GCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGT
GCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAA
CCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAA
GAGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGAG
GTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGT
TCCCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAAC
GCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGA
GTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGA
TTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGAT
AGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAG
ACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTA
CGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAG
TACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAG
CACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTA
CTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGAC
CAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCT
GTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGG
AACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCA
AAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAG
AACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 66 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG
VL7-46(13)) GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv
4.32.63 AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC cleavable
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC linker
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH3_23-VH12
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 FolR1
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT 16D5 VH CH1
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC hum Fc knob
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT PG LALA,
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG pETR15603
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG (FIG. 45J)
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGG
GGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAG
GTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGG
ATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC
CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGA
ATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTAC
TACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGA
CAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA
GGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAA
CAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGT
GACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCT
GGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGG
GCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTT
GGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCC
GTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACC
GTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCC
AAGAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGCTCCGA
GGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGG
TTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAAC
GCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGA
GTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGA
TTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGAT
AGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAG
ACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTA
CGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAG
TACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAG
CACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTA
CTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGAC
CAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGC
AGCGGCCTGTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGC
TCCCTGGGAACACAGACATATATCTGTAATGTCAATCACAAGCCTTC
CAACACCAAAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAA
AACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG
GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAAC
AGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC
CTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAG
CCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAG
CTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGG
CAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT GA anti CD3
CAGATCCAGCTGGTGCAGAGCGGCCCTGAGCTGAAGAAACCCGGC 67 (CH2527
GAGACAGTGCGGATCAGCTGCAAGGCCAGCGGCTACACCTTCACC VH_3-23(12)
GACTACAGCATCCACTGGGTCAAGCAGGCCCCTGGCAAGTGCCTG VL7-46(13))
AAGTGGATGGGCTGGATCAACACCGAGACAGGCGAGCCCGCCTAC scFv15 non-
GCCGACGATTTCAAGGGCAGATTCGCCTTCAGCCTGGAAACCAGC cleavable
GCCAGCACCGCCTACCTGCAGATCAACAACCTGAAGAACGAGGAC linker
ACCGCCACCTTTTTCTGCGCCCACCCCTACGACTACGACGTGCTG CH2527
GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGCGGAGGC VH3_23-VH12
GGAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGG CH1 FolR1
GAGGCGGATCTGATACCGTGCTGACACAGAGCCCTGCCAGCCTGG 16D5 VH CH1
GAGTGTCCCTGGGACAGAGAGCCACCATCAGCTGTCGGGCCAGCA hum Fc knob
AGAGCGTGTCCACCAGCAACTACAGCTATATCCACTGGTATCAGCA PG LALA,
GAAGCCCGGCCAGCCCCCCAAGCTGCTGATCAAATACGTGTCCTA pETR14759
CCTGGAAAGCGGCGTGCCCGCCAGATTTTCTGGCTCTGGCAGCGG (FIG. 45K)
CACCGACTTCACCCTGAACATCCACCCCGTGGAAGAGGAAGATGC
CGCCACCTACTACTGCCAGCACAGCAGAGAGTTCCCTTGGACCTTC
GGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGG
AGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGG
GGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGG
TGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGA
TCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACC
TACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAA
TGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACT
ACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGAC
AGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAG
GACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAAC
AGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTG
ACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTG
GCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGG
CTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTG
GAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGT
GCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGT
GCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAA
CCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAA
GAGCTGTGATGGCGGAGGAGGGTCCGGAGGCGGAGGCTCCGAGG
TGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC
CCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCCAACGC
GTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGT
GGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTA
CGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGC
AAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACAC
CGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGAT
TATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACC
AAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACA
TCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTC
CCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGC
GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTAC
TCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACA
CAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGT
CGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA MK062
GGCGGGGGAGGCTCCGGAGGCGGCGGAAGTAGACAGGCCAGAG 68 Protease
TCGTGAACGGGGGAGGGGGGGGAAGTGGGGGCGGAGGCAGTGG linker GGGCGGAGGATCC
anti HER1 CAGGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAGAAACCCGG 69 (GA201
heavy GTCCTCTGTCAAGGTGTCATGCAAGGCTAGCGGATTCACCTTTACA chain, pUC-
GACTACAAAATCCACTGGGTTAGGCAGGCACCTGGCCAAGGACTC Exp-GA201-
GAATGGATGGGGTATTTCAACCCAAATTCCGGCTACTCTACCTATG HC)
CCCAGAAGTTTCAGGGAAGAGTGACTATTACAGCTGATAAGAGTAC
CAGCACTGCATACATGGAGCTGTCCTCTCTTCGCTCAGAGGACACC
GCCGTCTACTATTGTGCTCGGCTGAGCCCCGGTGGCTACTATGTG
ATGGATGCATGGGGGCAGGGAACAACCGTAACAGTGTCCTCTGCG
TCGACTAAGGGCCCTTCAGTTTTTCCACTCGCCCCCAGTAGCAAGT
CCACATCTGGGGGTACCGCTGCCCTGGGCTGCCTTGTGAAAGACT
ATTTCCCTGAACCAGTCACTGTGTCATGGAATAGCGGAGCCCTGAC
CTCCGGTGTACACACATTCCCCGCTGTGTTGCAGTCTAGTGGCCTG
TACAGCCTCTCCTCTGTTGTGACCGTCCCTTCAAGCTCCCTGGGGA
CACAGACCTATATCTGTAACGTGAATCATAAGCCATCTAACACTAAA
GTAGATAAAAAAGTGGAGCCCAAGAGTTGCGACAAAACACACACCT
GTCCCCCTTGCCCAGCCCCCGAGCTTCTGGGAGGCCCTAGCGTCT
TTCTCTTCCCACCCAAGCCTAAGGATACTCTGATGATATCCAGGAC
CCCAGAAGTTACATGCGTGGTCGTGGACGTCTCACACGAGGACCC
CGAAGTGAAATTTAACTGGTACGTTGATGGTGTGGAAGTCCATAAT
GCCAAGACCAAGCCTAGAGAGGAGCAATACAACAGTACATATCGC
GTGGTAAGCGTGTTGACCGTTCTCCACCAGGACTGGCTCAATGGG
AAAGAATACAAGTGTAAAGTGTCCAACAAAGCTCTGCCAGCACCCA
TCGAGAAGACTATTTCTAAGGCCAAAGGCCAGCCCCGGGAGCCTC
AGGTCTATACACTTCCACCCTCAAGGGATGAACTGACCAAGAACCA
AGTGAGCTTGACTTGCCTGGTAAAGGGGTTCTACCCTTCCGACATC
GCTGTGGAGTGGGAGTCTAATGGACAACCAGAAAACAATTACAAAA
CCACACCCCCTGTCCTCGACAGTGATGGCAGCTTTTTCCTGTATAG
CAAACTTACCGTTGACAAGTCCAGATGGCAGCAGGGAAACGTGTTC
TCATGTAGCGTCATGCACGAAGCTTTGCATAACCACTACACACAGA
AAAGCCTCAGCCTGAGTCCAGGGAAG anti HER1
GACATCCAAATGACCCAGTCACCTAGTAGCCTCTCCGCCTCTGTTG 70 (GA201 light
GCGACAGGGTGACAATTACATGCAGAGCTTCACAGGGTATCAACAA chain, pUC-
TTACCTGAACTGGTATCAGCAGAAACCAGGGAAGGCCCCCAAGCG Exp-GA201-
CTTGATATATAACACCAATAACCTGCAAACTGGCGTCCCTAGCCGG LC)
TTCTCCGGATCTGGTAGTGGCACCGAATTTACACTCACCATCAGCT
CCCTGCAGCCAGAGGATTTCGCCACATACTATTGTCTTCAGCATAA
TTCTTTCCCCACCTTTGGGCAAGGAACTAAACTGGAGATTAAGCGT
ACTGTCGCCGCTCCCTCTGTGTTCATTTTTCCTCCAAGTGATGAGC
AGCTCAAAAGCGGTACCGCATCCGTTGTGTGCCTGCTTAACAACTT
CTATCCCCGGGAAGCCAAGGTCCAATGGAAGGTGGACAATGCTCT
GCAGTCAGGAAACAGTCAGGAGAGCGTAACCGAGCAGGATTCCAA
AGACTCTACTTACTCATTGAGCTCCACCCTGACACTCTCTAAGGCA
GACTATGAAAAGCATAAAGTGTACGCCTGTGAGGTTACCCACCAGG
GCCTGAGTAGCCCTGTGACAAAGTCCTTCAATAGGGGAGAGTGC HER1 (anti-
GAGGTTCAGCTGGAGCAGTCAGGACCTGTGCTGGTGAAGCCTGGG 71 GA201 VH-
ACTTCAGTGAAGATGTCCTGTAAGGCTTCTGGATACACATTCACTG VL scFv MMP
ACTACTATATAAACTGGATAATACAGAGCCATGGAAAGTGTCTTGAG cleavable
TGGATTGGAGTTATTAATCCTGACAGCGGTGGTACTGACTACAACC linker G4S
AGAACTTCAAGGGCAAGGCCACATTGACTGTTGACAAGTCCTCCAC GA201 light
CACAGCCTACATGGAACTCACTAGCCTGACATCTGAGGACTCTGCA chain, pUC-
GTCTATTATTGTGCAAGAAGGGATTCTTACGGCTTTGACTACTGGG I_GA201_MMP_
GCCAAGGCACCACTCTCACAGTCTCCTCAGGCGGAGGTGGCTCAG LC)
GGGGAGGCGGTAGCGGCGGAGGTGGCTCAGGGGGAGGCGGTAG
CGACATTGTGCTGACCCAGACTCCCAAATTCCTGCTTGTGCCAGCA
GGAGACAGGATTACCATGACCTGCAAGGCCAGTCTGAGTGTGACT
AATGATGTAGCTTGGTATCAACAGAAACCAGGGCAGTCTCCTAAAC
TGCTGTTATACTATGCATCCAATCGCAACGCTGGAGTCCCTGATCG
CTTCACTGGCAGTGGATATGGGACGGATTTCACTTTCACCATCACC
ACTTTGCAGGCTGAAGACCTGGCAGTTTATTTCTGTCAGCAGGATT
ATACCTCTCCTCCGACGTTCGGTTGTGGCACCAAGCTAGAAATCCG
TGGTGGCGGCGGTTCTGGCGGAGGGGGTTCTGGCCCCCTGGGGC
TATGGAGCCAGGGTGGCGGCGGTTCTGGCGGAGGGGGTTCTGGC
GGTGGTGGCTCTGGCGGTGACATCCAAATGACCCAGTCACCTAGT
AGCCTCTCCGCCTCTGTTGGCGACAGGGTGACAATTACATGCAGA
GCTTCACAGGGTATCAACAATTACCTGAACTGGTATCAGCAGAAAC
CAGGGAAGGCCCCCAAGCGCTTGATATATAACACCAATAACCTGCA
AACTGGCGTCCCTAGCCGGTTCTCCGGATCTGGTAGTGGCACCGA
ATTTACACTCACCATCAGCTCCCTGCAGCCAGAGGATTTCGCCACA
TACTATTGTCTTCAGCATAATTCTTTCCCCACCTTTGGGCAAGGAAC
TAAACTGGAGATTAAGCGTACTGTCGCCGCTCCCTCTGTGTTCATT
TTTCCTCCAAGTGATGAGCAGCTCAAAAGCGGTACCGCATCCGTTG
TGTGCCTGCTTAACAACTTCTATCCCCGGGAAGCCAAGGTCCAATG
GAAGGTGGACAATGCTCTGCAGTCAGGAAACAGTCAGGAGAGCGT
AACCGAGCAGGATTCCAAAGACTCTACTTACTCATTGAGCTCCACC
CTGACACTCTCTAAGGCAGACTATGAAAAGCATAAAGTGTACGCCT
GTGAGGTTACCCACCAGGGCCTGAGTAGCCCTGTGACAAAGTCCT TCAATAGGGGAGAGTGC SEQ
ID Construct Amino acid Sequence No anti CD3 (CH2527
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 72 VH_3-23(12) VL7-
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG MMP9 Matriptase
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 CH2527
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS VH3_23-VH12 CH1
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL FolR1 16D5 VH CH1
EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSEVQ hum Fc knob PG LALA,
LLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK pETR16546 (FIG. 45L)
GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVES
GGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE
WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNS
LKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA
PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 16D5 HC
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQA 73 CH2527-VH3_23-12
PGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTL HC Fc knob PG LALA,
YLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSA pCON999 (FIG. 45M)
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGG
GLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVS
RIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAE
DTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALG
APIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKG
FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK anti ID CD3 scFv
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 74 4.32.63 MK062
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS protease site CD3 VL
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG CLambda, pETR16544
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL
EIKGGGGSGGGGSRQARVVNGGGGGSGGGGSGGGGSQA
VVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG
QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED
EAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSS
EELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTP
SKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS anti ID CD3 scFv
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 75 4.32.63 non-cleavable
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS linker CD3 VL
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG CLambda, pETR16545
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL
EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP
GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPE
DEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPS
SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT
PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS aMSLN RG7787 VH
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA 76 CH1 EE CD3 CH2527-
PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVY VH3_23-12 VL CH1 Fc
MELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSA knob PG LALA,
STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWN pETR15445
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSL
TVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIG
GTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCAL
WYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK aMSLN
RG7787 VH QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA 77 CH1 EE Fc
hole P329G PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVY LALA, pETR15444
MELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
VNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS
CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aMSLN RG7787 VL Ck
DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGK 78 RK, pETR15443
APKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT
YYCQQWSKHPLTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKS
GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC anti ID CH2527
4.32.63 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 79 CD3 CH2527 VH
23-12 GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS Ck, MMP9-MK062
site, LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG pETR16758
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL
EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSEVQ
LLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGK
GLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
anti ID CH2527 4.32.63 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 80
CD3 CH2527 VH 23-12 GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS Ck,
non-cleavable LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG linker,
pETR16759 GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL
EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSEV
QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG
KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW
KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC
CD3 CH2527 VH 23- EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQA 81
12-Ck, pETR13811 PGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLY
LQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVT
VSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE
KHKVYACEVTHQGLSSPVTKSFNRGEC anti CD3 (CH2527
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 82 VH 3-23(12) VL7-
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG MMP9 Matriptase
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 aMSLN VH
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 EE CH2527-
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL VL7_46-13 CH1 hum Fc
EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV knob PG LALA,
VTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG pETR16751
QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED
EAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSC
KASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYNQ
KFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYDG
RGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQV
YTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK anti
CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 83
VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG
non-cleavable linker GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT aMSLN
VH CH1 EE CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS
CH2527-VL7_46-13 GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum
Fc knob PG EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ LALA, pETR16752
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP
GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPE
DEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS
CKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSYN
QKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGYD
GRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKT
HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK CH2527
XFab aMSLN QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 84 RG7787 HC
EE Fc PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP knob PG LALA,
EDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLA pETR16764
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKV
SCKASGYSFTGYTMNWVRQAPGQGLEWMGLITPYNGASSY
NQKFRGKATMTVDTSTSTVYMELSSLRSEDTAVYYCARGGY
DGRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK anti
CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 85
VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG
Cathepsin S/B site GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT CH2527
VH3_23-VH12 CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 FolR1
16D5 VH GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum Fc knob
PG EIKGGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGSEV LALA, pETR16550
QLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPG
KGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQ
MNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVS
SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTY1
CNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVES
GGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLE
WVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNS
LKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS
NTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGA
PIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Combined MMP9
GGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGS 86 MK062, 33 AA for CD3 Combined
MMP9 GGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSGG 87 MK062, 35 AA for Her1
Cathepsin S/B GGGGSGGGGSGGGGSFVGGTGGGGSGGGGSGGS 88 KKAAPVNG
GGGGSGGGGSKKAAPVNGGGGGSGGGGSGGGGS 89 PMAKKVNG
GGGGSGGGGSPMAKKVNGGGGGSGGGGSGGGGS 90 QARAKVNG
GGGGSGGGGSQARAKVNGGGGGSGGGGSGGGGS 91 MMP9
GGGGSGGGGSVHMPLGFLGPGGGGSGGGGSGGS 92 QARAK
GGGGSGGGGSQARAKGGGGSGGGGSGGGGSGGS 93 MMP9-PMAKK
GGGGSVHMPLGFLGPPMAKKGGGGSGGGGSGGS 94 KKAAP
GGGGSGGGGSKKAAPGGGGSGGGGSGGGGSGGS 95 PMAKK
GGGGSGGGGSPMAKKGGGGSGGGGSGGGGSGGS 96 Protease recognition
VHMPLGFLGPRQARVVNG 97 site 6 Protease recognition FVGGTG 98 site 7
Protease recognition KKAAPVNG 99 site 8 Protease recognition
PMAKKVNG 100 site 9 Protease recognition QARAKVNG 101 site 10
Protease recognition VHMPLGFLGP 102 site 11 Protease recognition
QARAK 103 site 12 Protease recognition VHMPLGFLGPPMAKK 104 site 13
Protease recognition KKAAP 105
site 14 Protease recognition PMAKK 106 site 15 aMSLN CDR H1 Kabat
GYTMN 107 aMSLN CDR H2 Kabat LITPYNGASSYNQKFRG 108 aMSLN CDR H3
Kabat GGYDGRGFDY 109 aMSLN CDR L1 Kabat SASSSVSYMH 110 aMSLN CDR L2
Kabat DTSKLAS 111 aMSLN CDR L3 Kabat QQWSKHPLT 112 aMSLN VH
QVQLVQSGAEVKKPGASVKVSCKASGYSFTGYTMNWVRQA 113
PGQGLEWMGLITPYNGASSYNQKFRGKATMTVDTSTSTVY
MELSSLRSEDTAVYYCARGGYDGRGFDYWGQGTLVTVSS aMSLN VL
DIQMTQSPSSLSASVGDRVTITCSASSSVSYMHWYQQKSGK 114
APKLLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQWSKHPLTFGQGTKLEIK
aHER1 VH QVQLVQSGAEVKKPGSSVKVSCKASGFTFTDYKIHWVRQAP 115
GQGLEWMGYFNPNSGYSTYAQKFQGRVTITADKSTSTAYM
ELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSS aHER1 VL
DIQMTQSPSSLSASVGDRVTITCRASQGINNYLNWYQQKPGKA 116
PKRLIYNTNNLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYC LQHNSFPTFGQGTKLEIK SEQ
ID Construct DNA Sequence No LC Common
CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 117 light chain
GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC pETR13197
ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC
TTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC
CCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTG
ACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGC
GCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAG
CTGACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTG
TTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTG
GTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCC
TGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCAC
CACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTA
CCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAG
CTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGG CCCCCACCGAGTGCAGCTGA
anti CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 118 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC Matriptase
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH3_23-VH12
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 FolR1
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT 16D5 VH CH1
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC hum Fc knob
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT PG LALA,
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG pETR16546
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG (FIG. 45N)
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTG
TGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCG
TGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGT
GCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGAT
CTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCT
ACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAAT
GGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTA
CGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACA
GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACA
GCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA
CCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCTGG
CCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGGGC
TGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGG
AACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCCGTG
CTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACCGTG
CCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAAC
CACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCCAAG
AGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGAGGT
GCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGGTTC
CCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAACGC
GTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGAGT
GGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGATTA
CGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGATAGC
AAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAGACAC
CGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTACGAT
TATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAGTACC
AAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAGCACA
TCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTACTTC
CCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGACCAGC
GGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTAC
TCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGGAACA
CAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCAAAGT
CGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACACATG
CCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 16D5
GAGGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGC 119 HC cH2527-
GGTTCCCTGCGTCTGAGCTGCGCGGCTTCCGGATTCACCTTCTCC VH3_23-12
AACGCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCT HC Fc knob
CGAGTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCAC PG LALA,
GGATTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGAC pCON999
GATAGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGA
AGACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGG
TACGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTTCCGCT
AGCACAAAGGGCCCTAGCGTGTTCCCTCTGGCCCCCAGCAGCAAG
AGCACAAGCGGCGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGA
CTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACAGCGGAGCCCT
GACAAGCGGCGTGCACACTTTCCCTGCCGTGCTGCAGAGCAGCGG
CCTGTACTCCCTGAGCAGCGTGGTCACCGTGCCTAGCAGCAGCCT
GGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAA
CACCAAAGTGGACAAGAAGGTGGAGCCCAAGAGCTGTGATGGCGG
AGGAGGGTCCGGAGGCGGAGGATCCGAGGTGCAGCTGCTGGAAT
CTGGCGGCGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCT
GTGCCGCCAGCGGCTTCACCTTCAGCACCTACGCCATGAACTGGG
TGCGCCAGGCCCCTGGCAAAGGCCTGGAATGGGTGTCCCGGATCA
GAAGCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGA
AGGGCCGGTTCACCATCAGCCGGGACGACAGCAAGAACACCCTGT
ACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACT
ATTGTGTGCGGCACGGCAACTTCGGCAACAGCTATGTGTCTTGGTT
TGCCTACTGGGGCCAGGGCACCCTCGTGACCGTGTCAAGCGCTAG
TACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAG
CACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTA
CTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGAC
CAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCT
GTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGG
AACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCA
AAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAG
AACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti ID CD3
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 120 scFv 4.32.63
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC MK062
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG protease site
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC CD3 VL
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG CLambda,
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC pETR16544
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTAGACAGGCCAGAGTCGTGAACGGGGGAGGG
GGGGGAAGTGGGGGCGGAGGCAGTGGGGGCGGAGGATCCCAGG
CCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCA
CCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCA
GCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCA
GAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCT
GCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACA
CTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCC
CTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTG
ACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTC
CCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGT
CTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTG
GAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCA
CCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACC
TGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCT
GCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCC CCCACCGAGTGCAGCTGA
anti ID CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 121 scFv
4.32.63 CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC non-cleavable
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG linker CD3 VL
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC CLambda,
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG pETR16545
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGG
GGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAG
GCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGC
ACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACC
AGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC
AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCT
GCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACA
CTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCC
CTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTG
ACAGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTC
CCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGT
CTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTG
GAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCA
CCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACC
TGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCT
GCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCC CCCACCGAGTGCAGCTGA
aMSLN CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGC 122 RG7787 VH
GCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACC CH1 EE CD3
GGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTG cH2527-
GAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTAC VH3_23-12
AACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGC VL CH1 Fc
ACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGAC knob PG
ACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGC LALA,
TTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTA pETR15445
GCACCAAGGGCCCCTCCGTGTTTCCTCTGGCCCCTTCCAGCAAGT
CCACCTCTGGCGGAACTGCCGCTCTGGGCTGCCTGGTGGAAGATT
ACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCTCTGA
CCTCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCC
TGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAGCTCTCTGGG
CACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACC
AAGGTGGACGAGAAGGTGGAACCCAAGTCCTGCGACGGTGGCGG
AGGTTCCGGAGGCGGAGGATCCCAGGCTGTCGTGACCCAGGAAC
CCTCCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTGACCTGTG
GATCTTCTACCGGCGCTGTGACCACCTCCAACTACGCCAATTGGGT
GCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATCGGCGGCA
CCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCTCCGGTTCTC
TGCTGGGCGGCAAGGCTGCCCTGACTCTGTCTGGTGCTCAGCCTG
AGGACGAGGCCGAGTACTACTGCGCCCTGTGGTACTCCAACCTGT
GGGTGTTCGGCGGAGGCACCAAGCTGACCGTGCTGTCCAGCGCTT
CCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGCTCCAAGT
CTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTGAAGGACT
ACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAGCCCTGA
CCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCAGCGGCC
TGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTAGCCTGG
GAACACAGACATATATCTGTAATGTGAATCATAAGCCCAGTAATACC
AAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATAAGACCCACA
CCTGTCCCCCCTGCCCTGCTCCTGAAGCTGCTGGTGGCCCTAGCG
TGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTCCCG
GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGA
CCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCA
CAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTA
CCGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGACTGGCTGAA
CGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGGGCGC
TCCCATCGAAAAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGA
ACCCCAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAA
GAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAG
CGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACA
ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA aMSLN
CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGC 123 RG7787 VH
GCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACC CH1 EE Fc
GGCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTG hole P329G
GAATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTAC LALA,
AACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGC pETR15444
ACCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGAC
ACCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGC
TTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTA
GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGA
GCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGAC
TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGC
CTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTG
GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC
ACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA aMSLN
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTG 124 RG7787 VL
GGCGACAGAGTGACCATCACCTGTAGCGCCAGCAGCAGCGTGTCC Ck RK,
TACATGCACTGGTATCAGCAGAAGTCCGGCAAGGCCCCCAAGCTG pETR15443
CTGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCCAGCAGA
TTTTCTGGCAGCGGCTCCGGCACCGACTTCACCCTGACAATCAGCT
CCCTCCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTGGT
CCAAGCACCCCCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCA
AGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGA
TCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAAT
AACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG
CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACA
GCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA
AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCA
TCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGA GTGTTAG anti ID
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 125 CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC 4.32.63 CD3
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG CH2527 VH
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC 23-12 Ck,
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9-MK062
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC site,
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC pETR16758
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTG
TGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCG
TGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAGGT
GCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGGAT
CTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCACCT
ACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGAAT
GGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTACTA
CGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGACA
GCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGG
ACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAACA
GCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGTGA
CCGTGTCAAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTCC
CACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGT
GCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGA
AGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTGA
CCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCC
TGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCT
GCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTT TCAACCGGGGCGAGTGCTGA
anti ID CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 126 CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC 4.32.63 CD3
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG CH2527 VH
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC 23-12 Ck,
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-cleavable
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC linker,
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC pETR16759
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGG
GGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGAG
GTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGG
ATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC
CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGA
ATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTAC
TACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGA
CAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA
GGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAA
CAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGT
GACCGTGTCAAGCGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTT
CCCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGT
GTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTG
GAAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGT
GACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCAC
CCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGC
CTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTC
TTTCAACCGGGGCGAGTGCTGA CD3 CH2527
GAAGTGCAGCTGCTGGAATCCGGCGGAGGACTGGTGCAGCCTGG 127 VH 23-12-
CGGATCTCTGAGACTGTCTTGTGCCGCCTCCGGCTTCACCTTCTCC Ck,
ACCTACGCCATGAACTGGGTGCGACAGGCTCCTGGCAAGGGCCTG pETR13811
GAATGGGTGTCCCGGATCAGATCCAAGTACAACAACTACGCCACCT
ACTACGCCGACTCCGTGAAGGGCCGGTTCACCATCTCTCGGGACG
ACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGGCCG
AGGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCA
ACTCCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGT
GACCGTGTCATCTGCTAGCGTGGCCGCTCCCTCCGTGTTCATCTTC
CCACCTTCCGACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTG
TGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGG
AAGGTGGACAACGCCCTGCAGTCCGGCAACAGCCAGGAATCCGTG
ACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACC
CTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCC
TGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCT TTCAACCGGGGCGAGTGCTGA
anti CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 128 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC Matriptase
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aMSLN VH
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 EE
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum Fc
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PG
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALA,
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG pETR16751
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTG
TGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCG
TGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGC
CGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC
CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAG
CAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAG
AGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGC
CAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT
GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCT
GTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGAC
AGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCT
GGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGG
ATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGG
AACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTG
CTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTG
CCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCA
TAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGC
TGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCA
GCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCG
TGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCGGCTACA
CCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGA
TGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACAACCAGA
AGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCACCTCCA
CCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCG
TGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCGATT
ATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCACCA
AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCA
GCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTC
CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCC
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTAT
AGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACC
CAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAG
GTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGA
ACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCG
ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC
TCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 129 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG non-cleavable
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC linker aMSLN
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC VH CH1 EE
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH2527-
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VL7_46-13
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 hum Fc
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT knob PG
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC LALA,
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT pETR16752
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGG
GGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAG
GCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGC
ACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACC
AGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC
AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCT
GCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACA
CTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCC
CTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTG
ACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCC
CTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTG
GGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTT
GGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAG
TGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCG
TGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAAT
CATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGA
GCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGT
GCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCA
GCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCGGCT
ACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAAT
GGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACAACC
AGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCACCT
CCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACACCG
CCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCTTCG
ATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTAGCA
CCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCA
CCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTAC
TTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACC
TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGC
ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCA
AGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACA
CATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGC
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA
GAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA CH2527 XFab
CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 130 aMSLN
GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC RG7787 HC
ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC EE Fc knob
TTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC PG LALA,
CCTGCCAGATTCTCCGGTTCTCTGCTGGGCGGCAAGGCTGCCCTG pETR16764
ACTCTGTCTGGTGCTCAGCCTGAGGACGAGGCCGAGTACTACTGC
GCCCTGTGGTACTCCAACCTGTGGGTGTTCGGCGGAGGCACCAAG
CTGACCGTGCTGTCCAGCGCTTCCACCAAGGGACCCAGTGTGTTC
CCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCC
CTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGT
CTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTG
CAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGA
CCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGT
GAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCT
AAGAGCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCA
GGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCG
CCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACAGCTTCACCG
GCTACACCATGAACTGGGTGCGCCAGGCTCCTGGACAGGGCCTGG
AATGGATGGGCCTGATCACCCCCTACAACGGCGCCAGCAGCTACA
ACCAGAAGTTCCGGGGCAAGGCCACCATGACCGTGGACACCAGCA
CCTCCACCGTGTATATGGAACTGAGCAGCCTGCGGAGCGAGGACA
CCGCCGTGTACTATTGTGCCAGAGGCGGCTACGACGGCAGAGGCT
TCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTA
GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGA
GCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGAC
TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGC
CTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTG
GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC
ACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 131 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG Cathepsin S/B
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC site CH2527
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC VH3_23-VH12
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG CH1 FolR1
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG 16D5 VH CH1
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC hum Fc knob
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT PG LALA,
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC pETR16550
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTTTCGTGGGGGGGAC
CGGGGGCGGAGGCAGTGGGGGGGGAGGATCCGGGGGATCCGAG
GTGCAGCTGCTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCGG
ATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCAGCAC
CTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGCCTGGA
ATGGGTGTCCCGGATCAGAAGCAAGTACAACAACTACGCCACCTAC
TACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACGA
CAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGCGGGCCGA
GGACACCGCCGTGTACTATTGTGTGCGGCACGGCAACTTCGGCAA
CAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACCCTCGT
GACCGTGTCAAGCGCTAGCACAAAGGGCCCTAGCGTGTTCCCTCT
GGCCCCCAGCAGCAAGAGCACAAGCGGCGGAACAGCCGCCCTGG
GCTGCCTCGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTT
GGAACAGCGGAGCCCTGACAAGCGGCGTGCACACCTTCCCTGCC
GTGCTGCAGAGCAGCGGCCTGTACTCCCTGAGCAGCGTGGTCACC
GTGCCTAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTG
AACCACAAGCCCAGCAACACCAAAGTGGACAAGAAGGTGGAGCCC
AAGAGCTGTGATGGCGGAGGAGGGTCCGGGGGCGGAGGATCCGA
GGTGCAATTGGTTGAATCTGGTGGTGGTCTGGTAAAACCGGGCGG
TTCCCTGCGTCTGAGCTGCGCGGCTTCCGGGTTCACCTTCTCCAAC
GCGTGGATGAGCTGGGTTCGCCAGGCCCCGGGCAAAGGCCTCGA
GTGGGTTGGTCGTATCAAGTCTAAAACTGACGGTGGCACCACGGA
TTACGCGGCTCCAGTTAAAGGTCGTTTTACCATTTCCCGCGACGAT
AGCAAAAACACTCTGTATCTGCAGATGAACTCTCTGAAAACTGAAG
ACACCGCAGTCTACTACTGTACTACCCCGTGGGAATGGTCTTGGTA
CGATTATTGGGGCCAGGGCACGCTGGTTACGGTGTCTAGCGCTAG
TACCAAGGGCCCCAGCGTGTTCCCCCTGGCACCCAGCAGCAAGAG
CACATCTGGCGGAACAGCCGCTCTGGGCTGTCTGGTGAAAGACTA
CTTCCCCGAGCCCGTGACCGTGTCTTGGAACTCTGGCGCCCTGAC
CAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCT
GTACTCCCTGTCCTCCGTGGTCACCGTGCCCTCTAGCTCCCTGGG
AACACAGACATATATCTGTAATGTCAATCACAAGCCTTCCAACACCA
AAGTCGATAAGAAAGTCGAGCCCAAGAGCTGCGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAG
AACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGG
GAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID Construct Amino acid
Sequence No pETR16859 Omnitarg
EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQA 132 aff.mat variant Fab
cv- PGKGLEWVADVNPNSGGSIVNRRFKGRFTLSVDRSKNTLYL Fc hole PG LALA
QMNSLRAEDTAVYYCARNLGPFFYFDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS
CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK pETR16860 Herceptarg
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPG 133 common CLkRK
KAPKLLIYSASFRYTGVPSRFSGSRSGTDFTLTISSLQPEDFA
TYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDRKLK
SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC pETR17605 CD3 X
Fab QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 134 Herceptin HC
charged PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP variants Fc knob
PG EDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLA LALA
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRL
SCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYA
DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGE
GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT
AALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDK
THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP
QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK
pETR17606 anti CD3 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 135
(CH2527 VH_3-23(12) GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
VL7-46(13)) scFv LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG 4.32.63
non cleavable GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT linker
aHerceptin VH CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS CH1 EE
CH2527- GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL VL7_46-13 CH1 hum
Fc EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ knob PG LALA
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP
GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPE
DEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLS
CAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD
SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEG
FYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTH
TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK
pETR17607 anti CD3 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 136
(CH2527 VH_3-23(12) GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
VL7-46(13)) scFv LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG 4.32.63
MMP9 GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT Matriptase MK062
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS aHerceptin VH CH1 EE
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH2527-VL7_46-13
EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV CH1 hurn Fc knob PG
VTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG LALA
QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED
EAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSC
AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS
VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGEGF
YAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQV
YTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK FolR1
36F2 VH CH1 EE QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQA 137 Fc hole
PG LALA PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVY pETR14797
MELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSSAS
TKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV
NHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLF
PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVS
NKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLS
CAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FolR1 36F2 VL Ck RK,
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP 138 pETR14798
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYTNEHYYTFGQGTKVEIKRTVAAPSVFIFPPSDRK
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC anti CD3 (CH2527
QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 139 VH_3-23(12) VL7-
GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS 46(13)) scFv 4.32.63
LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG MMP9 Matriptase
GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT MK062 aFolR1 36F2
CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS VH CH1 EE CH2527-
GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL VL7 46-13 CH1 hum Fc
EIKGGGGSVHMPLGFLGPRQARVVNGGGGGSGGGGSQAV knob PG LALA
VTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPG pETR17621
QAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPED
EAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPS
SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK
VEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSC
KASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQ
KFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFTG
FHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
SVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHT
CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQV
YTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGK anti
CD3 (CH2527 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPP 140
VH_3-23(12) VL7- GKCLEWLGIIWGDGSTNYHSALISRLSISKDNSKSQVFLKLNS
46(13)) scFv 4.32.63 LQTDDTATYYCAKGITTVVDDYYAMDYWGQGTSVTVSSGG non
cleavable linker GGSGGGGSGGGGSGGGGSDIQMTQSPASLSASVGETVTIT aFolR1
36F2 VH CH1 CRASENIDSYLAWYQQKQGKSPQLLVYAATFLADDVPSRFS EE
CH2527-VL7_46-13 GSGSGTQYSLKINSLQSEDVARYYCQHYYSTPYTFGCGTKL CH1 hum
Fc knob PG EIKGGGGSGGGGSGGGGSGGGGGGGSGGGGSGGGGSQ LALA pETR17622
AVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKP
GQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPE
DEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAP
SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS
CKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYA
QKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFFT
GFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA
LGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL
SSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTH
TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL
TVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK FolR1
36F2 classic QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEK 141 format:
CH2527 XFab PGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQP 36F2 HC EE Fc
knob EDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLA PG LALA pETR17623
PSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD
KKVEPKSCDGGGGSGGGGSQVQLVQSGAEVKKPGASVKV
SCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSY
AQKFQGRVTMTHDTSTSTVYMELSSLRSEDTAVYYCARSFF
TGFHLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKT
HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV
LTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQ
VYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK
Herceptin/Omnitarg DYTMD 142 CDR H1 Kabat Herceptin/Omnitarg
DVNPNSGGSIVNRRFKG 143 CDR H2 Kabat Herceptin/Omnitarg NLGPFFYFDY
144 CDR H3 Kabat Perjeta CDR H1 Kabat TSNYANW 145 Perjeta CDR H2
Kabat GTNKRAPGTPARFSGSLLGG 146 Perjeta CDR H3 Kabat TKLTV 147 CLC
CDR L1 Kabat KASQDVSTAVA 148 CLC CDR L2 Kabat SASFRYT 149 CLC CDR
L3 Kabat QQHYTTPPT 150 36F2 CDR H1 Kabat SYYMH 151 36F2 CDR H2
Kabat IINPSGGSTSYAQKFQG 152 36F2 CDR H3 Kabat SFFTGFHLDY 153 36F2
CDR L1 Kabat RASQSVSSSYLA 154 36F2 CDR L2 Kabat GASSRAT 155 36F2
CDR L3 Kabat QQYTNEHYYT 156 Anti-FolR1 36F2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQA 157 variable region VH
PGQGLEWMGIINPSGGSTSYAQKFQGRVTMTHDTSTSTVY
MELSSLRSEDTAVYYCARSFFTGFHLDYWGQGTLVTVSS Anti-FolR1 36F2
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKP 158 variable region VL
GQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDF
AVYYCQQYTNEHYYTFGQGTKVEIK Herceptarg variable
EVQLVESGGGLVQPGGSLRLSCAASGFTFNDYTMDWVRQA 159 region VH1
PGKGLEWVADVNPNSGGSIVNRRFKGRFTLSVDRSKNTLYL
QMNSLRAEDTAVYYCARNLGPFFYFDYWGQGTLVTVSS Herceptarg variable
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAP 160 region VH2
GKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQ
MNSLRAEDTAVYYCSRWGGEGFYAMDYWGQGTLVTVSS Herceptarg common
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPG 161 variable region VL
KAPKLLIYSASFRYTGVPSRFSGSRSGTDFTLTISSLQPEDFA TYYCQQHYTTPPTFGQGTKVEIK
SEQ ID Construct DNA Sequence No pETR16859
GAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGT 162 Omnitarg
GGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTACCTTTAACG aff.mat variant
ATTATACCATGGATTGGGTTCGTCAGGCACCGGGTAAAGGTCTGGA Fab cv-Fc
ATGGGTTGCAGATGTTAATCCGAATAGCGGTGGTAGCATTGTTAAC hole PG LALA
CGTCGTTTTAAAGGTCGTTTTACCCTGAGCGTTGATCGTAGCAAAA
ATACCCTGTATCTGCAAATGAATAGTCTGCGTGCAGAGGATACCGC
AGTGTATTATTGTGCACGTAACCTGGGTCCGTTCTTCTACTTTGATT
ATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGCGCTAGCACCA
AGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCA
GCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTC
CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCC
GGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTAT
AGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACC
CAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAG
GTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACA
TGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTC
TTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA
CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACC
GTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAAC
CACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGA
ACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCG
ACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACT
ACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCC
TCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG
AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACT
ACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA pETR16860
GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTG 163 Herceptarg
GGCGACAGAGTGACCATCACATGCAAGGCCAGCCAGGACGTGTCC common
ACAGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAG CLkRK
CTGCTGATCTACAGCGCCAGCTTCCGGTACACCGGCGTGCCCAGC
AGATTCAGCGGCAGCAGATCCGGCACCGACTTCACCCTGACCATC
AGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAG
CACTACACCACCCCCCCCACATTTGGCCAGGGCACCAAGGTGGAA
ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT
CTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCT
GAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT
AACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG
GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTG
AGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCA
CCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGTTAG pETR17606
CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 164 anti CD3
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC (CH2527
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VH_3-23(12)
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC VL7-46(13))
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG scFv 4.32.63
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC non cleavable
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC linker
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aHerceptin VH
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG CH1 EE
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum Fc
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PG
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALA
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGCGGGGGAGGCTCCG
GAGGCGGCGGAAGTGGAGGCGGCGGAAGTGGCGGAGGCGGAGG
GGGGGGAAGTGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAG
GCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGC
ACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACC
AGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTC
AGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCT
GCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACA
CTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCC
CTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTG
ACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCC
CTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTG
GGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTT
GGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAG
TGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCG
TGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAAT
CATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGA
GCTGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCGAGGT
CCAGCTGGTCGAGTCTGGAGGAGGACTGGTGCAGCCAGGCGGAT
CTCTGAGACTGAGCTGCGCCGCCAGCGGATTCAACATCAAGGACA
CCTACATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGT
GGGTGGCCAGAATCTACCCCACCAACGGCTACACAAGATACGCCG
ACAGCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGA
ACACCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAG
CCGTGTACTACTGCTCTAGATGGGGAGGCGAGGGCTTCTACGCCA
TGGACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGCTA
GCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGA
GCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGAC
TACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTG
ACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGC
CTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTG
GGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAAC
ACCAAGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACT
CACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACC
GTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCAC
GAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC
ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG
CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC
GGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCC
CGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTG
ACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATC
CCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAG
AACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
pETR17607 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 165 anti
CD3 CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC (CH2527
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VH_3-23(12)
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC VL7-46(13))
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG scFv 4.32.63
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC MMP9
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC Matriptase
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG MK062
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG aHerceptin VH
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH1 EE
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT CH2527-
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC VL7_46-13
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT CH1 hum Fc
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG knob PG
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG LALA
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTG
TGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCG
TGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGC
CGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC
CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAG
CAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAG
AGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGC
CAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT
GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCT
GTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGAC
AGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCT
GGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGG
ATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGG
AACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTG
CTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTG
CCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCA
TAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGC
TGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCGAGGTCCA
GCTGGTCGAGTCTGGAGGAGGACTGGTGCAGCCAGGCGGATCTCT
GAGACTGAGCTGCGCCGCCAGCGGATTCAACATCAAGGACACCTA
CATCCACTGGGTGAGGCAGGCCCCTGGAAAGGGACTGGAGTGGG
TGGCCAGAATCTACCCCACCAACGGCTACACAAGATACGCCGACA
GCGTGAAGGGCAGATTCACCATCAGCGCCGACACCAGCAAGAACA
CCGCCTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCG
TGTACTACTGCTCTAGATGGGGAGGCGAGGGCTTCTACGCCATGG
ACTACTGGGGACAGGGCACACTGGTGACCGTGTCCAGCGCTAGCA
CCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCA
CCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTAC
TTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACC
TCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTG
TATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGC
ACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCA
AGGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACA
CATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCA
GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAA
GACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTG
CATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG
AATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGC
GCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGA
GAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACC
AAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCA
GCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTC
TTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACC
ACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 36F2
CAGGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCG 166 VH CH1 EE
CTTCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTC Fc hole PG
CTATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGA LALA
ATGGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGC pETR14797
GCAGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCAC
CTCTACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACT
GCAGTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGG
ACTATTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCAC
CAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCAC
CAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTT
CCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTC
CGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTA
TAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCAC
CCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAA
GGTGGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACAC
ATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGT
CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG
ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCAT
AATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC
CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAAT
GGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCC
CCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA
CCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAG
AACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGC
GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTT
CCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA
CTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA FolR1 36F2
GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAG 167 VL Ck RK,
GGGAAAGAGCCACCCTCTCTTGCAGGGCCAGTCAGAGTGTTAGCA pETR14798
GCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCA
GGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAG
ACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCAT
CAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAG
TATACCAACGAACATTATTATACGTTCGGCCAGGGGACCAAAGTGG
AAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCC
ATCTGATCGGAAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG
CTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGG
ATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGC
AGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGC
TGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGTTAG anti
CD3 CAAGTGCAGCTGAAAGAGTCCGGCCCTGGACTGGTGGCCCCTAGC 168 (CH2527
CAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACC VH_3-23(12)
AGCTACGGCGTGTCATGGGTGCGCCAGCCTCCAGGCAAGTGTCTG VL7-46(13))
GAATGGCTGGGCATCATCTGGGGCGACGGCAGCACCAATTACCAC scFv 4.32.63
AGCGCCCTGATCAGCAGACTGAGCATCTCCAAGGACAACAGCAAG MMP9
AGCCAGGTGTTCCTGAAGCTGAACAGCCTGCAGACCGACGACACC Matriptase
GCCACCTACTACTGCGCCAAGGGCATCACCACCGTGGTGGACGAC MK062
TACTACGCTATGGACTACTGGGGCCAGGGCACCAGCGTGACAGTG aFolR1 36F2
TCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGAGG VH CH1 EE
GGGATCTGGGGGAGGCGGAAGCGATATCCAGATGACCCAGAGCC CH2527-
CTGCCAGCCTGTCTGCCTCTGTGGGCGAGACAGTGACCATCACAT VL7_46-13
GCCGGGCCAGCGAGAACATCGACAGCTACCTGGCCTGGTATCAGC CH1 hum Fc
AGAAGCAGGGCAAGAGCCCCCAGCTGCTGGTGTACGCCGCCACCT knob PG
TTCTGGCCGACGATGTGCCCAGCAGATTCAGCGGCAGCGGAAGCG LALA
GCACACAGTACAGCCTGAAGATCAACTCCCTGCAGAGCGAGGACG pETR17621
TGGCCCGGTACTACTGCCAGCACTACTACAGCACCCCCTACACCTT
CGGCTGCGGCACCAAGCTGGAAATCAAAGGAGGCGGCGGAAGTG
TGCACATGCCCCTGGGCTTCCTGGGCCCCAGACAGGCCAGAGTCG
TGAACGGGGGGGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGC
CGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCAC
CGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACCACCAG
CAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAG
AGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGC
CAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACT
GTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCT
GTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGAC
AGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCT
GGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGG
ATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGG
AACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTGCAGTG
CTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTG
CCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGTGAATCA
TAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGC
TGCGATGGCGGAGGAGGGTCCGGAGGCGGAGGGTCCCAGGTGCA
ATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTT
AAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCTATTACAT
GCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGG
GCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATT
CCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTCTACCGT
TTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTAC
TACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGG
GTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCC
CCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGC
GGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAG
CCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGT
GCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCT
GAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGAC
CTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGA
CGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCC
ACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCT
CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCT
GAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAG
GTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATC
GAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAG
GTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC
GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAA
GACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTA
CAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACG
TCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC
GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA anti CD3
AGAGTCCGGCCCTGGACTGGTGGCCCCTAGCCAGAGCCTGAGCAT 169 (CH2527
CACCTGTACCGTGTCCGGCTTCAGCCTGACCAGCTACGGCGTGTC VH_3-23(12)
ATGGGTGCGCCAGCCTCCAGGCAAGTGTCTGGAATGGCTGGGCAT VL7-46(13))
CATCTGGGGCGACGGCAGCACCAATTACCACAGCGCCCTGATCAG scFv 4.32.63
CAGACTGAGCATCTCCAAGGACAACAGCAAGAGCCAGGTGTTCCT non cleavable
GAAGCTGAACAGCCTGCAGACCGACGACACCGCCACCTACTACTG linker aFolR1
CGCCAAGGGCATCACCACCGTGGTGGACGACTACTACGCTATGGA 36F2 VH CH1
CTACTGGGGCCAGGGCACCAGCGTGACAGTGTCTAGCGGAGGCG EE CH2527-
GAGGATCTGGCGGCGGAGGAAGTGGCGGAGGGGGATCTGGGGG VL7_46-13
AGGCGGAAGCGATATCCAGATGACCCAGAGCCCTGCCAGCCTGTC CH1 hum Fc
TGCCTCTGTGGGCGAGACAGTGACCATCACATGCCGGGCCAGCGA knob PG
GAACATCGACAGCTACCTGGCCTGGTATCAGCAGAAGCAGGGCAA LALA
GAGCCCCCAGCTGCTGGTGTACGCCGCCACCTTTCTGGCCGACGA pETR17622
TGTGCCCAGCAGATTCAGCGGCAGCGGAAGCGGCACACAGTACAG
CCTGAAGATCAACTCCCTGCAGAGCGAGGACGTGGCCCGGTACTA
CTGCCAGCACTACTACAGCACCCCCTACACCTTCGGCTGCGGCAC
CAAGCTGGAAATCAAAGGCGGGGGAGGCTCCGGAGGCGGCGGAA
GTGGAGGCGGCGGAAGTGGCGGAGGCGGAGGGGGGGGAAGTGG
GGGCGGAGGCAGTGGGGGGGGAGGATCCCAGGCCGTCGTGACC
CAGGAACCCAGCCTGACAGTGTCTCCTGGCGGCACCGTGACCCTG
ACATGTGGCAGTTCTACAGGCGCCGTGACCACCAGCAACTACGCC
AACTGGGTGCAGGAAAAGCCCGGCCAGGCCTTCAGAGGACTGATC
GGCGGCACCAACAAGAGAGCCCCTGGCACCCCTGCCAGATTCAGC
GGATCTCTGCTGGGAGGAAAGGCCGCCCTGACACTGTCTGGCGCC
CAGCCAGAAGATGAGGCCGAGTACTACTGCGCCCTGTGGTACAGC
AACCTGTGGGTGTTCGGCGGAGGCACCAAGCTGACAGTGCTGAGC
AGCGCTTCCACCAAGGGACCCAGTGTGTTCCCCCTGGCCCCCAGC
TCCAAGTCTACATCCGGTGGCACAGCTGCCCTGGGATGTCTCGTG
AAGGACTACTTTCCTGAGCCTGTGACAGTGTCTTGGAACAGCGGAG
CCCTGACCAGCGGAGTGCACACATTCCCTGCAGTGCTGCAGAGCA
GCGGCCTGTATAGCCTGAGCAGCGTCGTGACCGTGCCTTCCTCTA
GCCTGGGAACACAGACATATATCTGTAATGTGAATCATAAGCCCAG
TAATACCAAAGTGGATAAGAAAGTGGAACCTAAGAGCTGCGATGGC
GGAGGAGGGTCTGGAGGCGGAGGGTCCCAGGTGCAATTGGTTCA
ATCTGGTGCTGAAGTAAAAAAACCGGGCGCTTCCGTTAAAGTGAGC
TGCAAAGCATCCGGATACACCTTCACTTCCTATTACATGCACTGGG
TTCGTCAAGCCCCGGGCCAGGGTCTGGAATGGATGGGCATCATTA
ACCCAAGCGGTGGCTCTACCTCCTACGCGCAGAAATTCCAGGGTC
GCGTCACGATGACCCATGACACTAGCACCTCTACCGTTTATATGGA
GCTGTCCAGCCTGCGTTCTGAAGATACTGCAGTGTACTACTGTGCA
CGCTCTTTCTTCACTGGTTTCCATCTGGACTATTGGGGTCAAGGCA
CCCTCGTAACGGTTTCTTCTGCTAGCACCAAGGGCCCCTCCGTGTT
CCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCG
CTCTGGGCTGCCTGGTCGAGGACTACTTCCCCGAGCCCGTGACCG
TGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCC
CCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGG
TCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAA
CGTGAACCACAAGCCCAGCAACACCAAGGTGGACGAGAAGGTGGA
GCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGC
ACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAA
ACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATG
CGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA
CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC
GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCT
CACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTG
CAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCAT
CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCT
GCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTG
GTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC
CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAATGA
FolR1 36F2 CAGGCCGTCGTGACCCAGGAACCCAGCCTGACAGTGTCTCCTGGC 170
classic format: GGCACCGTGACCCTGACATGTGGCAGTTCTACAGGCGCCGTGACC
CH2527 XFab ACCAGCAACTACGCCAACTGGGTGCAGGAAAAGCCCGGCCAGGCC 36F2 HC
EE TTCAGAGGACTGATCGGCGGCACCAACAAGAGAGCCCCTGGCACC Fc knob PG
CCTGCCAGATTCAGCGGATCTCTGCTGGGAGGAAAGGCCGCCCTG LALA
ACACTGTCTGGCGCCCAGCCAGAAGATGAGGCCGAGTACTACTGC pETR17623
GCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAG
CTGACAGTGCTGAGCAGCGCTTCCACCAAGGGACCCAGTGTGTTC
CCCCTGGCCCCCAGCTCCAAGTCTACATCCGGTGGCACAGCTGCC
CTGGGATGTCTCGTGAAGGACTACTTTCCTGAGCCTGTGACAGTGT
CTTGGAACAGCGGAGCCCTGACCAGCGGAGTGCACACATTCCCTG
CAGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTCGTGA
CCGTGCCTTCCTCTAGCCTGGGAACACAGACATATATCTGTAATGT
GAATCATAAGCCCAGTAATACCAAAGTGGATAAGAAAGTGGAACCT
AAGAGCTGCGATGGCGGAGGAGGGTCTGGAGGCGGAGGGTCCCA
GGTGCAATTGGTTCAATCTGGTGCTGAAGTAAAAAAACCGGGCGCT
TCCGTTAAAGTGAGCTGCAAAGCATCCGGATACACCTTCACTTCCT
ATTACATGCACTGGGTTCGTCAAGCCCCGGGCCAGGGTCTGGAAT
GGATGGGCATCATTAACCCAAGCGGTGGCTCTACCTCCTACGCGC
AGAAATTCCAGGGTCGCGTCACGATGACCCATGACACTAGCACCTC
TACCGTTTATATGGAGCTGTCCAGCCTGCGTTCTGAAGATACTGCA
GTGTACTACTGTGCACGCTCTTTCTTCACTGGTTTCCATCTGGACTA
TTGGGGTCAAGGCACCCTCGTAACGGTTTCTTCTGCTAGCACCAAG
GGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGC
GGCGGCACAGCCGCTCTGGGCTGCCTGGTCGAGGACTACTTCCCC
GAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGG
CGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAG
CCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCA
GACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGT
GGACGAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATG
CCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACC
CCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCT
GAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGT
GTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCC
ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCA
CAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC
AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCT
ACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAAC
GTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
[0834] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
1701215PRTArtificial SequenceSynthetic Construct 1Gln Ala Val Val
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr
Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr
Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55
60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65
70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
Gln Pro 100 105 110Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser
Ser Glu Glu Leu 115 120 125Gln Ala Asn Lys Ala Thr Leu Val Cys Leu
Ile Ser Asp Phe Tyr Pro 130 135 140Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro Val Lys Ala145 150 155 160Gly Val Glu Thr Thr
Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175Ala Ser Ser
Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190Ser
Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200
205Val Ala Pro Thr Glu Cys Ser 210 2152971PRTArtificial
SequenceSynthetic Construct 2Gln Ile Gln Leu Val Gln Ser Gly Pro
Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Arg Ile Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ser Ile His Trp Val Lys Gln
Ala Pro Gly Lys Cys Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Glu
Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe 50 55 60Lys Gly Arg Phe Ala
Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile
Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Phe Phe Cys 85 90 95Ala His
Pro Tyr Asp Tyr Asp Val Leu Asp Tyr Trp Gly Gln Gly Thr 100 105
110Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Thr Val Leu
Thr Gln 130 135 140Ser Pro Ala Ser Leu Gly Val Ser Leu Gly Gln Arg
Ala Thr Ile Ser145 150 155 160Cys Arg Ala Ser Lys Ser Val Ser Thr
Ser Asn Tyr Ser Tyr Ile His 165 170 175Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro Lys Leu Leu Ile Lys Tyr 180 185 190Val Ser Tyr Leu Glu
Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 195 200 205Ser Gly Thr
Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp 210 215 220Ala
Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Phe Pro Trp Thr Phe225 230
235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly
Gly 245 250 255Gly Gly Ser Arg Gln Ala Arg Val Val Asn Gly Gly Gly
Gly Gly Ser 260 265 270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
Val Gln Leu Leu Glu 275 280 285Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser Gly Phe Thr Phe
Ser Thr Tyr Ala Met Asn Trp Val Arg305 310 315 320Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys 325 330 335Tyr Asn
Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 340 345
350Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
355 360 365Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg
His Gly 370 375 380Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr
Trp Gly Gln Gly385 390 395 400Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 405 410 415Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 420 425 430Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 435 440 445Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 450 455 460Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser465 470
475 480Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 485 490 495Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Gly 500 505 510Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Val Glu Ser 515 520 525Gly Gly Gly Leu Val Lys Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala 530 535 540Ala Ser Gly Phe Thr Phe Ser
Asn Ala Trp Met Ser Trp Val Arg Gln545 550 555 560Ala Pro Gly Lys
Gly Leu Glu Trp Val Gly Arg Ile Lys Ser Lys Thr 565 570 575Asp Gly
Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg Phe Thr 580 585
590Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
595 600 605Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Pro
Trp Glu 610 615 620Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser625 630 635 640Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser 645 650 655Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp 660 665 670Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 675 680 685Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 690 695 700Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln705 710
715 720Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp 725 730 735Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro 740 745 750Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe Leu Phe Pro 755 760 765Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr 770 775 780Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn785 790 795 800Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 805 810 815Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 820 825
830Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
835 840 845Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys 850 855 860Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys Arg Asp865 870 875 880Glu Leu Thr Lys Asn Gln Val Ser Leu
Trp Cys Leu Val Lys Gly Phe 885 890 895Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu 900 905 910Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 915 920 925Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 930 935 940Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr945 950
955 960Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 965
9703450PRTArtificial SequenceSynthetic Construct 3Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55
60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 4504971PRTArtificial SequenceSynthetic Construct 4Gln
Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10
15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr
20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp
Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala
Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln
Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala
Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr
Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln
Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val
Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170
175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala
180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly
Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu
Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr
Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu
Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser Arg Gln
Ala Arg Val Val Asn Gly Gly Gly Gly Gly Ser 260 265 270Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 275 280 285Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295
300Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val
Arg305 310 315 320Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg
Ile Arg Ser Lys 325 330 335Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
Ser Val Lys Gly Arg Phe 340 345 350Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn 355 360 365Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Val Arg His Gly 370 375 380Asn Phe Gly Asn
Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly385 390 395 400Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 405 410
415Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
420 425 430Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 435 440 445Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 450 455 460Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser465 470 475 480Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 485 490 495Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly 500 505 510Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 515 520 525Gly
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 530 535
540Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg
Gln545 550 555 560Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile
Lys Ser Lys Thr 565 570 575Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
Val Lys Gly Arg Phe Thr 580 585 590Ile Ser Arg Asp Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser 595 600 605Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr Cys Thr Thr Pro Trp Glu 610 615 620Trp Ser Trp Tyr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser625 630 635 640Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 645 650
655Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
660 665 670Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr 675 680 685Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 690 695 700Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln705 710 715 720Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp 725 730 735Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 740 745 750Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 755 760 765Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 770 775
780Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn785 790 795 800Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg
805 810 815Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val 820 825 830Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 835 840 845Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 850 855 860Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Cys Arg Asp865 870 875 880Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 885 890 895Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 900 905 910Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 915 920
925Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
930 935 940Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr945 950 955 960Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 9705971PRTArtificial SequenceSynthetic Construct 5Gln Val Gln
Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly
Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40
45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr
Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala
Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile
Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185
190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly
195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr
Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 275 280 285Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala
Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg305 310
315 320Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser
Lys 325 330 335Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys
Gly Arg Phe 340 345 350Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu
Tyr Leu Gln Met Asn 355 360 365Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Val Arg His Gly 370 375 380Asn Phe Gly Asn Ser Tyr Val
Ser Trp Phe Ala Tyr Trp Gly Gln Gly385 390 395 400Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 405 410 415Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 420 425
430Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
435 440 445Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 450 455 460Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser465 470 475 480Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 485 490 495Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Gly 500 505 510Gly Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 515 520 525Gly Gly Gly
Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 530 535 540Ala
Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln545 550
555 560Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Lys Ser Lys
Thr 565 570 575Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys Gly
Arg Phe Thr 580 585 590Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr
Leu Gln Met Asn Ser 595 600 605Leu Lys Thr Glu Asp Thr Ala Val Tyr
Tyr Cys Thr Thr Pro Trp Glu 610 615 620Trp Ser Trp Tyr Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser625 630 635 640Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 645 650 655Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 660 665
670Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
675 680 685Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr 690 695 700Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln705 710 715 720Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp 725 730 735Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro 740 745 750Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 755 760 765Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 770 775 780Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn785 790
795 800Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg 805 810 815Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val 820 825 830Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 835 840 845Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys 850 855 860Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Cys Arg Asp865 870 875 880Glu Leu Thr Lys
Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 885 890 895Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 900 905
910Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
915 920 925Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly 930 935 940Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr945 950 955 960Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 965 9706971PRTArtificial SequenceSynthetic Construct 6Gln
Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10
15Thr Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30Ser Ile His Trp Val Lys Gln Ala Pro Gly Lys Cys Leu Lys Trp
Met 35 40 45Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Ala Tyr Ala Asp
Asp Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr
Ala Thr Phe Phe Cys 85 90 95Ala His Pro Tyr Asp Tyr Asp Val Leu Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Thr Val Leu Thr Gln 130 135 140Ser Pro Ala Ser
Leu Gly Val Ser Leu Gly Gln Arg Ala Thr Ile Ser145 150 155 160Cys
Arg Ala Ser Lys Ser Val Ser Thr Ser Asn Tyr Ser Tyr Ile His 165 170
175Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr
180 185 190Val Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly
Ser Gly 195 200 205Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val
Glu Glu Glu Asp 210 215 220Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg
Glu Phe Pro Trp Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu
Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser Gly Gly
Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 275 280 285Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295
300Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val
Arg305 310 315 320Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg
Ile Arg Ser Lys 325 330 335Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
Ser Val Lys Gly Arg Phe 340 345 350Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr Leu Tyr Leu Gln Met Asn 355 360 365Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys Val Arg His Gly 370 375 380Asn Phe Gly Asn
Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly385 390 395 400Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 405 410
415Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
420 425 430Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp 435 440 445Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 450 455 460Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser465 470 475 480Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 485 490 495Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly 500 505 510Gly Gly Gly
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 515 520 525Gly
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 530 535
540Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg
Gln545 550 555 560Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile
Lys Ser Lys Thr 565 570 575Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
Val Lys Gly Arg Phe Thr 580 585 590Ile Ser Arg Asp Asp Ser Lys Asn
Thr Leu Tyr Leu Gln Met Asn Ser 595 600 605Leu Lys Thr Glu Asp Thr
Ala Val Tyr Tyr Cys Thr Thr Pro Trp Glu 610 615 620Trp Ser Trp Tyr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser625 630 635 640Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 645 650
655Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
660 665 670Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr 675 680 685Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr 690 695 700Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln705 710 715 720Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp 725 730 735Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 740 745 750Cys Pro Ala
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro 755 760 765Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 770 775
780Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn785 790 795 800Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg 805 810 815Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val 820 825 830Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser 835 840 845Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 850 855 860Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp865 870 875 880Glu
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 885 890
895Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
900 905 910Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 915 920 925Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly 930 935 940Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr945 950 955 960Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 965 970733PRTArtificial SequenceSynthetic Construct
7Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Ala Arg Val Val1 5
10 15Asn Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly 20 25 30Ser828PRTArtificial SequenceSynthetic Construct 8Gly
Gly Gly Gly Ser Val His Met Pro Leu Gly Phe Leu Gly Pro Gly1 5 10
15Arg Ser Arg Gly Ser Phe Pro Gly Gly Gly Gly Ser 20
25941PRTArtificial SequenceSynthetic Construct 9Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Arg Gln Ala Arg Val Val1 5 10 15Asn Gly Gly Gly
Gly Gly Ser Val Pro Leu Ser Leu Tyr Ser Gly Gly 20 25 30Gly Gly Gly
Ser Gly Gly Gly Gly Ser 35 401036PRTArtificial SequenceSynthetic
Construct 10Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Gln Ala Arg
Val Val1 5 10 15Asn Gly Val Pro Leu Ser Leu Tyr Ser Gly Gly Gly Gly
Gly Ser Gly 20 25 30Gly Gly Gly Ser 35115PRTArtificial
SequenceSynthetic Construct 11Thr Tyr Ala Met Asn1
51219PRTArtificial SequenceSynthetic Construct 12Arg Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1 5 10 15Val Lys
Gly1314PRTArtificial SequenceSynthetic Construct 13His Gly Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5 10145PRTArtificial
SequenceSynthetic Construct 14Asn Ala Trp Met Ser1
51519PRTArtificial SequenceSynthetic Construct 15Arg Ile Lys Ser
Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro1 5 10 15Val Lys
Gly169PRTArtificial SequenceSynthetic Construct 16Pro Trp Glu Trp
Ser Trp Tyr Asp Tyr1 51714PRTArtificial SequenceSynthetic Construct
17Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn1 5
10187PRTArtificial SequenceSynthetic Construct 18Gly Thr Asn Lys
Arg Ala Pro1 5199PRTArtificial SequenceSynthetic Construct 19Ala
Leu Trp Tyr Ser Asn Leu Trp Val1 5205PRTArtificial
SequenceSynthetic Construct 20Asp Tyr Ser Ile His1
52117PRTArtificial SequenceSynthetic Construct 21Trp Ile Asn Thr
Glu Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe Lys1 5 10
15Gly229PRTArtificial SequenceSynthetic Construct
22Pro Tyr Asp Tyr Asp Val Leu Asp Tyr1 52315PRTArtificial
SequenceSynthetic Construct 23Arg Ala Ser Lys Ser Val Ser Thr Ser
Asn Tyr Ser Tyr Ile His1 5 10 15247PRTArtificial SequenceSynthetic
Construct 24Tyr Val Ser Tyr Leu Glu Ser1 5259PRTArtificial
SequenceSynthetic Construct 25Gln His Ser Arg Glu Phe Pro Trp Thr1
5265PRTArtificial SequenceSynthetic Construct 26Ser Tyr Gly Val
Ser1 52716PRTArtificial SequenceSynthetic Construct 27Ile Ile Trp
Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile Ser1 5 10
152814PRTArtificial SequenceSynthetic Construct 28Gly Ile Thr Thr
Val Val Asp Asp Tyr Tyr Ala Met Asp Tyr1 5 102911PRTArtificial
SequenceSynthetic Construct 29Arg Ala Ser Glu Asn Ile Asp Ser Tyr
Leu Ala1 5 10307PRTArtificial SequenceSynthetic Construct 30Ala Ala
Thr Phe Leu Ala Asp1 5319PRTArtificial SequenceSynthetic Construct
31Gln His Tyr Tyr Ser Thr Pro Tyr Thr1 532450PRTArtificial
SequenceSynthetic Construct 32Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn
Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230
235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345
350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly Lys
45033213PRTArtificial SequenceSynthetic Construct 33Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr
Asn Thr Asn Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65
70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Phe Pro
Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala Pro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200
205Asn Arg Gly Glu Cys 21034492PRTArtificial SequenceSynthetic
Construct 34Glu Val Gln Leu Glu Gln Ser Gly Pro Val Leu Val Lys Pro
Gly Thr1 5 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30Tyr Ile Asn Trp Ile Ile Gln Ser His Gly Lys Cys
Leu Glu Trp Ile 35 40 45Gly Val Ile Asn Pro Asp Ser Gly Gly Thr Asp
Tyr Asn Gln Asn Phe 50 55 60Lys Gly Lys Ala Thr Leu Thr Val Asp Lys
Ser Ser Thr Thr Ala Tyr65 70 75 80Met Glu Leu Thr Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Asp Ser Tyr Gly
Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110Leu Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Thr 130 135 140Pro
Lys Phe Leu Leu Val Pro Ala Gly Asp Arg Ile Thr Met Thr Cys145 150
155 160Lys Ala Ser Leu Ser Val Thr Asn Asp Val Ala Trp Tyr Gln Gln
Lys 165 170 175Pro Gly Gln Ser Pro Lys Leu Leu Leu Tyr Tyr Ala Ser
Asn Arg Asn 180 185 190Ala Gly Val Pro Asp Arg Phe Thr Gly Ser Gly
Tyr Gly Thr Asp Phe 195 200 205Thr Phe Thr Ile Thr Thr Leu Gln Ala
Glu Asp Leu Ala Val Tyr Phe 210 215 220Cys Gln Gln Asp Tyr Thr Ser
Pro Pro Thr Phe Gly Cys Gly Thr Lys225 230 235 240Leu Glu Ile Arg
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro 245 250 255Leu Gly
Leu Trp Ser Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 260 265
270Gly Gly Gly Gly Ser Gly Gly Asp Ile Gln Met Thr Gln Ser Pro Ser
275 280 285Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala 290 295 300Ser Gln Gly Ile Asn Asn Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro Gly305 310 315 320Lys Ala Pro Lys Arg Leu Ile Tyr Asn
Thr Asn Asn Leu Gln Thr Gly 325 330 335Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Glu Phe Thr Leu 340 345 350Thr Ile Ser Ser Leu
Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu 355 360 365Gln His Asn
Ser Phe Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 370 375 380Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp385 390
395 400Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn 405 410 415Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 420 425 430Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 435 440 445Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 450 455 460Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser465 470 475 480Ser Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 485 4903535PRTArtificial
SequenceSynthetic Construct 35Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Pro Leu Gly Leu Trp1 5 10 15Ser Gln Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30Ser Gly Gly
35368PRTArtificial SequenceSynthetic Construct 36Arg Gln Ala Arg
Val Val Asn Gly1 53718PRTArtificial SequenceSynthetic Construct
37Val His Met Pro Leu Gly Phe Leu Gly Pro Gly Arg Ser Arg Gly Ser1
5 10 15Phe Pro3821PRTArtificial SequenceSynthetic
ConstructX(9)..(13)X is an amino acidX(9)..(13)X or Xaa is an amino
acid 38Arg Gln Ala Arg Val Val Asn Gly Xaa Xaa Xaa Xaa Xaa Val Pro
Leu1 5 10 15Ser Leu Tyr Ser Gly 203916PRTArtificial
SequenceSynthetic Construct 39Arg Gln Ala Arg Val Val Asn Gly Val
Pro Leu Ser Leu Tyr Ser Gly1 5 10 15407PRTArtificial
SequenceSynthetic Construct 40Pro Leu Gly Leu Trp Ser Gln1
541249PRTArtificial SequenceSynthetic Construct 41Gln Ile Gln Leu
Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val Arg
Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Ser Ile
His Trp Val Lys Gln Ala Pro Gly Lys Cys Leu Lys Trp Met 35 40 45Gly
Trp Ile Asn Thr Glu Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe 50 55
60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr65
70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Phe Phe
Cys 85 90 95Ala His Pro Tyr Asp Tyr Asp Val Leu Asp Tyr Trp Gly Gln
Gly Thr 100 105 110Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser 115 120 125Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Thr Val Leu Thr Gln 130 135 140Ser Pro Ala Ser Leu Gly Val Ser
Leu Gly Gln Arg Ala Thr Ile Ser145 150 155 160Cys Arg Ala Ser Lys
Ser Val Ser Thr Ser Asn Tyr Ser Tyr Ile His 165 170 175Trp Tyr Gln
Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr 180 185 190Val
Ser Tyr Leu Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 195 200
205Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp
210 215 220Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg Glu Phe Pro Trp
Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys
24542249PRTArtificial SequenceSynthetic Construct 42Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val
Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly
Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65
70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys
Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala
Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200
205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp
210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr
Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys
24543125PRTArtificial SequenceSynthetic Construct 43Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30Ala Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser
Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 125445PRTArtificial SequenceSynthetic Construct 44Thr
Tyr Ala Met Asn1 54519PRTArtificial SequenceSynthetic Construct
45Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser1
5 10 15Val Lys Gly4614PRTArtificial SequenceSynthetic Construct
46His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr1 5
1047120PRTArtificial SequenceSynthetic Construct 47Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly
Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55
60Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65
70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val
Tyr 85 90 95Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120485PRTArtificial SequenceSynthetic Construct 48Asp Tyr Tyr Ile
Asn1 54917PRTArtificial SequenceSynthetic Construct 49Val Ile Asn
Pro Asp Ser Gly Gly Thr Asp Tyr Asn Gln Asn Phe Lys1 5 10
15Gly508PRTArtificial SequenceSynthetic Construct 50Arg Asp Ser Tyr
Gly Phe Asp Tyr1 55111PRTArtificial SequenceSynthetic Construct
51Lys Ala Ser Leu Ser Val Thr Asn Asp Val Ala1 5 10527PRTArtificial
SequenceSynthetic Construct 52Tyr Ala Ser Asn Arg Asn Ala1
5539PRTArtificial SequenceSynthetic Construct 53Gln Gln Asp Tyr Thr
Ser Pro Pro Thr1 554207PRTHomo sapiens 54Met Gln Ser Gly Thr His
Trp Arg Val Leu Gly Leu Cys Leu Leu Ser1 5 10 15Val Gly Val Trp Gly
Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30Gln Thr Pro Tyr
Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45Cys Pro Gln
Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60Asn Ile
Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp65
70 75 80His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr
Tyr 85 90 95Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe
Tyr Leu 100 105 110Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu
Met Asp Val Met 115 120 125Ser Val Ala Thr Ile Val Ile Val Asp Ile
Cys Ile Thr Gly Gly Leu 130 135 140Leu Leu Leu Val Tyr Tyr Trp Ser
Lys Asn Arg Lys Ala Lys Ala Lys145 150 155 160Pro Val Thr Arg Gly
Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175Lys Glu Arg
Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190Lys
Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200
20555109PRTArtificial SequenceSynthetic Construct 55Gln Ala Val Val
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr
Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr
Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55
60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65
70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100
105565PRTArtificial SequenceSynthetic Construct 56Asp Tyr Lys Ile
His1 55717PRTArtificial SequenceSynthetic Construct 57Tyr Phe Asn
Pro Asn Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe Gln1 5 10
15Gly5811PRTArtificial SequenceSynthetic Construct 58Leu Ser Pro
Gly Gly Tyr Tyr Val Met Asp Ala1 5 105911PRTArtificial
SequenceSynthetic Construct 59Arg Ala Ser Gln Gly Ile Asn Asn Tyr
Leu Asn1 5 10607PRTArtificial SequenceSynthetic Construct 60Asn Thr
Asn Asn Leu Gln Thr1 5618PRTArtificial SequenceSynthetic Construct
61Leu Gln His Asn Ser Phe Pro Thr1 562648DNAArtificial
SequenceSynthetic Construct 62caggccgtcg tgacccagga acccagcctg
acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc
accagcaact acgccaactg ggtgcaggaa 120aagcccggcc aggccttcag
aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat
tcagcggatc tctgctggga ggaaaggccg ccctgacact gtctggcgcc
240cagccagaag atgaggccga gtactactgc gccctgtggt acagcaacct
gtgggtgttc 300ggcggaggca ccaagctgac agtcctaggt caacccaagg
ctgcccccag cgtgaccctg 360ttccccccca gcagcgagga actgcaggcc
aacaaggcca ccctggtctg cctgatcagc 420gacttctacc caggcgccgt
gaccgtggcc tggaaggccg acagcagccc cgtgaaggcc 480ggcgtggaga
ccaccacccc cagcaagcag agcaacaaca agtacgccgc cagcagctac
540ctgagcctga cccccgagca gtggaagagc cacaggtcct acagctgcca
ggtgacccac 600gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagctga
648632916DNAArtificial SequenceSynthetic Construct 63cagatccagc
tggtgcagag cggccctgag ctgaagaaac ccggcgagac agtgcggatc 60agctgcaagg
ccagcggcta caccttcacc gactacagca tccactgggt caagcaggcc
120cctggcaagt gcctgaagtg gatgggctgg atcaacaccg agacaggcga
gcccgcctac 180gccgacgatt tcaagggcag attcgccttc agcctggaaa
ccagcgccag caccgcctac 240ctgcagatca acaacctgaa gaacgaggac
accgccacct ttttctgcgc ccacccctac 300gactacgacg tgctggatta
ttggggccag ggcaccagcg tgaccgtgtc tagcggaggc 360ggaggatctg
gcggcggagg aagtggcgga gggggatctg ggggaggcgg atctgatacc
420gtgctgacac agagccctgc cagcctggga gtgtccctgg gacagagagc
caccatcagc 480tgtcgggcca gcaagagcgt gtccaccagc aactacagct
atatccactg gtatcagcag 540aagcccggcc agccccccaa gctgctgatc
aaatacgtgt cctacctgga aagcggcgtg 600cccgccagat tttctggctc
tggcagcggc accgacttca ccctgaacat ccaccccgtg 660gaagaggaag
atgccgccac ctactactgc cagcacagca gagagttccc ttggaccttc
720ggctgcggca ccaagctgga aatcaaaggc gggggaggct ccggaggcgg
cggaagtaga 780caggccagag tcgtgaacgg gggagggggg ggaagtgggg
gcggaggcag tgggggggga 840ggatccgagg tgcagctgct ggaatctggc
ggcggactgg tgcagcctgg cggatctctg 900agactgagct gtgccgccag
cggcttcacc ttcagcacct acgccatgaa ctgggtgcgc 960caggcccctg
gcaaaggcct ggaatgggtg tcccggatca gaagcaagta caacaactac
1020gccacctact acgccgacag cgtgaagggc cggttcacca tcagccggga
cgacagcaag 1080aacaccctgt acctgcagat gaacagcctg cgggccgagg
acaccgccgt gtactattgt 1140gtgcggcacg gcaacttcgg caacagctat
gtgtcttggt ttgcctactg gggccagggc 1200accctcgtga ccgtgtcaag
cgctagcaca aagggcccta gcgtgttccc tctggccccc 1260agcagcaaga
gcacaagcgg cggaacagcc gccctgggct gcctcgtgaa ggactacttc
1320cccgagcccg tgacagtgtc ttggaacagc ggagccctga caagcggcgt
gcacaccttc 1380cctgccgtgc tgcagagcag cggcctgtac tccctgagca
gcgtggtcac cgtgcctagc 1440agcagcctgg gcacccagac ctacatctgc
aacgtgaacc acaagcccag caacaccaaa 1500gtggacaaga aggtggagcc
caagagctgt gatggcggag gagggtccgg aggcggagga 1560tccgaggtgc
aattggttga atctggtggt ggtctggtaa aaccgggcgg ttccctgcgt
1620ctgagctgcg cggcttccgg attcaccttc tccaacgcgt ggatgagctg
ggttcgccag 1680gccccgggca aaggcctcga gtgggttggt cgtatcaagt
ctaaaactga cggtggcacc 1740acggattacg cggctccagt taaaggtcgt
tttaccattt cccgcgacga tagcaaaaac 1800actctgtatc tgcagatgaa
ctctctgaaa actgaagaca ccgcagtcta ctactgtact 1860accccgtggg
aatggtcttg gtacgattat tggggccagg gcacgctggt tacggtgtct
1920agcgctagta ccaagggccc cagcgtgttc cccctggcac ccagcagcaa
gagcacatct 1980ggcggaacag ccgctctggg ctgtctggtg aaagactact
tccccgagcc cgtgaccgtg 2040tcttggaact ctggcgccct gaccagcggc
gtgcacacct ttccagccgt gctgcagagc 2100agcggcctgt actccctgtc
ctccgtggtc accgtgccct ctagctccct gggaacacag 2160acatatatct
gtaatgtcaa tcacaagcct tccaacacca aagtcgataa gaaagtcgag
2220cccaagagct gcgacaaaac tcacacatgc ccaccgtgcc cagcacctga
agctgcaggg 2280ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 2340cctgaggtca catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac 2400tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 2460aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
2520aaggagtaca agtgcaaggt ctccaacaaa gccctcggcg cccccatcga
gaaaaccatc 2580tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atgccgggat 2640gagctgacca agaaccaggt cagcctgtgg
tgcctggtca aaggcttcta tcccagcgac 2700atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 2760gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
2820tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 2880acgcagaaga gcctctccct gtctccgggt aaatga
2916641353DNAArtificial SequenceSynthetic Construct 64gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc
120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc
gcgacgatag caaaaacact 240ctgtatctgc agatgaactc tctgaaaact
gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacca
agggcccctc cgtgttcccc ctggccccca gcagcaagag caccagcggc
420ggcacagccg ctctgggctg cctggtcaag gactacttcc ccgagcccgt
gaccgtgtcc 480tggaacagcg gagccctgac ctccggcgtg cacaccttcc
ccgccgtgct gcagagttct 540ggcctgtata gcctgagcag cgtggtcacc
gtgccttcta gcagcctggg cacccagacc 600tacatctgca acgtgaacca
caagcccagc aacaccaagg tggacaagaa ggtggagccc 660aagagctgcg
acaaaactca cacatgccca ccgtgcccag cacctgaagc tgcaggggga
720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt
gcaaggtctc caacaaagcc ctcggcgccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtgcaccc tgcccccatc
ccgggatgag 1080ctgaccaaga accaggtcag cctctcgtgc gcagtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctcgtg agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccgcttcacg
1320cagaagagcc tctccctgtc tccgggtaaa tga 1353652916DNAArtificial
SequenceSynthetic Construct 65caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtaga 780caggccagag
tcgtgaacgg gggagggggg ggaagtgggg gcggaggcag tgggggcgga
840ggatccgagg tgcagctgct ggaatctggc ggcggactgg tgcagcctgg
cggatctctg 900agactgagct gtgccgccag cggcttcacc ttcagcacct
acgccatgaa ctgggtgcgc 960caggcccctg gcaaaggcct ggaatgggtg
tcccggatca gaagcaagta caacaactac 1020gccacctact acgccgacag
cgtgaagggc cggttcacca tcagccggga cgacagcaag 1080aacaccctgt
acctgcagat gaacagcctg cgggccgagg acaccgccgt gtactattgt
1140gtgcggcacg gcaacttcgg caacagctat gtgtcttggt ttgcctactg
gggccagggc 1200accctcgtga ccgtgtcaag cgctagcaca aagggcccta
gcgtgttccc tctggccccc 1260agcagcaaga gcacaagcgg cggaacagcc
gccctgggct gcctcgtgaa ggactacttc 1320cccgagcccg tgacagtgtc
ttggaacagc ggagccctga caagcggcgt gcacaccttc 1380cctgccgtgc
tgcagagcag cggcctgtac tccctgagca gcgtggtcac cgtgcctagc
1440agcagcctgg gcacccagac ctacatctgc aacgtgaacc acaagcccag
caacaccaaa 1500gtggacaaga aggtggagcc caagagctgt gatggcggag
gagggtccgg gggcggagga 1560tccgaggtgc aattggttga atctggtggt
ggtctggtaa aaccgggcgg ttccctgcgt 1620ctgagctgcg cggcttccgg
gttcaccttc tccaacgcgt ggatgagctg ggttcgccag 1680gccccgggca
aaggcctcga gtgggttggt cgtatcaagt ctaaaactga cggtggcacc
1740acggattacg cggctccagt taaaggtcgt tttaccattt cccgcgacga
tagcaaaaac 1800actctgtatc tgcagatgaa ctctctgaaa actgaagaca
ccgcagtcta ctactgtact 1860accccgtggg aatggtcttg gtacgattat
tggggccagg gcacgctggt tacggtgtct 1920agcgctagta ccaagggccc
cagcgtgttc cccctggcac ccagcagcaa gagcacatct 1980ggcggaacag
ccgctctggg ctgtctggtg aaagactact tccccgagcc cgtgaccgtg
2040tcttggaact ctggcgccct gaccagcggc gtgcacacct ttccagccgt
gctgcagagc 2100agcggcctgt actccctgtc ctccgtggtc accgtgccct
ctagctccct gggaacacag 2160acatatatct gtaatgtcaa tcacaagcct
tccaacacca aagtcgataa gaaagtcgag 2220cccaagagct gcgacaaaac
tcacacatgc ccaccgtgcc cagcacctga agctgcaggg 2280ggaccgtcag
tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
2340cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt
caagttcaac 2400tggtacgtgg acggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagtac 2460aacagcacgt accgtgtggt cagcgtcctc
accgtcctgc accaggactg gctgaatggc 2520aaggagtaca agtgcaaggt
ctccaacaaa gccctcggcg cccccatcga gaaaaccatc 2580tccaaagcca
aagggcagcc ccgagaacca caggtgtaca ccctgccccc atgccgggat
2640gagctgacca agaaccaggt cagcctgtgg tgcctggtca aaggcttcta
tcccagcgac 2700atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 2760gtgctggact ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga caagagcagg 2820tggcagcagg ggaacgtctt
ctcatgctcc gtgatgcatg aggctctgca caaccactac 2880acgcagaaga
gcctctccct gtctccgggt aaatga 2916662916DNAArtificial
SequenceSynthetic Construct 66caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtgga 780ggcggcggaa
gtggcggagg cggagggggg ggaagtgggg gcggaggcag tgggggggga
840ggatccgagg tgcagctgct ggaatctggc ggcggactgg tgcagcctgg
cggatctctg 900agactgagct gtgccgccag cggcttcacc ttcagcacct
acgccatgaa ctgggtgcgc 960caggcccctg gcaaaggcct ggaatgggtg
tcccggatca gaagcaagta caacaactac 1020gccacctact acgccgacag
cgtgaagggc cggttcacca tcagccggga cgacagcaag 1080aacaccctgt
acctgcagat gaacagcctg cgggccgagg acaccgccgt gtactattgt
1140gtgcggcacg gcaacttcgg caacagctat gtgtcttggt ttgcctactg
gggccagggc 1200accctcgtga ccgtgtcaag cgctagcaca aagggcccta
gcgtgttccc tctggccccc 1260agcagcaaga gcacaagcgg cggaacagcc
gccctgggct gcctcgtgaa ggactacttc 1320cccgagcccg tgacagtgtc
ttggaacagc ggagccctga caagcggcgt gcacaccttc 1380cctgccgtgc
tgcagagcag cggcctgtac tccctgagca gcgtggtcac cgtgcctagc
1440agcagcctgg gcacccagac ctacatctgc aacgtgaacc acaagcccag
caacaccaaa 1500gtggacaaga aggtggagcc caagagctgt gatggcggag
gagggtccgg aggcggaggc 1560tccgaggtgc aattggttga atctggtggt
ggtctggtaa aaccgggcgg ttccctgcgt 1620ctgagctgcg cggcttccgg
attcaccttc tccaacgcgt ggatgagctg ggttcgccag 1680gccccgggca
aaggcctcga gtgggttggt cgtatcaagt ctaaaactga cggtggcacc
1740acggattacg cggctccagt taaaggtcgt tttaccattt cccgcgacga
tagcaaaaac 1800actctgtatc tgcagatgaa ctctctgaaa actgaagaca
ccgcagtcta ctactgtact 1860accccgtggg aatggtcttg gtacgattat
tggggccagg gcacgctggt tacggtgtct 1920agcgctagta ccaagggccc
cagcgtgttc cccctggcac ccagcagcaa gagcacatct 1980ggcggaacag
ccgctctggg ctgtctggtg aaagactact tccccgagcc cgtgaccgtg
2040tcttggaact ctggcgccct gaccagcggc gtgcacacct ttccagccgt
gctgcagagc 2100agcggcctgt actccctgtc ctccgtggtc accgtgccct
ctagctccct gggaacacag 2160acatatatct gtaatgtcaa tcacaagcct
tccaacacca aagtcgataa gaaagtcgag 2220cccaagagct gcgacaaaac
tcacacatgc ccaccgtgcc cagcacctga agctgcaggg 2280ggaccgtcag
tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc
2340cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt
caagttcaac 2400tggtacgtgg acggcgtgga ggtgcataat gccaagacaa
agccgcggga ggagcagtac 2460aacagcacgt accgtgtggt cagcgtcctc
accgtcctgc accaggactg gctgaatggc 2520aaggagtaca agtgcaaggt
ctccaacaaa gccctcggcg cccccatcga gaaaaccatc 2580tccaaagcca
aagggcagcc ccgagaacca caggtgtaca ccctgccccc atgccgggat
2640gagctgacca agaaccaggt cagcctgtgg tgcctggtca aaggcttcta
tcccagcgac 2700atcgccgtgg agtgggagag caatgggcag ccggagaaca
actacaagac cacgcctccc 2760gtgctggact ccgacggctc cttcttcctc
tacagcaagc tcaccgtgga caagagcagg 2820tggcagcagg ggaacgtctt
ctcatgctcc gtgatgcatg aggctctgca caaccactac 2880acgcagaaga
gcctctccct gtctccgggt aaatga 2916672916DNAArtificial
SequenceSynthetic Construct 67cagatccagc tggtgcagag cggccctgag
ctgaagaaac ccggcgagac agtgcggatc 60agctgcaagg ccagcggcta caccttcacc
gactacagca tccactgggt caagcaggcc 120cctggcaagt gcctgaagtg
gatgggctgg atcaacaccg agacaggcga gcccgcctac 180gccgacgatt
tcaagggcag attcgccttc agcctggaaa ccagcgccag caccgcctac
240ctgcagatca acaacctgaa gaacgaggac accgccacct ttttctgcgc
ccacccctac 300gactacgacg tgctggatta ttggggccag ggcaccagcg
tgaccgtgtc tagcggaggc 360ggaggatctg gcggcggagg aagtggcgga
gggggatctg ggggaggcgg atctgatacc 420gtgctgacac agagccctgc
cagcctggga gtgtccctgg gacagagagc caccatcagc 480tgtcgggcca
gcaagagcgt gtccaccagc aactacagct atatccactg gtatcagcag
540aagcccggcc agccccccaa gctgctgatc aaatacgtgt cctacctgga
aagcggcgtg 600cccgccagat tttctggctc tggcagcggc accgacttca
ccctgaacat ccaccccgtg 660gaagaggaag atgccgccac ctactactgc
cagcacagca gagagttccc ttggaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtgga 780ggcggcggaa
gtggcggagg cggagggggg ggaagtgggg gcggaggcag tgggggggga
840ggatccgagg tgcagctgct ggaatctggc ggcggactgg tgcagcctgg
cggatctctg 900agactgagct gtgccgccag cggcttcacc ttcagcacct
acgccatgaa ctgggtgcgc 960caggcccctg gcaaaggcct ggaatgggtg
tcccggatca gaagcaagta caacaactac 1020gccacctact acgccgacag
cgtgaagggc cggttcacca tcagccggga cgacagcaag 1080aacaccctgt
acctgcagat gaacagcctg cgggccgagg acaccgccgt gtactattgt
1140gtgcggcacg gcaacttcgg caacagctat gtgtcttggt ttgcctactg
gggccagggc 1200accctcgtga ccgtgtcaag cgctagcaca aagggcccta
gcgtgttccc tctggccccc 1260agcagcaaga gcacaagcgg cggaacagcc
gccctgggct gcctcgtgaa ggactacttc 1320cccgagcccg tgacagtgtc
ttggaacagc ggagccctga caagcggcgt gcacaccttc 1380cctgccgtgc
tgcagagcag cggcctgtac tccctgagca gcgtggtcac cgtgcctagc
1440agcagcctgg gcacccagac ctacatctgc aacgtgaacc acaagcccag
caacaccaaa 1500gtggacaaga aggtggagcc caagagctgt gatggcggag
gagggtccgg aggcggaggc 1560tccgaggtgc aattggttga atctggtggt
ggtctggtaa aaccgggcgg ttccctgcgt 1620ctgagctgcg cggcttccgg
attcaccttc tccaacgcgt ggatgagctg ggttcgccag 1680gccccgggca
aaggcctcga gtgggttggt cgtatcaagt ctaaaactga cggtggcacc
1740acggattacg
cggctccagt taaaggtcgt tttaccattt cccgcgacga tagcaaaaac
1800actctgtatc tgcagatgaa ctctctgaaa actgaagaca ccgcagtcta
ctactgtact 1860accccgtggg aatggtcttg gtacgattat tggggccagg
gcacgctggt tacggtgtct 1920agcgctagta ccaagggccc cagcgtgttc
cccctggcac ccagcagcaa gagcacatct 1980ggcggaacag ccgctctggg
ctgtctggtg aaagactact tccccgagcc cgtgaccgtg 2040tcttggaact
ctggcgccct gaccagcggc gtgcacacct ttccagccgt gctgcagagc
2100agcggcctgt actccctgtc ctccgtggtc accgtgccct ctagctccct
gggaacacag 2160acatatatct gtaatgtcaa tcacaagcct tccaacacca
aagtcgataa gaaagtcgag 2220cccaagagct gcgacaaaac tcacacatgc
ccaccgtgcc cagcacctga agctgcaggg 2280ggaccgtcag tcttcctctt
ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 2340cctgaggtca
catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac
2400tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga
ggagcagtac 2460aacagcacgt accgtgtggt cagcgtcctc accgtcctgc
accaggactg gctgaatggc 2520aaggagtaca agtgcaaggt ctccaacaaa
gccctcggcg cccccatcga gaaaaccatc 2580tccaaagcca aagggcagcc
ccgagaacca caggtgtaca ccctgccccc atgccgggat 2640gagctgacca
agaaccaggt cagcctgtgg tgcctggtca aaggcttcta tcccagcgac
2700atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac
cacgcctccc 2760gtgctggact ccgacggctc cttcttcctc tacagcaagc
tcaccgtgga caagagcagg 2820tggcagcagg ggaacgtctt ctcatgctcc
gtgatgcatg aggctctgca caaccactac 2880acgcagaaga gcctctccct
gtctccgggt aaatga 29166899DNAArtificial SequenceSynthetic Construct
68ggcgggggag gctccggagg cggcggaagt agacaggcca gagtcgtgaa cgggggaggg
60gggggaagtg ggggcggagg cagtgggggc ggaggatcc 99691350DNAArtificial
SequenceSynthetic Construct 69caggtgcagc tggtccagag cggcgccgag
gtgaagaaac ccgggtcctc tgtcaaggtg 60tcatgcaagg ctagcggatt cacctttaca
gactacaaaa tccactgggt taggcaggca 120cctggccaag gactcgaatg
gatggggtat ttcaacccaa attccggcta ctctacctat 180gcccagaagt
ttcagggaag agtgactatt acagctgata agagtaccag cactgcatac
240atggagctgt cctctcttcg ctcagaggac accgccgtct actattgtgc
tcggctgagc 300cccggtggct actatgtgat ggatgcatgg gggcagggaa
caaccgtaac agtgtcctct 360gcgtcgacta agggcccttc agtttttcca
ctcgccccca gtagcaagtc cacatctggg 420ggtaccgctg ccctgggctg
ccttgtgaaa gactatttcc ctgaaccagt cactgtgtca 480tggaatagcg
gagccctgac ctccggtgta cacacattcc ccgctgtgtt gcagtctagt
540ggcctgtaca gcctctcctc tgttgtgacc gtcccttcaa gctccctggg
gacacagacc 600tatatctgta acgtgaatca taagccatct aacactaaag
tagataaaaa agtggagccc 660aagagttgcg acaaaacaca cacctgtccc
ccttgcccag cccccgagct tctgggaggc 720cctagcgtct ttctcttccc
acccaagcct aaggatactc tgatgatatc caggacccca 780gaagttacat
gcgtggtcgt ggacgtctca cacgaggacc ccgaagtgaa atttaactgg
840tacgttgatg gtgtggaagt ccataatgcc aagaccaagc ctagagagga
gcaatacaac 900agtacatatc gcgtggtaag cgtgttgacc gttctccacc
aggactggct caatgggaaa 960gaatacaagt gtaaagtgtc caacaaagct
ctgccagcac ccatcgagaa gactatttct 1020aaggccaaag gccagccccg
ggagcctcag gtctatacac ttccaccctc aagggatgaa 1080ctgaccaaga
accaagtgag cttgacttgc ctggtaaagg ggttctaccc ttccgacatc
1140gctgtggagt gggagtctaa tggacaacca gaaaacaatt acaaaaccac
accccctgtc 1200ctcgacagtg atggcagctt tttcctgtat agcaaactta
ccgttgacaa gtccagatgg 1260cagcagggaa acgtgttctc atgtagcgtc
atgcacgaag ctttgcataa ccactacaca 1320cagaaaagcc tcagcctgag
tccagggaag 135070639DNAArtificial SequenceSynthetic Construct
70gacatccaaa tgacccagtc acctagtagc ctctccgcct ctgttggcga cagggtgaca
60attacatgca gagcttcaca gggtatcaac aattacctga actggtatca gcagaaacca
120gggaaggccc ccaagcgctt gatatataac accaataacc tgcaaactgg
cgtccctagc 180cggttctccg gatctggtag tggcaccgaa tttacactca
ccatcagctc cctgcagcca 240gaggatttcg ccacatacta ttgtcttcag
cataattctt tccccacctt tgggcaagga 300actaaactgg agattaagcg
tactgtcgcc gctccctctg tgttcatttt tcctccaagt 360gatgagcagc
tcaaaagcgg taccgcatcc gttgtgtgcc tgcttaacaa cttctatccc
420cgggaagcca aggtccaatg gaaggtggac aatgctctgc agtcaggaaa
cagtcaggag 480agcgtaaccg agcaggattc caaagactct acttactcat
tgagctccac cctgacactc 540tctaaggcag actatgaaaa gcataaagtg
tacgcctgtg aggttaccca ccagggcctg 600agtagccctg tgacaaagtc
cttcaatagg ggagagtgc 639711476DNAArtificial SequenceSynthetic
Construct 71gaggttcagc tggagcagtc aggacctgtg ctggtgaagc ctgggacttc
agtgaagatg 60tcctgtaagg cttctggata cacattcact gactactata taaactggat
aatacagagc 120catggaaagt gtcttgagtg gattggagtt attaatcctg
acagcggtgg tactgactac 180aaccagaact tcaagggcaa ggccacattg
actgttgaca agtcctccac cacagcctac 240atggaactca ctagcctgac
atctgaggac tctgcagtct attattgtgc aagaagggat 300tcttacggct
ttgactactg gggccaaggc accactctca cagtctcctc aggcggaggt
360ggctcagggg gaggcggtag cggcggaggt ggctcagggg gaggcggtag
cgacattgtg 420ctgacccaga ctcccaaatt cctgcttgtg ccagcaggag
acaggattac catgacctgc 480aaggccagtc tgagtgtgac taatgatgta
gcttggtatc aacagaaacc agggcagtct 540cctaaactgc tgttatacta
tgcatccaat cgcaacgctg gagtccctga tcgcttcact 600ggcagtggat
atgggacgga tttcactttc accatcacca ctttgcaggc tgaagacctg
660gcagtttatt tctgtcagca ggattatacc tctcctccga cgttcggttg
tggcaccaag 720ctagaaatcc gtggtggcgg cggttctggc ggagggggtt
ctggccccct ggggctatgg 780agccagggtg gcggcggttc tggcggaggg
ggttctggcg gtggtggctc tggcggtgac 840atccaaatga cccagtcacc
tagtagcctc tccgcctctg ttggcgacag ggtgacaatt 900acatgcagag
cttcacaggg tatcaacaat tacctgaact ggtatcagca gaaaccaggg
960aaggccccca agcgcttgat atataacacc aataacctgc aaactggcgt
ccctagccgg 1020ttctccggat ctggtagtgg caccgaattt acactcacca
tcagctccct gcagccagag 1080gatttcgcca catactattg tcttcagcat
aattctttcc ccacctttgg gcaaggaact 1140aaactggaga ttaagcgtac
tgtcgccgct ccctctgtgt tcatttttcc tccaagtgat 1200gagcagctca
aaagcggtac cgcatccgtt gtgtgcctgc ttaacaactt ctatccccgg
1260gaagccaagg tccaatggaa ggtggacaat gctctgcagt caggaaacag
tcaggagagc 1320gtaaccgagc aggattccaa agactctact tactcattga
gctccaccct gacactctct 1380aaggcagact atgaaaagca taaagtgtac
gcctgtgagg ttacccacca gggcctgagt 1440agccctgtga caaagtcctt
caatagggga gagtgc 147672971PRTArtificial SequenceSynthetic
Construct 72Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro
Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
Thr Ser Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys
Leu Glu Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr
His Ser Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser
Lys Ser Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp
Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val
Asp Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150
155 160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu
Ala 165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu
Val Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg
Phe Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile
Asn Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln
His Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Val His 245 250 255Met Pro
Leu Gly Phe Leu Gly Pro Arg Gln Ala Arg Val Val Asn Gly 260 265
270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
275 280 285Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys 290 295 300Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met
Asn Trp Val Arg305 310 315 320Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Ser Arg Ile Arg Ser Lys 325 330 335Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe 340 345 350Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 355 360 365Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly 370 375 380Asn
Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly385 390
395 400Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 405 410 415Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 420 425 430Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 435 440 445Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 450 455 460Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser465 470 475 480Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 485 490 495Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly 500 505
510Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser
515 520 525Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
Cys Ala 530 535 540Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp Met Ser
Trp Val Arg Gln545 550 555 560Ala Pro Gly Lys Gly Leu Glu Trp Val
Gly Arg Ile Lys Ser Lys Thr 565 570 575Asp Gly Gly Thr Thr Asp Tyr
Ala Ala Pro Val Lys Gly Arg Phe Thr 580 585 590Ile Ser Arg Asp Asp
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser 595 600 605Leu Lys Thr
Glu Asp Thr Ala Val Tyr Tyr Cys Thr Thr Pro Trp Glu 610 615 620Trp
Ser Trp Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser625 630
635 640Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser 645 650 655Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp 660 665 670Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr 675 680 685Ser Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr 690 695 700Ser Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln705 710 715 720Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 725 730 735Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 740 745
750Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
755 760 765Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
Val Thr 770 775 780Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn785 790 795 800Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg 805 810 815Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val 820 825 830Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 835 840 845Asn Lys Ala
Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 850 855 860Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp865 870
875 880Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
Phe 885 890 895Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu 900 905 910Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe 915 920 925Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly 930 935 940Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr945 950 955 960Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 965 97073689PRTArtificial
SequenceSynthetic Construct 73Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Arg Ile Lys Ser Lys
Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60Pro Val Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr65 70 75 80Leu Tyr Leu
Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys
Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu225 230
235 240Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
Cys 245 250 255Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn
Trp Val Arg 260 265 270Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
Arg Ile Arg Ser Lys 275 280 285Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp Ser Val Lys Gly Arg Phe 290 295 300Thr Ile Ser Arg Asp Asp Ser
Lys Asn Thr Leu Tyr Leu Gln Met Asn305 310 315 320Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly 325 330 335Asn Phe
Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly 340 345
350Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
355 360 365Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu 370 375 380Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp385 390 395 400Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu 405 410 415Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser 420 425 430Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 435 440 445Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 450 455 460Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro465 470
475 480Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser 485 490 495Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp 500 505 510Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn 515 520 525Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Thr Tyr Arg Val 530 535 540Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu545 550 555 560Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys 565 570 575Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 580 585
590Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
595 600 605Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu 610 615 620Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu625 630 635 640Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys 645 650 655Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser
Cys Ser Val Met His Glu 660 665 670Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly 675 680 685Lys74497PRTArtificial
SequenceSynthetic Construct 74Gln Val Gln Leu Lys Glu Ser Gly Pro
Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val
Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val Ser Trp Val Arg Gln
Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp
Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile
Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Leu Asn
Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly
Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105
110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile 130 135 140Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser
Val Gly Glu Thr145 150 155 160Val Thr Ile Thr Cys Arg Ala Ser Glu
Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln Gln Lys Gln Gly
Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala Thr Phe Leu Ala
Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205Ser Gly Thr
Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp 210 215 220Val
Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230
235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly
Gly 245 250 255Gly Gly Ser Arg Gln Ala Arg Val Val Asn Gly Gly Gly
Gly Gly Ser 260 265 270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
Ala Val Val Thr Gln 275 280 285Glu Pro Ser Leu Thr Val Ser Pro Gly
Gly Thr Val Thr Leu Thr Cys 290 295 300Gly Ser Ser Thr Gly Ala Val
Thr Thr Ser Asn Tyr Ala Asn Trp Val305 310 315 320Gln Glu Lys Pro
Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn 325 330 335Lys Arg
Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly 340 345
350Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala
355 360 365Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe
Gly Gly 370 375 380Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala
Ala Pro Ser Val385 390 395 400Thr Leu Phe Pro Pro Ser Ser Glu Glu
Leu Gln Ala Asn Lys Ala Thr 405 410 415Leu Val Cys Leu Ile Ser Asp
Phe Tyr Pro Gly Ala Val Thr Val Ala 420 425 430Trp Lys Ala Asp Ser
Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr 435 440 445Pro Ser Lys
Gln Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser 450 455 460Leu
Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val465 470
475 480Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu
Cys 485 490 495Ser75497PRTArtificial SequenceSynthetic Construct
75Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1
5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu
Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser
Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser
Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr
Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155
160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala
165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe
Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His
Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 275 280
285Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
290 295 300Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn
Trp Val305 310 315 320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu
Ile Gly Gly Thr Asn 325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg
Phe Ser Gly Ser Leu Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala Gln Pro Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys
Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val385 390 395
400Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr
405 410 415Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala Val Thr
Val Ala 420 425 430Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val
Glu Thr Thr Thr 435 440 445Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
Ala Ser Ser Tyr Leu Ser 450 455 460Leu Thr Pro Glu Gln Trp Lys Ser
His Arg Ser Tyr Ser Cys Gln Val465 470 475 480Thr His Glu Gly Ser
Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys 485 490
495Ser76674PRTArtificial SequenceSynthetic Construct 76Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Thr
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe
50 55 60Arg Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
Asp Gly 210 215 220Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val
Val Thr Gln Glu225 230 235 240Pro Ser Leu Thr Val Ser Pro Gly Gly
Thr Val Thr Leu Thr Cys Gly 245 250 255Ser Ser Thr Gly Ala Val Thr
Thr Ser Asn Tyr Ala Asn Trp Val Gln 260 265 270Glu Lys Pro Gly Gln
Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys 275 280 285Arg Ala Pro
Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly 290 295 300Lys
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu305 310
315 320Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly
Gly 325 330 335Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 340 345 350Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 355 360 365Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser 370 375 380Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val385 390 395 400Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 405 410 415Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 420 425
430Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
435 440 445Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
Gly Gly 450 455 460Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile465 470 475 480Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 485 490 495Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 500 505 510Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 515 520 525Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu545 550
555 560Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 565 570 575Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu 580 585 590Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp 595 600 605Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val 610 615 620Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp625 630 635 640Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 660 665
670Gly Lys77449PRTArtificial SequenceSynthetic Construct 77Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25
30Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys
Phe 50 55 60Arg Gly Lys Ala Thr Met Thr Val Asp Thr Ser Thr Ser Thr
Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Glu
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Cys Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Ser 355 360 365Cys Ala Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp
Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410
415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly 435 440 445Lys78213PRTArtificial SequenceSynthetic
Construct 78Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser Gly Lys Ala Pro Lys
Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Trp Ser Lys His Pro Leu Thr 85 90 95Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110Ser Val Phe Ile Phe
Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr 115 120 125Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu145 150
155 160Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser 165 170 175Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala 180 185 190Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe 195 200 205Asn Arg Gly Glu Cys
21079514PRTArtificial SequenceSynthetic Construct 79Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val
Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly
Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65
70 75 80Lys Leu Asn Ser
Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile
Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly
Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120
125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
130 135 140Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly
Glu Thr145 150 155 160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile
Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser
Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp
Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr
Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg
Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235
240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Val His
245 250 255Met Pro Leu Gly Phe Leu Gly Pro Arg Gln Ala Arg Val Val
Asn Gly 260 265 270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Leu Glu 275 280 285Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser Gly Phe Thr Phe Ser
Thr Tyr Ala Met Asn Trp Val Arg305 310 315 320Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys 325 330 335Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe 340 345 350Thr
Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 355 360
365Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly
370 375 380Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly
Gln Gly385 390 395 400Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala
Ala Pro Ser Val Phe 405 410 415Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly Thr Ala Ser Val 420 425 430Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp 435 440 445Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 450 455 460Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr465 470 475
480Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val
485 490 495Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
Arg Gly 500 505 510Glu Cys80514PRTArtificial SequenceSynthetic
Construct 80Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro
Ser Gln1 5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu
Thr Ser Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys
Leu Glu Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr
His Ser Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser
Lys Ser Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp
Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val
Asp Asp Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln
Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150
155 160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu
Ala 165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu
Val Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg
Phe Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile
Asn Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln
His Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr
Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265
270Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
275 280 285Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu
Ser Cys 290 295 300Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met
Asn Trp Val Arg305 310 315 320Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val Ser Arg Ile Arg Ser Lys 325 330 335Tyr Asn Asn Tyr Ala Thr Tyr
Tyr Ala Asp Ser Val Lys Gly Arg Phe 340 345 350Thr Ile Ser Arg Asp
Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn 355 360 365Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Gly 370 375 380Asn
Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp Gly Gln Gly385 390
395 400Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val
Phe 405 410 415Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
Ala Ser Val 420 425 430Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
Ala Lys Val Gln Trp 435 440 445Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr 450 455 460Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr465 470 475 480Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 485 490 495Thr His
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 500 505
510Glu Cys81232PRTArtificial SequenceSynthetic Construct 81Glu Val
Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Asn Thr65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Val 115 120 125Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg145 150 155 160Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170
175Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
180 185 190Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys 195 200 205Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr 210 215 220Lys Ser Phe Asn Arg Gly Glu Cys225
23082956PRTArtificial SequenceSynthetic Construct 82Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val
Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly
Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65
70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys
Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala
Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200
205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp
210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr
Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly
Gly Gly Ser Val His 245 250 255Met Pro Leu Gly Phe Leu Gly Pro Arg
Gln Ala Arg Val Val Asn Gly 260 265 270Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Ala Val Val Thr Gln 275 280 285Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys 290 295 300Gly Ser Ser
Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val305 310 315
320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn
325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro
Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys Leu Thr Val Leu Ser
Ser Ala Ser Thr Lys Gly Pro Ser385 390 395 400Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 405 410 415Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 420 425 430Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 435 440
445Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
450 455 460Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His465 470 475 480Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys 485 490 495Asp Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln Val Gln Leu Val 500 505 510Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala Ser Val Lys Val Ser 515 520 525Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 530 535 540Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Thr Pro545 550 555
560Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr
565 570 575Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
Ser Ser 580 585 590Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Gly Gly Tyr 595 600 605Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val 610 615 620Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser625 630 635 640Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu 645 650 655Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 660 665 670Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 675 680
685Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
690 695 700Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val705 710 715 720Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro 725 730 735Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe 740 745 750Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 755 760 765Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 770 775 780Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro785 790 795
800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
805 810 815Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val 820 825 830Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala 835 840 845Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Cys Arg 850 855 860Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Trp Cys Leu Val Lys Gly865 870 875 880Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 885 890 895Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 900 905 910Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 915 920
925Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
930 935 940Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys945 950
95583956PRTArtificial SequenceSynthetic Construct 83Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val
Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly
Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65
70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys
Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala
Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200
205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp
210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr
Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly
Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Gln Ala Val Val Thr Gln 275 280 285Glu Pro Ser Leu Thr
Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys 290 295 300Gly Ser Ser
Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val305 310 315
320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn
325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu
Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro
Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys Leu Thr Val Leu Ser
Ser Ala Ser Thr Lys Gly Pro Ser385 390 395 400Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 405 410 415Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 420 425 430Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 435 440
445Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
450 455 460Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His465 470 475 480Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys 485 490 495Asp Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gln Val Gln Leu Val 500 505 510Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala Ser Val Lys Val Ser 515 520 525Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 530 535 540Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met Gly Leu Ile Thr Pro545 550 555
560Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr
565 570 575Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu
Ser Ser 580 585 590Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Gly Gly Tyr 595 600 605Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val 610 615 620Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Ser625 630 635 640Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu 645 650 655Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 660 665 670Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 675 680
685Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
690 695 700Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val705 710 715 720Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro 725 730 735Pro Cys Pro Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe 740 745 750Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val 755 760 765Thr Cys Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 770 775 780Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro785 790 795
800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
805 810 815Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val 820 825 830Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala 835 840 845Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Cys Arg 850 855 860Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Trp Cys Leu Val Lys Gly865 870 875 880Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 885 890 895Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 900 905 910Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 915 920
925Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
930 935 940Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys945 950
95584674PRTArtificial SequenceSynthetic Construct 84Gln Ala Val Val
Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val Thr
Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn Tyr
Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40 45Leu
Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe 50 55
60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala65
70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser
Ser Ala 100 105 110Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser 115 120 125Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe 130 135 140Pro Glu Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly145 150 155 160Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170 175Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 180 185 190Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 195 200
205Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly
210 215 220Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly225 230 235 240Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly
Tyr Ser Phe Thr Gly 245 250 255Tyr Thr Met Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp 260 265 270Met Gly Leu Ile Thr Pro Tyr
Asn Gly Ala Ser Ser Tyr Asn Gln Lys 275 280 285Phe Arg Gly Lys Ala
Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val 290 295 300Tyr Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr305 310 315
320Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln
325 330 335Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 340 345 350Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 355 360 365Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser 370 375 380Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val385 390 395 400Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 405 410 415Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 420 425 430Pro
Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp 435 440
445Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
450 455 460Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile465 470 475 480Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His Glu 485 490 495Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His 500 505 510Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 515 520 525Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 530 535 540Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu545 550 555
560Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
565 570 575Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 580 585 590Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 595 600 605Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val 610 615 620Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp625 630 635 640Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 645 650 655Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 660 665 670Gly
Lys85971PRTArtificial SequenceSynthetic Construct 85Gln Val Gln Leu
Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser
Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val
Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40 45Gly
Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile 50 55
60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65
70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr Tyr Cys
Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile Thr Cys
Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp Tyr Gln
Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185 190Ala
Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly 195 200
205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser Glu Asp
210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr Pro Tyr
Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly
Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser Gly Gly Gly Gly Ser Phe
Val Gly Gly Thr Gly Gly Gly 260 265 270Gly Ser Gly Gly Gly Gly Ser
Gly Gly Ser Glu Val Gln Leu Leu Glu 275 280 285Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 290 295 300Ala Ala Ser
Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn Trp Val Arg305 310 315
320Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys
325 330 335Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly
Arg Phe 340 345 350Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr
Leu Gln Met Asn 355 360 365Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Val Arg His Gly 370 375 380Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr Trp Gly Gln Gly385 390 395 400Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 405 410 415Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 420 425 430Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 435 440
445Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
450 455 460Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser465 470 475 480Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 485 490 495Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Gly 500 505 510Gly Gly Gly Ser Gly Gly Gly
Gly Ser Glu Val Gln Leu Val Glu Ser 515 520 525Gly Gly Gly Leu Val
Lys Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 530 535 540Ala Ser Gly
Phe Thr Phe Ser Asn Ala Trp Met Ser Trp Val Arg Gln545 550 555
560Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile Lys Ser Lys Thr
565 570 575Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro Val Lys Gly Arg
Phe Thr 580 585 590Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu
Gln Met Asn Ser 595 600 605Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
Cys Thr Thr Pro Trp Glu 610 615 620Trp Ser Trp Tyr Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser625 630 635 640Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 645 650 655Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 660 665 670Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 675 680
685Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
690 695 700Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln705 710 715 720Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp 725 730 735Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro 740 745 750Cys Pro Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro 755 760 765Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 770 775 780Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn785 790 795
800Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
805 810 815Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val 820 825 830Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 835 840 845Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 850 855 860Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Cys Arg Asp865 870 875 880Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe 885 890 895Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 900 905 910Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 915 920
925Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
930 935 940Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr945 950 955 960Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
965 9708633PRTArtificial SequenceSynthetic Construct 86Gly Gly Gly
Gly Ser Val His Met Pro Leu Gly Phe Leu Gly Pro Arg1 5 10 15Gln Ala
Arg Val Val Asn Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25
30Ser8735PRTArtificial SequenceSynthetic Construct 87Gly Gly Gly
Gly Ser Val His Met Pro Leu Gly Phe Leu Gly Pro Arg1 5 10 15Gln Ala
Arg Val Val Asn Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25 30Ser
Gly Gly 358833PRTArtificial SequenceSynthetic Construct 88Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe1 5 10 15Val
Gly Gly Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser8933PRTArtificial SequenceSynthetic Construct 89Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Lys Lys Ala Ala Pro Val1 5 10 15Asn Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 20 25
30Ser9033PRTArtificial SequenceSynthetic Construct 90Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Pro Met Ala Lys Lys Val1 5
10 15Asn Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly 20 25 30Ser9133PRTArtificial SequenceSynthetic Construct 91Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Arg Ala Lys Val1 5 10
15Asn Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
20 25 30Ser9233PRTArtificial SequenceSynthetic Construct 92Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Val His Met Pro Leu Gly1 5 10 15Phe
Leu Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser9333PRTArtificial SequenceSynthetic Construct 93Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gln Ala Arg Ala Lys Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser9433PRTArtificial SequenceSynthetic Construct 94Gly Gly Gly
Gly Ser Val His Met Pro Leu Gly Phe Leu Gly Pro Pro1 5 10 15Met Ala
Lys Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser9533PRTArtificial SequenceSynthetic Construct 95Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Lys Lys Ala Ala Pro Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser9633PRTArtificial SequenceSynthetic Construct 96Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Pro Met Ala Lys Lys Gly1 5 10 15Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25
30Ser9718PRTArtificial SequenceSynthetic Construct 97Val His Met
Pro Leu Gly Phe Leu Gly Pro Arg Gln Ala Arg Val Val1 5 10 15Asn
Gly986PRTArtificial SequenceSynthetic Construct 98Phe Val Gly Gly
Thr Gly1 5998PRTArtificial SequenceSynthetic Construct 99Lys Lys
Ala Ala Pro Val Asn Gly1 51008PRTArtificial SequenceSynthetic
Construct 100Pro Met Ala Lys Lys Val Asn Gly1 51018PRTArtificial
SequenceSynthetic Construct 101Gln Ala Arg Ala Lys Val Asn Gly1
510210PRTArtificial SequenceSynthetic Construct 102Val His Met Pro
Leu Gly Phe Leu Gly Pro1 5 101035PRTArtificial SequenceSynthetic
Construct 103Gln Ala Arg Ala Lys1 510415PRTArtificial
SequenceSynthetic Construct 104Val His Met Pro Leu Gly Phe Leu Gly
Pro Pro Met Ala Lys Lys1 5 10 151055PRTArtificial SequenceSynthetic
Construct 105Lys Lys Ala Ala Pro1 51065PRTArtificial
SequenceSynthetic Construct 106Pro Met Ala Lys Lys1
51075PRTArtificial SequenceSynthetic Construct 107Gly Tyr Thr Met
Asn1 510817PRTArtificial SequenceSynthetic Construct 108Leu Ile Thr
Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10
15Gly10910PRTArtificial SequenceSynthetic Construct 109Gly Gly Tyr
Asp Gly Arg Gly Phe Asp Tyr1 5 1011010PRTArtificial
SequenceSynthetic Construct 110Ser Ala Ser Ser Ser Val Ser Tyr Met
His1 5 101117PRTArtificial SequenceSynthetic Construct 111Asp Thr
Ser Lys Leu Ala Ser1 51129PRTArtificial SequenceSynthetic Construct
112Gln Gln Trp Ser Lys His Pro Leu Thr1 5113119PRTArtificial
SequenceSynthetic Construct 113Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30Thr Met Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Leu Ile Thr Pro Tyr
Asn Gly Ala Ser Ser Tyr Asn Gln Lys Phe 50 55 60Arg Gly Lys Ala Thr
Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly Gln Gly 100 105
110Thr Leu Val Thr Val Ser Ser 115114106PRTArtificial
SequenceSynthetic Construct 114Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Ser
Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr 85 90 95Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105115120PRTArtificial
SequenceSynthetic Construct 115Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Lys Ile His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Phe Asn Pro Asn
Ser Gly Tyr Ser Thr Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr
Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Leu Ser Pro Gly Gly Tyr Tyr Val Met Asp Ala Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser 115 120116106PRTArtificial
SequenceSynthetic Construct 116Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45Tyr Asn Thr Asn Asn Leu
Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Phe Pro Thr 85 90 95Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105117648DNAArtificial
SequenceSynthetic Construct 117caggccgtcg tgacccagga acccagcctg
acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc
accagcaact acgccaactg ggtgcaggaa 120aagcccggcc aggccttcag
aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat
tcagcggatc tctgctggga ggaaaggccg ccctgacact gtctggcgcc
240cagccagaag atgaggccga gtactactgc gccctgtggt acagcaacct
gtgggtgttc 300ggcggaggca ccaagctgac agtcctaggt caacccaagg
ctgcccccag cgtgaccctg 360ttccccccca gcagcgagga actgcaggcc
aacaaggcca ccctggtctg cctgatcagc 420gacttctacc caggcgccgt
gaccgtggcc tggaaggccg acagcagccc cgtgaaggcc 480ggcgtggaga
ccaccacccc cagcaagcag agcaacaaca agtacgccgc cagcagctac
540ctgagcctga cccccgagca gtggaagagc cacaggtcct acagctgcca
ggtgacccac 600gagggcagca ccgtggagaa aaccgtggcc cccaccgagt gcagctga
6481182916DNAArtificial SequenceSynthetic Construct 118caagtgcagc
tgaaagagtc cggccctgga ctggtggccc ctagccagag cctgagcatc 60acctgtaccg
tgtccggctt cagcctgacc agctacggcg tgtcatgggt gcgccagcct
120ccaggcaagt gtctggaatg gctgggcatc atctggggcg acggcagcac
caattaccac 180agcgccctga tcagcagact gagcatctcc aaggacaaca
gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac cgacgacacc
gccacctact actgcgccaa gggcatcacc 300accgtggtgg acgactacta
cgctatggac tactggggcc agggcaccag cgtgacagtg 360tctagcggag
gcggaggatc tggcggcgga ggaagtggcg gagggggatc tgggggaggc
420ggaagcgata tccagatgac ccagagccct gccagcctgt ctgcctctgt
gggcgagaca 480gtgaccatca catgccgggc cagcgagaac atcgacagct
acctggcctg gtatcagcag 540aagcagggca agagccccca gctgctggtg
tacgccgcca cctttctggc cgacgatgtg 600cccagcagat tcagcggcag
cggaagcggc acacagtaca gcctgaagat caactccctg 660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaagga ggcggcggaa gtgtgcacat
gcccctgggc 780ttcctgggcc ccagacaggc cagagtcgtg aacggggggg
gcggaggcag tgggggggga 840ggatccgagg tgcagctgct ggaatctggc
ggcggactgg tgcagcctgg cggatctctg 900agactgagct gtgccgccag
cggcttcacc ttcagcacct acgccatgaa ctgggtgcgc 960caggcccctg
gcaaaggcct ggaatgggtg tcccggatca gaagcaagta caacaactac
1020gccacctact acgccgacag cgtgaagggc cggttcacca tcagccggga
cgacagcaag 1080aacaccctgt acctgcagat gaacagcctg cgggccgagg
acaccgccgt gtactattgt 1140gtgcggcacg gcaacttcgg caacagctat
gtgtcttggt ttgcctactg gggccagggc 1200accctcgtga ccgtgtcaag
cgctagcaca aagggcccta gcgtgttccc tctggccccc 1260agcagcaaga
gcacaagcgg cggaacagcc gccctgggct gcctcgtgaa ggactacttc
1320cccgagcccg tgacagtgtc ttggaacagc ggagccctga caagcggcgt
gcacaccttc 1380cctgccgtgc tgcagagcag cggcctgtac tccctgagca
gcgtggtcac cgtgcctagc 1440agcagcctgg gcacccagac ctacatctgc
aacgtgaacc acaagcccag caacaccaaa 1500gtggacaaga aggtggagcc
caagagctgt gatggcggag gagggtccgg gggcggagga 1560tccgaggtgc
aattggttga atctggtggt ggtctggtaa aaccgggcgg ttccctgcgt
1620ctgagctgcg cggcttccgg gttcaccttc tccaacgcgt ggatgagctg
ggttcgccag 1680gccccgggca aaggcctcga gtgggttggt cgtatcaagt
ctaaaactga cggtggcacc 1740acggattacg cggctccagt taaaggtcgt
tttaccattt cccgcgacga tagcaaaaac 1800actctgtatc tgcagatgaa
ctctctgaaa actgaagaca ccgcagtcta ctactgtact 1860accccgtggg
aatggtcttg gtacgattat tggggccagg gcacgctggt tacggtgtct
1920agcgctagta ccaagggccc cagcgtgttc cccctggcac ccagcagcaa
gagcacatct 1980ggcggaacag ccgctctggg ctgtctggtg aaagactact
tccccgagcc cgtgaccgtg 2040tcttggaact ctggcgccct gaccagcggc
gtgcacacct ttccagccgt gctgcagagc 2100agcggcctgt actccctgtc
ctccgtggtc accgtgccct ctagctccct gggaacacag 2160acatatatct
gtaatgtcaa tcacaagcct tccaacacca aagtcgataa gaaagtcgag
2220cccaagagct gcgacaaaac tcacacatgc ccaccgtgcc cagcacctga
agctgcaggg 2280ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca
ccctcatgat ctcccggacc 2340cctgaggtca catgcgtggt ggtggacgtg
agccacgaag accctgaggt caagttcaac 2400tggtacgtgg acggcgtgga
ggtgcataat gccaagacaa agccgcggga ggagcagtac 2460aacagcacgt
accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc
2520aaggagtaca agtgcaaggt ctccaacaaa gccctcggcg cccccatcga
gaaaaccatc 2580tccaaagcca aagggcagcc ccgagaacca caggtgtaca
ccctgccccc atgccgggat 2640gagctgacca agaaccaggt cagcctgtgg
tgcctggtca aaggcttcta tcccagcgac 2700atcgccgtgg agtgggagag
caatgggcag ccggagaaca actacaagac cacgcctccc 2760gtgctggact
ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg
2820tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca
caaccactac 2880acgcagaaga gcctctccct gtctccgggt aaatga
29161192070DNAArtificial SequenceSynthetic Construct 119gaggtgcaat
tggttgaatc tggtggtggt ctggtaaaac cgggcggttc cctgcgtctg 60agctgcgcgg
cttccggatt caccttctcc aacgcgtgga tgagctgggt tcgccaggcc
120ccgggcaaag gcctcgagtg ggttggtcgt atcaagtcta aaactgacgg
tggcaccacg 180gattacgcgg ctccagttaa aggtcgtttt accatttccc
gcgacgatag caaaaacact 240ctgtatctgc agatgaactc tctgaaaact
gaagacaccg cagtctacta ctgtactacc 300ccgtgggaat ggtcttggta
cgattattgg ggccagggca cgctggttac ggtgtcttcc 360gctagcacaa
agggccctag cgtgttccct ctggccccca gcagcaagag cacaagcggc
420ggaacagccg ccctgggctg cctcgtgaag gactacttcc ccgagcccgt
gacagtgtct 480tggaacagcg gagccctgac aagcggcgtg cacactttcc
ctgccgtgct gcagagcagc 540ggcctgtact ccctgagcag cgtggtcacc
gtgcctagca gcagcctggg cacccagacc 600tacatctgca acgtgaacca
caagcccagc aacaccaaag tggacaagaa ggtggagccc 660aagagctgtg
atggcggagg agggtccgga ggcggaggat ccgaggtgca gctgctggaa
720tctggcggcg gactggtgca gcctggcgga tctctgagac tgagctgtgc
cgccagcggc 780ttcaccttca gcacctacgc catgaactgg gtgcgccagg
cccctggcaa aggcctggaa 840tgggtgtccc ggatcagaag caagtacaac
aactacgcca cctactacgc cgacagcgtg 900aagggccggt tcaccatcag
ccgggacgac agcaagaaca ccctgtacct gcagatgaac 960agcctgcggg
ccgaggacac cgccgtgtac tattgtgtgc ggcacggcaa cttcggcaac
1020agctatgtgt cttggtttgc ctactggggc cagggcaccc tcgtgaccgt
gtcaagcgct 1080agtaccaagg gccccagcgt gttccccctg gcacccagca
gcaagagcac atctggcgga 1140acagccgctc tgggctgtct ggtgaaagac
tacttccccg agcccgtgac cgtgtcttgg 1200aactctggcg ccctgaccag
cggcgtgcac acctttccag ccgtgctgca gagcagcggc 1260ctgtactccc
tgtcctccgt ggtcaccgtg ccctctagct ccctgggaac acagacatat
1320atctgtaatg tcaatcacaa gccttccaac accaaagtcg ataagaaagt
cgagcccaag 1380agctgcgaca aaactcacac atgcccaccg tgcccagcac
ctgaagctgc agggggaccg 1440tcagtcttcc tcttcccccc aaaacccaag
gacaccctca tgatctcccg gacccctgag 1500gtcacatgcg tggtggtgga
cgtgagccac gaagaccctg aggtcaagtt caactggtac 1560gtggacggcg
tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
1620acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 1680tacaagtgca aggtctccaa caaagccctc ggcgccccca
tcgagaaaac catctccaaa 1740gccaaagggc agccccgaga accacaggtg
tacaccctgc ccccatgccg ggatgagctg 1800accaagaacc aggtcagcct
gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1860gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1920gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1980caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacgcag 2040aagagcctct ccctgtctcc gggtaaatga
20701201494DNAArtificial SequenceSynthetic Construct 120caagtgcagc
tgaaagagtc cggccctgga ctggtggccc ctagccagag cctgagcatc 60acctgtaccg
tgtccggctt cagcctgacc agctacggcg tgtcatgggt gcgccagcct
120ccaggcaagt gtctggaatg gctgggcatc atctggggcg acggcagcac
caattaccac 180agcgccctga tcagcagact gagcatctcc aaggacaaca
gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac cgacgacacc
gccacctact actgcgccaa gggcatcacc 300accgtggtgg acgactacta
cgctatggac tactggggcc agggcaccag cgtgacagtg 360tctagcggag
gcggaggatc tggcggcgga ggaagtggcg gagggggatc tgggggaggc
420ggaagcgata tccagatgac ccagagccct gccagcctgt ctgcctctgt
gggcgagaca 480gtgaccatca catgccgggc cagcgagaac atcgacagct
acctggcctg gtatcagcag 540aagcagggca agagccccca gctgctggtg
tacgccgcca cctttctggc cgacgatgtg 600cccagcagat tcagcggcag
cggaagcggc acacagtaca gcctgaagat caactccctg 660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaaggc gggggaggct ccggaggcgg
cggaagtaga 780caggccagag tcgtgaacgg gggagggggg ggaagtgggg
gcggaggcag tgggggcgga 840ggatcccagg ccgtcgtgac ccaggaaccc
agcctgacag tgtctcctgg cggcaccgtg 900accctgacat gtggcagttc
tacaggcgcc gtgaccacca gcaactacgc caactgggtg 960caggaaaagc
ccggccaggc cttcagagga ctgatcggcg gcaccaacaa gagagcccct
1020ggcacccctg ccagattcag cggatctctg ctgggaggaa aggccgccct
gacactgtct 1080ggcgcccagc cagaagatga ggccgagtac tactgcgccc
tgtggtacag caacctgtgg 1140gtgttcggcg gaggcaccaa gctgacagtc
ctaggtcaac ccaaggctgc ccccagcgtg 1200accctgttcc cccccagcag
cgaggaactg caggccaaca aggccaccct ggtctgcctg 1260atcagcgact
tctacccagg cgccgtgacc gtggcctgga aggccgacag cagccccgtg
1320aaggccggcg tggagaccac cacccccagc aagcagagca acaacaagta
cgccgccagc 1380agctacctga gcctgacccc cgagcagtgg aagagccaca
ggtcctacag ctgccaggtg 1440acccacgagg gcagcaccgt ggagaaaacc
gtggccccca ccgagtgcag ctga 14941211494DNAArtificial
SequenceSynthetic Construct 121caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtgga 780ggcggcggaa
gtggcggagg cggagggggg ggaagtgggg gcggaggcag tgggggggga
840ggatcccagg ccgtcgtgac ccaggaaccc agcctgacag tgtctcctgg
cggcaccgtg 900accctgacat gtggcagttc tacaggcgcc gtgaccacca
gcaactacgc caactgggtg 960caggaaaagc ccggccaggc cttcagagga
ctgatcggcg gcaccaacaa gagagcccct 1020ggcacccctg ccagattcag
cggatctctg ctgggaggaa aggccgccct gacactgtct 1080ggcgcccagc
cagaagatga ggccgagtac tactgcgccc tgtggtacag caacctgtgg
1140gtgttcggcg gaggcaccaa gctgacagtc ctaggtcaac ccaaggctgc
ccccagcgtg 1200accctgttcc cccccagcag cgaggaactg caggccaaca
aggccaccct ggtctgcctg 1260atcagcgact tctacccagg cgccgtgacc
gtggcctgga aggccgacag cagccccgtg 1320aaggccggcg tggagaccac
cacccccagc aagcagagca acaacaagta cgccgccagc 1380agctacctga
gcctgacccc cgagcagtgg aagagccaca ggtcctacag ctgccaggtg
1440acccacgagg gcagcaccgt ggagaaaacc gtggccccca ccgagtgcag ctga
14941222025DNAArtificial SequenceSynthetic Construct
122caggtgcagc
tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 60tcctgcaagg
ccagcggcta cagcttcacc ggctacacca tgaactgggt gcgccaggct
120cctggacagg gcctggaatg gatgggcctg atcaccccct acaacggcgc
cagcagctac 180aaccagaagt tccggggcaa ggccaccatg accgtggaca
ccagcacctc caccgtgtat 240atggaactga gcagcctgcg gagcgaggac
accgccgtgt actattgtgc cagaggcggc 300tacgacggca gaggcttcga
ttattggggc cagggcaccc tcgtgaccgt gtcctctgct 360agcaccaagg
gcccctccgt gtttcctctg gccccttcca gcaagtccac ctctggcgga
420actgccgctc tgggctgcct ggtggaagat tacttccccg agcccgtgac
cgtgtcctgg 480aattctggcg ctctgacctc cggcgtgcac acctttccag
ctgtgctgca gtcctccggc 540ctgtactccc tgtcctccgt cgtgacagtg
ccctccagct ctctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gccctccaac accaaggtgg acgagaaggt ggaacccaag 660tcctgcgacg
gtggcggagg ttccggaggc ggaggatccc aggctgtcgt gacccaggaa
720ccctccctga cagtgtctcc tggcggcacc gtgaccctga cctgtggatc
ttctaccggc 780gctgtgacca cctccaacta cgccaattgg gtgcaggaaa
agcccggcca ggccttcaga 840ggactgatcg gcggcaccaa caagagagcc
cctggcaccc ctgccagatt ctccggttct 900ctgctgggcg gcaaggctgc
cctgactctg tctggtgctc agcctgagga cgaggccgag 960tactactgcg
ccctgtggta ctccaacctg tgggtgttcg gcggaggcac caagctgacc
1020gtgctgtcca gcgcttccac caagggaccc agtgtgttcc ccctggcccc
cagctccaag 1080tctacatccg gtggcacagc tgccctggga tgtctcgtga
aggactactt tcctgagcct 1140gtgacagtgt cttggaacag cggagccctg
accagcggag tgcacacatt ccctgcagtg 1200ctgcagagca gcggcctgta
tagcctgagc agcgtcgtga ccgtgccttc ctctagcctg 1260ggaacacaga
catatatctg taatgtgaat cataagccca gtaataccaa agtggataag
1320aaagtggaac ctaagagctg cgataagacc cacacctgtc ccccctgccc
tgctcctgaa 1380gctgctggtg gccctagcgt gttcctgttc cccccaaagc
ccaaggacac cctgatgatc 1440tcccggaccc ccgaagtgac ctgcgtggtg
gtggatgtgt cccacgagga ccctgaagtg 1500aagttcaatt ggtacgtgga
cggcgtggaa gtgcacaacg ccaagaccaa gcctagagag 1560gaacagtaca
actccaccta ccgggtggtg tccgtgctga cagtgctgca ccaggactgg
1620ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctgggcgc
tcccatcgaa 1680aagaccatct ccaaggccaa gggccagccc cgggaacccc
aggtgtacac cctgccccca 1740tgccgggatg agctgaccaa gaaccaggtc
agcctgtggt gcctggtcaa aggcttctat 1800cccagcgaca tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1860acgcctcccg
tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
1920aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac 1980aaccactaca cgcagaagag cctctccctg tctccgggta aatga
20251231350DNAArtificial SequenceSynthetic Construct 123caggtgcagc
tggtgcagtc tggcgccgaa gtgaagaaac caggcgccag cgtgaaggtg 60tcctgcaagg
ccagcggcta cagcttcacc ggctacacca tgaactgggt gcgccaggct
120cctggacagg gcctggaatg gatgggcctg atcaccccct acaacggcgc
cagcagctac 180aaccagaagt tccggggcaa ggccaccatg accgtggaca
ccagcacctc caccgtgtat 240atggaactga gcagcctgcg gagcgaggac
accgccgtgt actattgtgc cagaggcggc 300tacgacggca gaggcttcga
ttattggggc cagggcaccc tcgtgaccgt gtcctctgct 360agcaccaagg
gcccctccgt gttccccctg gcccccagca gcaagagcac cagcggcggc
420acagccgctc tgggctgcct ggtcgaggac tacttccccg agcccgtgac
cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg
ccgtgctgca gagttctggc 540ctgtatagcc tgagcagcgt ggtcaccgtg
ccttctagca gcctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gcccagcaac accaaggtgg acgagaaggt ggagcccaag 660agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg
720tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
aggtcaagtt caactggtac 840gtggacggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa
1020gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg
ggatgagctg 1080accaagaacc aggtcagcct ctcgtgcgca gtcaaaggct
tctatcccag cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 1200gactccgacg gctccttctt
cctcgtgagc aagctcaccg tggacaagag caggtggcag 1260caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320aagagcctct ccctgtctcc gggtaaatga 1350124642DNAArtificial
SequenceSynthetic Construct 124gacatccaga tgacccagag ccccagcagc
ctgtctgcca gcgtgggcga cagagtgacc 60atcacctgta gcgccagcag cagcgtgtcc
tacatgcact ggtatcagca gaagtccggc 120aaggccccca agctgctgat
ctacgacacc agcaagctgg cctccggcgt gcccagcaga 180ttttctggca
gcggctccgg caccgacttc accctgacaa tcagctccct ccagcccgag
240gacttcgcca cctactactg ccagcagtgg tccaagcacc ccctgacctt
tggccagggc 300accaagctgg aaatcaagcg tacggtggct gcaccatctg
tcttcatctt cccgccatct 360gatcggaagt tgaaatctgg aactgcctct
gttgtgtgcc tgctgaataa cttctatccc 420agagaggcca aagtacagtg
gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480agtgtcacag
agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg
540agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca
tcagggcctg 600agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ag
6421251545DNAArtificial SequenceSynthetic Construct 125caagtgcagc
tgaaagagtc cggccctgga ctggtggccc ctagccagag cctgagcatc 60acctgtaccg
tgtccggctt cagcctgacc agctacggcg tgtcatgggt gcgccagcct
120ccaggcaagt gtctggaatg gctgggcatc atctggggcg acggcagcac
caattaccac 180agcgccctga tcagcagact gagcatctcc aaggacaaca
gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac cgacgacacc
gccacctact actgcgccaa gggcatcacc 300accgtggtgg acgactacta
cgctatggac tactggggcc agggcaccag cgtgacagtg 360tctagcggag
gcggaggatc tggcggcgga ggaagtggcg gagggggatc tgggggaggc
420ggaagcgata tccagatgac ccagagccct gccagcctgt ctgcctctgt
gggcgagaca 480gtgaccatca catgccgggc cagcgagaac atcgacagct
acctggcctg gtatcagcag 540aagcagggca agagccccca gctgctggtg
tacgccgcca cctttctggc cgacgatgtg 600cccagcagat tcagcggcag
cggaagcggc acacagtaca gcctgaagat caactccctg 660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaagga ggcggcggaa gtgtgcacat
gcccctgggc 780ttcctgggcc ccagacaggc cagagtcgtg aacggggggg
gcggaggcag tgggggggga 840ggatccgagg tgcagctgct ggaatctggc
ggcggactgg tgcagcctgg cggatctctg 900agactgagct gtgccgccag
cggcttcacc ttcagcacct acgccatgaa ctgggtgcgc 960caggcccctg
gcaaaggcct ggaatgggtg tcccggatca gaagcaagta caacaactac
1020gccacctact acgccgacag cgtgaagggc cggttcacca tcagccggga
cgacagcaag 1080aacaccctgt acctgcagat gaacagcctg cgggccgagg
acaccgccgt gtactattgt 1140gtgcggcacg gcaacttcgg caacagctat
gtgtcttggt ttgcctactg gggccagggc 1200accctcgtga ccgtgtcaag
cgctagcgtg gccgctccct ccgtgttcat cttcccacct 1260tccgacgagc
agctgaagtc cggcaccgct tctgtcgtgt gcctgctgaa caacttctac
1320ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg
caacagccag 1380gaatccgtga ccgagcagga ctccaaggac agcacctact
ccctgtcctc caccctgacc 1440ctgtccaagg ccgactacga gaagcacaag
gtgtacgcct gcgaagtgac ccaccagggc 1500ctgtctagcc ccgtgaccaa
gtctttcaac cggggcgagt gctga 15451261545DNAArtificial
SequenceSynthetic Construct 126caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtgga 780ggcggcggaa
gtggcggagg cggagggggg ggaagtgggg gcggaggcag tgggggggga
840ggatccgagg tgcagctgct ggaatctggc ggcggactgg tgcagcctgg
cggatctctg 900agactgagct gtgccgccag cggcttcacc ttcagcacct
acgccatgaa ctgggtgcgc 960caggcccctg gcaaaggcct ggaatgggtg
tcccggatca gaagcaagta caacaactac 1020gccacctact acgccgacag
cgtgaagggc cggttcacca tcagccggga cgacagcaag 1080aacaccctgt
acctgcagat gaacagcctg cgggccgagg acaccgccgt gtactattgt
1140gtgcggcacg gcaacttcgg caacagctat gtgtcttggt ttgcctactg
gggccagggc 1200accctcgtga ccgtgtcaag cgctagcgtg gccgctccct
ccgtgttcat cttcccacct 1260tccgacgagc agctgaagtc cggcaccgct
tctgtcgtgt gcctgctgaa caacttctac 1320ccccgcgagg ccaaggtgca
gtggaaggtg gacaacgccc tgcagtccgg caacagccag 1380gaatccgtga
ccgagcagga ctccaaggac agcacctact ccctgtcctc caccctgacc
1440ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac
ccaccagggc 1500ctgtctagcc ccgtgaccaa gtctttcaac cggggcgagt gctga
1545127699DNAArtificial SequenceSynthetic Construct 127gaagtgcagc
tgctggaatc cggcggagga ctggtgcagc ctggcggatc tctgagactg 60tcttgtgccg
cctccggctt caccttctcc acctacgcca tgaactgggt gcgacaggct
120cctggcaagg gcctggaatg ggtgtcccgg atcagatcca agtacaacaa
ctacgccacc 180tactacgccg actccgtgaa gggccggttc accatctctc
gggacgactc caagaacacc 240ctgtacctgc agatgaactc cctgcgggcc
gaggacaccg ccgtgtacta ttgtgtgcgg 300cacggcaact tcggcaactc
ctatgtgtct tggtttgcct actggggcca gggcaccctc 360gtgaccgtgt
catctgctag cgtggccgct ccctccgtgt tcatcttccc accttccgac
420gagcagctga agtccggcac cgcttctgtc gtgtgcctgc tgaacaactt
ctacccccgc 480gaggccaagg tgcagtggaa ggtggacaac gccctgcagt
ccggcaacag ccaggaatcc 540gtgaccgagc aggactccaa ggacagcacc
tactccctgt cctccaccct gaccctgtcc 600aaggccgact acgagaagca
caaggtgtac gcctgcgaag tgacccacca gggcctgtct 660agccccgtga
ccaagtcttt caaccggggc gagtgctga 6991282871DNAArtificial
SequenceSynthetic Construct 128caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaagga ggcggcggaa gtgtgcacat gcccctgggc 780ttcctgggcc
ccagacaggc cagagtcgtg aacggggggg gcggaggcag tgggggggga
840ggatcccagg ccgtcgtgac ccaggaaccc agcctgacag tgtctcctgg
cggcaccgtg 900accctgacat gtggcagttc tacaggcgcc gtgaccacca
gcaactacgc caactgggtg 960caggaaaagc ccggccaggc cttcagagga
ctgatcggcg gcaccaacaa gagagcccct 1020ggcacccctg ccagattcag
cggatctctg ctgggaggaa aggccgccct gacactgtct 1080ggcgcccagc
cagaagatga ggccgagtac tactgcgccc tgtggtacag caacctgtgg
1140gtgttcggcg gaggcaccaa gctgacagtg ctgagcagcg cttccaccaa
gggacccagt 1200gtgttccccc tggcccccag ctccaagtct acatccggtg
gcacagctgc cctgggatgt 1260ctcgtgaagg actactttcc tgagcctgtg
acagtgtctt ggaacagcgg agccctgacc 1320agcggagtgc acacattccc
tgcagtgctg cagagcagcg gcctgtatag cctgagcagc 1380gtcgtgaccg
tgccttcctc tagcctggga acacagacat atatctgtaa tgtgaatcat
1440aagcccagta ataccaaagt ggataagaaa gtggaaccta agagctgcga
tggcggagga 1500gggtccggag gcggagggtc ccaggtgcag ctggtgcagt
ctggcgccga agtgaagaaa 1560ccaggcgcca gcgtgaaggt gtcctgcaag
gccagcggct acagcttcac cggctacacc 1620atgaactggg tgcgccaggc
tcctggacag ggcctggaat ggatgggcct gatcaccccc 1680tacaacggcg
ccagcagcta caaccagaag ttccggggca aggccaccat gaccgtggac
1740accagcacct ccaccgtgta tatggaactg agcagcctgc ggagcgagga
caccgccgtg 1800tactattgtg ccagaggcgg ctacgacggc agaggcttcg
attattgggg ccagggcacc 1860ctcgtgaccg tgtcctctgc tagcaccaag
ggcccctccg tgttccccct ggcccccagc 1920agcaagagca ccagcggcgg
cacagccgct ctgggctgcc tggtcgagga ctacttcccc 1980gagcccgtga
ccgtgtcctg gaacagcgga gccctgacct ccggcgtgca caccttcccc
2040gccgtgctgc agagttctgg cctgtatagc ctgagcagcg tggtcaccgt
gccttctagc 2100agcctgggca cccagaccta catctgcaac gtgaaccaca
agcccagcaa caccaaggtg 2160gacgagaagg tggagcccaa gagctgcgac
aaaactcaca catgcccacc gtgcccagca 2220cctgaagctg cagggggacc
gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 2280atgatctccc
ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct
2340gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagccg 2400cgggaggagc agtacaacag cacgtaccgt gtggtcagcg
tcctcaccgt cctgcaccag 2460gactggctga atggcaagga gtacaagtgc
aaggtctcca acaaagccct cggcgccccc 2520atcgagaaaa ccatctccaa
agccaaaggg cagccccgag aaccacaggt gtacaccctg 2580cccccatgcc
gggatgagct gaccaagaac caggtcagcc tgtggtgcct ggtcaaaggc
2640ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga
gaacaactac 2700aagaccacgc ctcccgtgct ggactccgac ggctccttct
tcctctacag caagctcacc 2760gtggacaaga gcaggtggca gcaggggaac
gtcttctcat gctccgtgat gcatgaggct 2820ctgcacaacc actacacgca
gaagagcctc tccctgtctc cgggtaaatg a 28711292871DNAArtificial
SequenceSynthetic Construct 129caagtgcagc tgaaagagtc cggccctgga
ctggtggccc ctagccagag cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc
agctacggcg tgtcatgggt gcgccagcct 120ccaggcaagt gtctggaatg
gctgggcatc atctggggcg acggcagcac caattaccac 180agcgccctga
tcagcagact gagcatctcc aaggacaaca gcaagagcca ggtgttcctg
240aagctgaaca gcctgcagac cgacgacacc gccacctact actgcgccaa
gggcatcacc 300accgtggtgg acgactacta cgctatggac tactggggcc
agggcaccag cgtgacagtg 360tctagcggag gcggaggatc tggcggcgga
ggaagtggcg gagggggatc tgggggaggc 420ggaagcgata tccagatgac
ccagagccct gccagcctgt ctgcctctgt gggcgagaca 480gtgaccatca
catgccgggc cagcgagaac atcgacagct acctggcctg gtatcagcag
540aagcagggca agagccccca gctgctggtg tacgccgcca cctttctggc
cgacgatgtg 600cccagcagat tcagcggcag cggaagcggc acacagtaca
gcctgaagat caactccctg 660cagagcgagg acgtggcccg gtactactgc
cagcactact acagcacccc ctacaccttc 720ggctgcggca ccaagctgga
aatcaaaggc gggggaggct ccggaggcgg cggaagtgga 780ggcggcggaa
gtggcggagg cggagggggg ggaagtgggg gcggaggcag tgggggggga
840ggatcccagg ccgtcgtgac ccaggaaccc agcctgacag tgtctcctgg
cggcaccgtg 900accctgacat gtggcagttc tacaggcgcc gtgaccacca
gcaactacgc caactgggtg 960caggaaaagc ccggccaggc cttcagagga
ctgatcggcg gcaccaacaa gagagcccct 1020ggcacccctg ccagattcag
cggatctctg ctgggaggaa aggccgccct gacactgtct 1080ggcgcccagc
cagaagatga ggccgagtac tactgcgccc tgtggtacag caacctgtgg
1140gtgttcggcg gaggcaccaa gctgacagtg ctgagcagcg cttccaccaa
gggacccagt 1200gtgttccccc tggcccccag ctccaagtct acatccggtg
gcacagctgc cctgggatgt 1260ctcgtgaagg actactttcc tgagcctgtg
acagtgtctt ggaacagcgg agccctgacc 1320agcggagtgc acacattccc
tgcagtgctg cagagcagcg gcctgtatag cctgagcagc 1380gtcgtgaccg
tgccttcctc tagcctggga acacagacat atatctgtaa tgtgaatcat
1440aagcccagta ataccaaagt ggataagaaa gtggaaccta agagctgcga
tggcggagga 1500gggtccggag gcggagggtc ccaggtgcag ctggtgcagt
ctggcgccga agtgaagaaa 1560ccaggcgcca gcgtgaaggt gtcctgcaag
gccagcggct acagcttcac cggctacacc 1620atgaactggg tgcgccaggc
tcctggacag ggcctggaat ggatgggcct gatcaccccc 1680tacaacggcg
ccagcagcta caaccagaag ttccggggca aggccaccat gaccgtggac
1740accagcacct ccaccgtgta tatggaactg agcagcctgc ggagcgagga
caccgccgtg 1800tactattgtg ccagaggcgg ctacgacggc agaggcttcg
attattgggg ccagggcacc 1860ctcgtgaccg tgtcctctgc tagcaccaag
ggcccctccg tgttccccct ggcccccagc 1920agcaagagca ccagcggcgg
cacagccgct ctgggctgcc tggtcgagga ctacttcccc 1980gagcccgtga
ccgtgtcctg gaacagcgga gccctgacct ccggcgtgca caccttcccc
2040gccgtgctgc agagttctgg cctgtatagc ctgagcagcg tggtcaccgt
gccttctagc 2100agcctgggca cccagaccta catctgcaac gtgaaccaca
agcccagcaa caccaaggtg 2160gacgagaagg tggagcccaa gagctgcgac
aaaactcaca catgcccacc gtgcccagca 2220cctgaagctg cagggggacc
gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 2280atgatctccc
ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct
2340gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa
gacaaagccg 2400cgggaggagc agtacaacag cacgtaccgt gtggtcagcg
tcctcaccgt cctgcaccag 2460gactggctga atggcaagga gtacaagtgc
aaggtctcca acaaagccct cggcgccccc 2520atcgagaaaa ccatctccaa
agccaaaggg cagccccgag aaccacaggt gtacaccctg 2580cccccatgcc
gggatgagct gaccaagaac caggtcagcc tgtggtgcct ggtcaaaggc
2640ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga
gaacaactac 2700aagaccacgc ctcccgtgct ggactccgac ggctccttct
tcctctacag caagctcacc 2760gtggacaaga gcaggtggca gcaggggaac
gtcttctcat gctccgtgat gcatgaggct 2820ctgcacaacc actacacgca
gaagagcctc tccctgtctc cgggtaaatg a 28711302025DNAArtificial
SequenceSynthetic Construct 130caggccgtcg tgacccagga acccagcctg
acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca gttctacagg cgccgtgacc
accagcaact acgccaactg ggtgcaggaa 120aagcccggcc aggccttcag
aggactgatc ggcggcacca acaagagagc ccctggcacc 180cctgccagat
tctccggttc tctgctgggc ggcaaggctg ccctgactct gtctggtgct
240cagcctgagg acgaggccga gtactactgc gccctgtggt actccaacct
gtgggtgttc 300ggcggaggca ccaagctgac cgtgctgtcc agcgcttcca
ccaagggacc cagtgtgttc 360cccctggccc ccagctccaa gtctacatcc
ggtggcacag ctgccctggg atgtctcgtg 420aaggactact ttcctgagcc
tgtgacagtg tcttggaaca gcggagccct gaccagcgga 480gtgcacacat
tccctgcagt gctgcagagc agcggcctgt atagcctgag cagcgtcgtg
540accgtgcctt cctctagcct gggaacacag acatatatct gtaatgtgaa
tcataagccc 600agtaatacca aagtggataa gaaagtggaa cctaagagct
gcgatggcgg aggagggtcc 660ggaggcggag ggtcccaggt gcagctggtg
cagtctggcg ccgaagtgaa gaaaccaggc 720gccagcgtga aggtgtcctg
caaggccagc ggctacagct tcaccggcta caccatgaac 780tgggtgcgcc
aggctcctgg acagggcctg gaatggatgg gcctgatcac cccctacaac
840ggcgccagca gctacaacca gaagttccgg ggcaaggcca ccatgaccgt
ggacaccagc 900acctccaccg
tgtatatgga actgagcagc ctgcggagcg aggacaccgc cgtgtactat
960tgtgccagag gcggctacga cggcagaggc ttcgattatt ggggccaggg
caccctcgtg 1020accgtgtcct ctgctagcac caagggcccc tccgtgttcc
ccctggcccc cagcagcaag 1080agcaccagcg gcggcacagc cgctctgggc
tgcctggtcg aggactactt ccccgagccc 1140gtgaccgtgt cctggaacag
cggagccctg acctccggcg tgcacacctt ccccgccgtg 1200ctgcagagtt
ctggcctgta tagcctgagc agcgtggtca ccgtgccttc tagcagcctg
1260ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa
ggtggacgag 1320aaggtggagc ccaagagctg cgacaaaact cacacatgcc
caccgtgccc agcacctgaa 1380gctgcagggg gaccgtcagt cttcctcttc
cccccaaaac ccaaggacac cctcatgatc 1440tcccggaccc ctgaggtcac
atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1500aagttcaact
ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag
1560gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg 1620ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag
ccctcggcgc ccccatcgag 1680aaaaccatct ccaaagccaa agggcagccc
cgagaaccac aggtgtacac cctgccccca 1740tgccgggatg agctgaccaa
gaaccaggtc agcctgtggt gcctggtcaa aggcttctat 1800cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1860acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct
caccgtggac 1920aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg
tgatgcatga ggctctgcac 1980aaccactaca cgcagaagag cctctccctg
tctccgggta aatga 20251312916DNAArtificial SequenceSynthetic
Construct 131caagtgcagc tgaaagagtc cggccctgga ctggtggccc ctagccagag
cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc agctacggcg tgtcatgggt
gcgccagcct 120ccaggcaagt gtctggaatg gctgggcatc atctggggcg
acggcagcac caattaccac 180agcgccctga tcagcagact gagcatctcc
aaggacaaca gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac
cgacgacacc gccacctact actgcgccaa gggcatcacc 300accgtggtgg
acgactacta cgctatggac tactggggcc agggcaccag cgtgacagtg
360tctagcggag gcggaggatc tggcggcgga ggaagtggcg gagggggatc
tgggggaggc 420ggaagcgata tccagatgac ccagagccct gccagcctgt
ctgcctctgt gggcgagaca 480gtgaccatca catgccgggc cagcgagaac
atcgacagct acctggcctg gtatcagcag 540aagcagggca agagccccca
gctgctggtg tacgccgcca cctttctggc cgacgatgtg 600cccagcagat
tcagcggcag cggaagcggc acacagtaca gcctgaagat caactccctg
660cagagcgagg acgtggcccg gtactactgc cagcactact acagcacccc
ctacaccttc 720ggctgcggca ccaagctgga aatcaaaggc gggggaggct
ccggaggcgg cggaagtgga 780ggcggcggaa gtttcgtggg ggggaccggg
ggcggaggca gtgggggggg aggatccggg 840ggatccgagg tgcagctgct
ggaatctggc ggcggactgg tgcagcctgg cggatctctg 900agactgagct
gtgccgccag cggcttcacc ttcagcacct acgccatgaa ctgggtgcgc
960caggcccctg gcaaaggcct ggaatgggtg tcccggatca gaagcaagta
caacaactac 1020gccacctact acgccgacag cgtgaagggc cggttcacca
tcagccggga cgacagcaag 1080aacaccctgt acctgcagat gaacagcctg
cgggccgagg acaccgccgt gtactattgt 1140gtgcggcacg gcaacttcgg
caacagctat gtgtcttggt ttgcctactg gggccagggc 1200accctcgtga
ccgtgtcaag cgctagcaca aagggcccta gcgtgttccc tctggccccc
1260agcagcaaga gcacaagcgg cggaacagcc gccctgggct gcctcgtgaa
ggactacttc 1320cccgagcccg tgacagtgtc ttggaacagc ggagccctga
caagcggcgt gcacaccttc 1380cctgccgtgc tgcagagcag cggcctgtac
tccctgagca gcgtggtcac cgtgcctagc 1440agcagcctgg gcacccagac
ctacatctgc aacgtgaacc acaagcccag caacaccaaa 1500gtggacaaga
aggtggagcc caagagctgt gatggcggag gagggtccgg gggcggagga
1560tccgaggtgc aattggttga atctggtggt ggtctggtaa aaccgggcgg
ttccctgcgt 1620ctgagctgcg cggcttccgg gttcaccttc tccaacgcgt
ggatgagctg ggttcgccag 1680gccccgggca aaggcctcga gtgggttggt
cgtatcaagt ctaaaactga cggtggcacc 1740acggattacg cggctccagt
taaaggtcgt tttaccattt cccgcgacga tagcaaaaac 1800actctgtatc
tgcagatgaa ctctctgaaa actgaagaca ccgcagtcta ctactgtact
1860accccgtggg aatggtcttg gtacgattat tggggccagg gcacgctggt
tacggtgtct 1920agcgctagta ccaagggccc cagcgtgttc cccctggcac
ccagcagcaa gagcacatct 1980ggcggaacag ccgctctggg ctgtctggtg
aaagactact tccccgagcc cgtgaccgtg 2040tcttggaact ctggcgccct
gaccagcggc gtgcacacct ttccagccgt gctgcagagc 2100agcggcctgt
actccctgtc ctccgtggtc accgtgccct ctagctccct gggaacacag
2160acatatatct gtaatgtcaa tcacaagcct tccaacacca aagtcgataa
gaaagtcgag 2220cccaagagct gcgacaaaac tcacacatgc ccaccgtgcc
cagcacctga agctgcaggg 2280ggaccgtcag tcttcctctt ccccccaaaa
cccaaggaca ccctcatgat ctcccggacc 2340cctgaggtca catgcgtggt
ggtggacgtg agccacgaag accctgaggt caagttcaac 2400tggtacgtgg
acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac
2460aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg
gctgaatggc 2520aaggagtaca agtgcaaggt ctccaacaaa gccctcggcg
cccccatcga gaaaaccatc 2580tccaaagcca aagggcagcc ccgagaacca
caggtgtaca ccctgccccc atgccgggat 2640gagctgacca agaaccaggt
cagcctgtgg tgcctggtca aaggcttcta tcccagcgac 2700atcgccgtgg
agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc
2760gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga
caagagcagg 2820tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg
aggctctgca caaccactac 2880acgcagaaga gcctctccct gtctccgggt aaatga
2916132449PRTArtificial SequenceSynthetic Construct 132Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30Thr
Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Val Asn Arg Arg Phe
50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Phe Phe Tyr Phe Asp Tyr
Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185
190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
195 200 205Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310
315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Cys Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Ser 355 360 365Cys Ala Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly
Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425
430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
435 440 445Lys133214PRTArtificial SequenceSynthetic Construct
133Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Ser Ala Ser Phe Arg Tyr Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
His Tyr Thr Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Arg Lys Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
210134675PRTArtificial SequenceSynthetic Construct 134Gln Ala Val
Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly1 5 10 15Thr Val
Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30Asn
Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly 35 40
45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly
Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp
Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val
Leu Ser Ser Ala 100 105 110Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser 115 120 125Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe 130 135 140Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly145 150 155 160Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 165 170 175Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 180 185
190Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
195 200 205Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly
Gly Gly 210 215 220Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly225 230 235 240Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp 245 250 255Thr Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp 260 265 270Val Ala Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser 275 280 285Val Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 290 295 300Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr305 310
315 320Cys Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp Tyr Trp
Gly 325 330 335Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 340 345 350Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 355 360 365Ala Leu Gly Cys Leu Val Glu Asp Tyr
Phe Pro Glu Pro Val Thr Val 370 375 380Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala385 390 395 400Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 405 410 415Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 420 425
430Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
435 440 445Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala
Ala Gly 450 455 460Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met465 470 475 480Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 485 490 495Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 500 505 510His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 515 520 525Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 530 535 540Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile545 550
555 560Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 565 570 575Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser 580 585 590Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 595 600 605Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro 610 615 620Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val625 630 635 640Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 645 650 655His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 660 665
670Pro Gly Lys 675135957PRTArtificial SequenceSynthetic Construct
135Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1
5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu
Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser
Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser
Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr
Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155
160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala
165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe
Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His
Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 275 280
285Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
290 295 300Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn
Trp Val305 310 315 320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu
Ile Gly Gly Thr Asn 325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg
Phe Ser Gly Ser Leu Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala Gln Pro Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys
Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser385 390 395
400Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
405 410 415Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 420 425 430Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 435 440 445Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 450 455 460Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His465 470
475 480Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys 485 490 495Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val
Gln Leu Val 500 505 510Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser 515 520 525Cys Ala Ala Ser Gly Phe Asn Ile Lys
Asp Thr Tyr Ile His Trp Val 530 535 540Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val Ala Arg Ile Tyr Pro545 550 555 560Thr Asn Gly Tyr
Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr 565 570 575Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 580 585
590Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
595 600 605Glu Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu
Val Thr 610 615 620Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro625 630 635 640Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val 645 650 655Glu Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp Asn Ser Gly Ala 660 665 670Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 675 680 685Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 690 695 700Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys705 710
715 720Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys 725 730 735Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
Val Phe Leu 740 745 750Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 755 760 765Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys 770 775 780Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys785 790 795 800Pro Arg Glu Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 805 810 815Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 820 825
830Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys
835 840 845Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Cys 850 855 860Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
Cys Leu Val Lys865 870 875 880Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 885 890 895Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 900 905 910Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 915 920 925Gln Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 930 935 940His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys945 950
955136957PRTArtificial SequenceSynthetic Construct 136Gln Val Gln
Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu
Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly
Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Trp Leu 35 40
45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Ile
50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr Ala Thr Tyr
Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp Tyr Tyr Ala
Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155 160Val Thr Ile
Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala 165 170 175Trp
Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Tyr Ala 180 185
190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe Ser Gly Ser Gly
195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser
Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His Tyr Tyr Ser Thr
Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys Leu Glu Ile Lys
Gly Gly Gly Gly Ser Val His 245 250 255Met Pro Leu Gly Phe Leu Gly
Pro Arg Gln Ala Arg Val Val Asn Gly 260 265 270Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 275 280 285Glu Pro Ser
Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys 290 295 300Gly
Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val305 310
315 320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr
Asn 325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser
Leu Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln
Pro Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser
Asn Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys Leu Thr Val Leu
Ser Ser Ala Ser Thr Lys Gly Pro Ser385 390 395 400Val Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 405 410 415Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 420 425
430Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
435 440 445Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 450 455 460Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His465 470 475 480Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys 485 490 495Asp Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Glu Val Gln Leu Val 500 505 510Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 515 520 525Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 530 535 540Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro545 550
555 560Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe
Thr 565 570 575Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
Met Asn Ser 580 585 590Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ser Arg Trp Gly Gly 595 600 605Glu Gly Phe Tyr Ala Met Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr 610 615 620Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro625 630 635 640Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 645 650 655Glu Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 660 665
670Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
675 680 685Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly 690 695 700Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys705 710 715 720Val Asp Glu Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys 725 730 735Pro Pro Cys Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser Val Phe Leu 740 745 750Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 755 760 765Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 770 775 780Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys785 790
795 800Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu 805 810 815Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys 820 825 830Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
Lys Thr Ile Ser Lys 835 840 845Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Cys 850 855 860Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu Trp Cys Leu Val Lys865 870 875 880Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 885 890 895Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 900 905
910Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
915 920 925Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn 930 935 940His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys945 950 955137449PRTArtificial SequenceSynthetic Construct
137Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr His Asp Thr Ser Thr
Ser Thr Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Phe Thr Gly Phe His
Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu
Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp145 150 155
160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val Asp Glu Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly
Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Cys Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu Ser 355 360 365Cys Ala Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro Gly 435 440 445Lys138216PRTArtificial SequenceSynthetic
Construct 138Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly
Ile Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Thr Asn Glu His 85 90 95Tyr Tyr Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys 115 120 125Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135
140Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
Asn145 150 155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys His Lys 180 185 190Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro Val Thr 195 200 205Lys Ser Phe Asn Arg Gly
Glu Cys 210 215139956PRTArtificial SequenceSynthetic Construct
139Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1
5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu
Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser
Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser
Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr
Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155
160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala
165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe
Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His
Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys
Leu Glu Ile Lys Gly Gly Gly Gly Ser Val His 245 250 255Met Pro Leu
Gly Phe Leu Gly Pro Arg Gln Ala Arg Val Val Asn Gly 260 265 270Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 275 280
285Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
290 295
300Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
Val305 310 315 320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile
Gly Gly Thr Asn 325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
Ser Gly Ser Leu Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu Ser
Gly Ala Gln Pro Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala Leu
Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys Leu
Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser385 390 395 400Val
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 405 410
415Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
420 425 430Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala 435 440 445Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val 450 455 460Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His465 470 475 480Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys 485 490 495Asp Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val 500 505 510Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser 515 520 525Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val 530 535
540Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile Asn
Pro545 550 555 560Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln
Gly Arg Val Thr 565 570 575Met Thr His Asp Thr Ser Thr Ser Thr Val
Tyr Met Glu Leu Ser Ser 580 585 590Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys Ala Arg Ser Phe Phe 595 600 605Thr Gly Phe His Leu Asp
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 610 615 620Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser625 630 635 640Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu 645 650
655Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
660 665 670Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu 675 680 685Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr 690 695 700Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val705 710 715 720Asp Glu Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro 725 730 735Pro Cys Pro Ala Pro
Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 740 745 750Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 755 760 765Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 770 775
780Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro785 790 795 800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 805 810 815Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val 820 825 830Ser Asn Lys Ala Leu Gly Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala 835 840 845Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg 850 855 860Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly865 870 875 880Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 885 890
895Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
900 905 910Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln 915 920 925Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His 930 935 940Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys945 950 955140956PRTArtificial SequenceSynthetic Construct
140Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1
5 10 15Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser
Tyr 20 25 30Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu
Trp Leu 35 40 45Gly Ile Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser
Ala Leu Ile 50 55 60Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser
Gln Val Phe Leu65 70 75 80Lys Leu Asn Ser Leu Gln Thr Asp Asp Thr
Ala Thr Tyr Tyr Cys Ala 85 90 95Lys Gly Ile Thr Thr Val Val Asp Asp
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140Gln Met Thr
Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Thr145 150 155
160Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Asp Ser Tyr Leu Ala
165 170 175Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val
Tyr Ala 180 185 190Ala Thr Phe Leu Ala Asp Asp Val Pro Ser Arg Phe
Ser Gly Ser Gly 195 200 205Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn
Ser Leu Gln Ser Glu Asp 210 215 220Val Ala Arg Tyr Tyr Cys Gln His
Tyr Tyr Ser Thr Pro Tyr Thr Phe225 230 235 240Gly Cys Gly Thr Lys
Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser 260 265 270Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln 275 280
285Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys
290 295 300Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn
Trp Val305 310 315 320Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu
Ile Gly Gly Thr Asn 325 330 335Lys Arg Ala Pro Gly Thr Pro Ala Arg
Phe Ser Gly Ser Leu Leu Gly 340 345 350Gly Lys Ala Ala Leu Thr Leu
Ser Gly Ala Gln Pro Glu Asp Glu Ala 355 360 365Glu Tyr Tyr Cys Ala
Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly 370 375 380Gly Thr Lys
Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser385 390 395
400Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
405 410 415Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val 420 425 430Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala 435 440 445Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val 450 455 460Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His465 470 475 480Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 485 490 495Asp Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val 500 505 510Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser 515 520
525Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val
530 535 540Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Ile Ile
Asn Pro545 550 555 560Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe
Gln Gly Arg Val Thr 565 570 575Met Thr His Asp Thr Ser Thr Ser Thr
Val Tyr Met Glu Leu Ser Ser 580 585 590Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Ser Phe Phe 595 600 605Thr Gly Phe His Leu
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 610 615 620Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser625 630 635
640Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu
645 650 655Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu 660 665 670Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu 675 680 685Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr 690 695 700Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val705 710 715 720Asp Glu Lys Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 725 730 735Pro Cys Pro
Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 740 745 750Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 755 760
765Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
770 775 780Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro785 790 795 800Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr 805 810 815Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val 820 825 830Ser Asn Lys Ala Leu Gly Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 835 840 845Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg 850 855 860Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly865 870 875
880Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
885 890 895Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser 900 905 910Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln 915 920 925Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu His Asn His 930 935 940Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys945 950 955141674PRTArtificial SequenceSynthetic
Construct 141Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala
Val Thr Thr Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly
Gln Ala Phe Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro
Gly Thr Pro Ala Arg Phe 50 55 60Ser Gly Ser Leu Leu Gly Gly Lys Ala
Ala Leu Thr Leu Ser Gly Ala65 70 75 80Gln Pro Glu Asp Glu Ala Glu
Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly
Gly Thr Lys Leu Thr Val Leu Ser Ser Ala 100 105 110Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 115 120 125Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 130 135
140Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly145 150 155 160Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu 165 170 175Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr Tyr 180 185 190Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys Lys 195 200 205Val Glu Pro Lys Ser Cys
Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly 210 215 220Ser Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly225 230 235 240Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser 245 250
255Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
260 265 270Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala
Gln Lys 275 280 285Phe Gln Gly Arg Val Thr Met Thr His Asp Thr Ser
Thr Ser Thr Val 290 295 300Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr305 310 315 320Cys Ala Arg Ser Phe Phe Thr
Gly Phe His Leu Asp Tyr Trp Gly Gln 325 330 335Gly Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 340 345 350Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 355 360 365Leu
Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 370 375
380Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val385 390 395 400Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro 405 410 415Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys 420 425 430Pro Ser Asn Thr Lys Val Asp Glu
Lys Val Glu Pro Lys Ser Cys Asp 435 440 445Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly 450 455 460Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile465 470 475 480Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 485 490
495Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
500 505 510Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 515 520 525Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys 530 535 540Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Gly Ala Pro Ile Glu545 550 555 560Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 565 570 575Thr Leu Pro Pro Cys
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 580 585 590Trp Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 595 600 605Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 610 615
620Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp625 630 635 640Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His 645 650 655Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro 660 665 670Gly Lys1425PRTArtificial
SequenceSynthetic Construct 142Asp Tyr Thr Met Asp1
514317PRTArtificial SequenceSynthetic Construct 143Asp Val Asn Pro
Asn Ser Gly Gly Ser Ile Val Asn Arg Arg Phe Lys1 5 10
15Gly14410PRTArtificial SequenceSynthetic Construct 144Asn Leu Gly
Pro Phe Phe Tyr Phe Asp Tyr1 5 101457PRTArtificial
SequenceSynthetic Construct 145Thr Ser Asn Tyr Ala Asn Trp1
514620PRTArtificial SequenceSynthetic Construct 146Gly Thr Asn Lys
Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser1 5 10 15Leu Leu Gly
Gly 201475PRTArtificial SequenceSynthetic Construct 147Thr Lys Leu
Thr Val1 514811PRTArtificial SequenceSynthetic Construct 148Lys Ala
Ser Gln Asp Val Ser Thr Ala Val Ala1 5 101497PRTArtificial
SequenceSynthetic Construct 149Ser Ala Ser Phe Arg Tyr Thr1
51509PRTArtificial SequenceSynthetic Construct 150Gln Gln His Tyr
Thr Thr Pro Pro Thr1 51515PRTArtificial SequenceSynthetic Construct
151Ser Tyr Tyr Met His1 515217PRTArtificial SequenceSynthetic
Construct 152Ile Ile Asn Pro Ser
Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln1 5 10
15Gly15310PRTArtificial SequenceSynthetic Construct 153Ser Phe Phe
Thr Gly Phe His Leu Asp Tyr1 5 1015412PRTArtificial
SequenceSynthetic Construct 154Arg Ala Ser Gln Ser Val Ser Ser Ser
Tyr Leu Ala1 5 101557PRTArtificial SequenceSynthetic Construct
155Gly Ala Ser Ser Arg Ala Thr1 515610PRTArtificial
SequenceSynthetic Construct 156Gln Gln Tyr Thr Asn Glu His Tyr Tyr
Thr1 5 10157119PRTArtificial SequenceSynthetic Construct 157Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys
Phe 50 55 60Gln Gly Arg Val Thr Met Thr His Asp Thr Ser Thr Ser Thr
Val Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Ser Phe Phe Thr Gly Phe His Leu Asp
Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115158109PRTArtificial SequenceSynthetic Construct 158Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40
45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu
Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Thr
Asn Glu His 85 90 95Tyr Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys 100 105159119PRTArtificial SequenceSynthetic Construct 159Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr
20 25 30Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Val Asn Arg
Arg Phe 50 55 60Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn
Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asn Leu Gly Pro Phe Phe Tyr Phe
Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
115160120PRTArtificial SequenceSynthetic Construct 160Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30Tyr
Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala
Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ser Arg Trp Gly Gly Glu Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
120161107PRTArtificial SequenceSynthetic Construct 161Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Ser Ala Ser Phe Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
Thr Pro Pro 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
1051621350DNAArtificial SequenceSynthetic Construct 162gaagttcagc
tggttgaaag cggtggtggt ctggttcagc ctggtggtag cctgcgtctg 60agctgtgcag
caagcggttt tacctttaac gattatacca tggattgggt tcgtcaggca
120ccgggtaaag gtctggaatg ggttgcagat gttaatccga atagcggtgg
tagcattgtt 180aaccgtcgtt ttaaaggtcg ttttaccctg agcgttgatc
gtagcaaaaa taccctgtat 240ctgcaaatga atagtctgcg tgcagaggat
accgcagtgt attattgtgc acgtaacctg 300ggtccgttct tctactttga
ttattggggt cagggcaccc tggttaccgt tagcagcgct 360agcaccaagg
gcccctccgt gttccccctg gcccccagca gcaagagcac cagcggcggc
420acagccgctc tgggctgcct ggtcgaggac tacttccccg agcccgtgac
cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg
ccgtgctgca gagttctggc 540ctgtatagcc tgagcagcgt ggtcaccgtg
ccttctagca gcctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gcccagcaac accaaggtgg acgagaaggt ggagcccaag 660agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg
720tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
aggtcaagtt caactggtac 840gtggacggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa
1020gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg
ggatgagctg 1080accaagaacc aggtcagcct ctcgtgcgca gtcaaaggct
tctatcccag cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 1200gactccgacg gctccttctt
cctcgtgagc aagctcaccg tggacaagag caggtggcag 1260caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320aagagcctct ccctgtctcc gggtaaatga 1350163645DNAArtificial
SequenceSynthetic Construct 163gacatccaga tgacccagag ccccagcagc
ctgtctgcca gcgtgggcga cagagtgacc 60atcacatgca aggccagcca ggacgtgtcc
acagccgtgg cctggtatca gcagaagcct 120ggcaaggccc ccaagctgct
gatctacagc gccagcttcc ggtacaccgg cgtgcccagc 180agattcagcg
gcagcagatc cggcaccgac ttcaccctga ccatcagctc cctgcagccc
240gaggacttcg ccacctacta ctgccagcag cactacacca ccccccccac
atttggccag 300ggcaccaagg tggaaatcaa gcgtacggtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatcgga agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg taactcccag 480gagagtgtca
cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg
540ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac
ccatcagggc 600ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
6451642874DNAArtificial SequenceSynthetic Construct 164caagtgcagc
tgaaagagtc cggccctgga ctggtggccc ctagccagag cctgagcatc 60acctgtaccg
tgtccggctt cagcctgacc agctacggcg tgtcatgggt gcgccagcct
120ccaggcaagt gtctggaatg gctgggcatc atctggggcg acggcagcac
caattaccac 180agcgccctga tcagcagact gagcatctcc aaggacaaca
gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac cgacgacacc
gccacctact actgcgccaa gggcatcacc 300accgtggtgg acgactacta
cgctatggac tactggggcc agggcaccag cgtgacagtg 360tctagcggag
gcggaggatc tggcggcgga ggaagtggcg gagggggatc tgggggaggc
420ggaagcgata tccagatgac ccagagccct gccagcctgt ctgcctctgt
gggcgagaca 480gtgaccatca catgccgggc cagcgagaac atcgacagct
acctggcctg gtatcagcag 540aagcagggca agagccccca gctgctggtg
tacgccgcca cctttctggc cgacgatgtg 600cccagcagat tcagcggcag
cggaagcggc acacagtaca gcctgaagat caactccctg 660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaaggc gggggaggct ccggaggcgg
cggaagtgga 780ggcggcggaa gtggcggagg cggagggggg ggaagtgggg
gcggaggcag tgggggggga 840ggatcccagg ccgtcgtgac ccaggaaccc
agcctgacag tgtctcctgg cggcaccgtg 900accctgacat gtggcagttc
tacaggcgcc gtgaccacca gcaactacgc caactgggtg 960caggaaaagc
ccggccaggc cttcagagga ctgatcggcg gcaccaacaa gagagcccct
1020ggcacccctg ccagattcag cggatctctg ctgggaggaa aggccgccct
gacactgtct 1080ggcgcccagc cagaagatga ggccgagtac tactgcgccc
tgtggtacag caacctgtgg 1140gtgttcggcg gaggcaccaa gctgacagtg
ctgagcagcg cttccaccaa gggacccagt 1200gtgttccccc tggcccccag
ctccaagtct acatccggtg gcacagctgc cctgggatgt 1260ctcgtgaagg
actactttcc tgagcctgtg acagtgtctt ggaacagcgg agccctgacc
1320agcggagtgc acacattccc tgcagtgctg cagagcagcg gcctgtatag
cctgagcagc 1380gtcgtgaccg tgccttcctc tagcctggga acacagacat
atatctgtaa tgtgaatcat 1440aagcccagta ataccaaagt ggataagaaa
gtggaaccta agagctgcga tggcggagga 1500gggtccggag gcggagggtc
cgaggtccag ctggtcgagt ctggaggagg actggtgcag 1560ccaggcggat
ctctgagact gagctgcgcc gccagcggat tcaacatcaa ggacacctac
1620atccactggg tgaggcaggc ccctggaaag ggactggagt gggtggccag
aatctacccc 1680accaacggct acacaagata cgccgacagc gtgaagggca
gattcaccat cagcgccgac 1740accagcaaga acaccgccta cctgcagatg
aacagcctga gagccgagga cacagccgtg 1800tactactgct ctagatgggg
aggcgagggc ttctacgcca tggactactg gggacagggc 1860acactggtga
ccgtgtccag cgctagcacc aagggcccct ccgtgttccc cctggccccc
1920agcagcaaga gcaccagcgg cggcacagcc gctctgggct gcctggtcga
ggactacttc 1980cccgagcccg tgaccgtgtc ctggaacagc ggagccctga
cctccggcgt gcacaccttc 2040cccgccgtgc tgcagagttc tggcctgtat
agcctgagca gcgtggtcac cgtgccttct 2100agcagcctgg gcacccagac
ctacatctgc aacgtgaacc acaagcccag caacaccaag 2160gtggacgaga
aggtggagcc caagagctgc gacaaaactc acacatgccc accgtgccca
2220gcacctgaag ctgcaggggg accgtcagtc ttcctcttcc ccccaaaacc
caaggacacc 2280ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg
tggacgtgag ccacgaagac 2340cctgaggtca agttcaactg gtacgtggac
ggcgtggagg tgcataatgc caagacaaag 2400ccgcgggagg agcagtacaa
cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 2460caggactggc
tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcggcgcc
2520cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca
ggtgtacacc 2580ctgcccccat gccgggatga gctgaccaag aaccaggtca
gcctgtggtg cctggtcaaa 2640ggcttctatc ccagcgacat cgccgtggag
tgggagagca atgggcagcc ggagaacaac 2700tacaagacca cgcctcccgt
gctggactcc gacggctcct tcttcctcta cagcaagctc 2760accgtggaca
agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag
2820gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga
28741652874DNAArtificial SequenceSynthetic Construct 165caagtgcagc
tgaaagagtc cggccctgga ctggtggccc ctagccagag cctgagcatc 60acctgtaccg
tgtccggctt cagcctgacc agctacggcg tgtcatgggt gcgccagcct
120ccaggcaagt gtctggaatg gctgggcatc atctggggcg acggcagcac
caattaccac 180agcgccctga tcagcagact gagcatctcc aaggacaaca
gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac cgacgacacc
gccacctact actgcgccaa gggcatcacc 300accgtggtgg acgactacta
cgctatggac tactggggcc agggcaccag cgtgacagtg 360tctagcggag
gcggaggatc tggcggcgga ggaagtggcg gagggggatc tgggggaggc
420ggaagcgata tccagatgac ccagagccct gccagcctgt ctgcctctgt
gggcgagaca 480gtgaccatca catgccgggc cagcgagaac atcgacagct
acctggcctg gtatcagcag 540aagcagggca agagccccca gctgctggtg
tacgccgcca cctttctggc cgacgatgtg 600cccagcagat tcagcggcag
cggaagcggc acacagtaca gcctgaagat caactccctg 660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaagga ggcggcggaa gtgtgcacat
gcccctgggc 780ttcctgggcc ccagacaggc cagagtcgtg aacggggggg
gcggaggcag tgggggggga 840ggatcccagg ccgtcgtgac ccaggaaccc
agcctgacag tgtctcctgg cggcaccgtg 900accctgacat gtggcagttc
tacaggcgcc gtgaccacca gcaactacgc caactgggtg 960caggaaaagc
ccggccaggc cttcagagga ctgatcggcg gcaccaacaa gagagcccct
1020ggcacccctg ccagattcag cggatctctg ctgggaggaa aggccgccct
gacactgtct 1080ggcgcccagc cagaagatga ggccgagtac tactgcgccc
tgtggtacag caacctgtgg 1140gtgttcggcg gaggcaccaa gctgacagtg
ctgagcagcg cttccaccaa gggacccagt 1200gtgttccccc tggcccccag
ctccaagtct acatccggtg gcacagctgc cctgggatgt 1260ctcgtgaagg
actactttcc tgagcctgtg acagtgtctt ggaacagcgg agccctgacc
1320agcggagtgc acacattccc tgcagtgctg cagagcagcg gcctgtatag
cctgagcagc 1380gtcgtgaccg tgccttcctc tagcctggga acacagacat
atatctgtaa tgtgaatcat 1440aagcccagta ataccaaagt ggataagaaa
gtggaaccta agagctgcga tggcggagga 1500gggtccggag gcggagggtc
cgaggtccag ctggtcgagt ctggaggagg actggtgcag 1560ccaggcggat
ctctgagact gagctgcgcc gccagcggat tcaacatcaa ggacacctac
1620atccactggg tgaggcaggc ccctggaaag ggactggagt gggtggccag
aatctacccc 1680accaacggct acacaagata cgccgacagc gtgaagggca
gattcaccat cagcgccgac 1740accagcaaga acaccgccta cctgcagatg
aacagcctga gagccgagga cacagccgtg 1800tactactgct ctagatgggg
aggcgagggc ttctacgcca tggactactg gggacagggc 1860acactggtga
ccgtgtccag cgctagcacc aagggcccct ccgtgttccc cctggccccc
1920agcagcaaga gcaccagcgg cggcacagcc gctctgggct gcctggtcga
ggactacttc 1980cccgagcccg tgaccgtgtc ctggaacagc ggagccctga
cctccggcgt gcacaccttc 2040cccgccgtgc tgcagagttc tggcctgtat
agcctgagca gcgtggtcac cgtgccttct 2100agcagcctgg gcacccagac
ctacatctgc aacgtgaacc acaagcccag caacaccaag 2160gtggacgaga
aggtggagcc caagagctgc gacaaaactc acacatgccc accgtgccca
2220gcacctgaag ctgcaggggg accgtcagtc ttcctcttcc ccccaaaacc
caaggacacc 2280ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg
tggacgtgag ccacgaagac 2340cctgaggtca agttcaactg gtacgtggac
ggcgtggagg tgcataatgc caagacaaag 2400ccgcgggagg agcagtacaa
cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 2460caggactggc
tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcggcgcc
2520cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca
ggtgtacacc 2580ctgcccccat gccgggatga gctgaccaag aaccaggtca
gcctgtggtg cctggtcaaa 2640ggcttctatc ccagcgacat cgccgtggag
tgggagagca atgggcagcc ggagaacaac 2700tacaagacca cgcctcccgt
gctggactcc gacggctcct tcttcctcta cagcaagctc 2760accgtggaca
agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag
2820gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa atga
28741661350DNAArtificial SequenceSynthetic Construct 166caggtgcaat
tggttcaatc tggtgctgaa gtaaaaaaac cgggcgcttc cgttaaagtg 60agctgcaaag
catccggata caccttcact tcctattaca tgcactgggt tcgtcaagcc
120ccgggccagg gtctggaatg gatgggcatc attaacccaa gcggtggctc
tacctcctac 180gcgcagaaat tccagggtcg cgtcacgatg acccatgaca
ctagcacctc taccgtttat 240atggagctgt ccagcctgcg ttctgaagat
actgcagtgt actactgtgc acgctctttc 300ttcactggtt tccatctgga
ctattggggt caaggcaccc tcgtaacggt ttcttctgct 360agcaccaagg
gcccctccgt gttccccctg gcccccagca gcaagagcac cagcggcggc
420acagccgctc tgggctgcct ggtcgaggac tacttccccg agcccgtgac
cgtgtcctgg 480aacagcggag ccctgacctc cggcgtgcac accttccccg
ccgtgctgca gagttctggc 540ctgtatagcc tgagcagcgt ggtcaccgtg
ccttctagca gcctgggcac ccagacctac 600atctgcaacg tgaaccacaa
gcccagcaac accaaggtgg acgagaaggt ggagcccaag 660agctgcgaca
aaactcacac atgcccaccg tgcccagcac ctgaagctgc agggggaccg
720tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg
gacccctgag 780gtcacatgcg tggtggtgga cgtgagccac gaagaccctg
aggtcaagtt caactggtac 840gtggacggcg tggaggtgca taatgccaag
acaaagccgc gggaggagca gtacaacagc 900acgtaccgtg tggtcagcgt
cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960tacaagtgca
aggtctccaa caaagccctc ggcgccccca tcgagaaaac catctccaaa
1020gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg
ggatgagctg 1080accaagaacc aggtcagcct ctcgtgcgca gtcaaaggct
tctatcccag cgacatcgcc 1140gtggagtggg agagcaatgg gcagccggag
aacaactaca agaccacgcc tcccgtgctg 1200gactccgacg gctccttctt
cctcgtgagc aagctcaccg tggacaagag caggtggcag 1260caggggaacg
tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag
1320aagagcctct ccctgtctcc gggtaaatga 1350167651DNAArtificial
SequenceSynthetic Construct 167gaaatcgtgt taacgcagtc tccaggcacc
ctgtctttgt ctccagggga aagagccacc 60ctctcttgca gggccagtca gagtgttagc
agcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggct
cctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttca
gtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag
240cctgaagatt ttgcagtgta ttactgtcag cagtatacca acgaacatta
ttatacgttc 300ggccagggga ccaaagtgga aatcaaacgt acggtggctg
caccatctgt cttcatcttc 360ccgccatctg atcggaagtt gaaatctgga
actgcctctg ttgtgtgcct gctgaataac 420ttctatccca gagaggccaa
agtacagtgg aaggtggata acgccctcca atcgggtaac 480tcccaggaga
gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc
540ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga
agtcacccat 600cagggcctga gctcgcccgt cacaaagagc ttcaacaggg
gagagtgtta g 6511682871DNAArtificial SequenceSynthetic Construct
168caagtgcagc tgaaagagtc cggccctgga ctggtggccc ctagccagag
cctgagcatc 60acctgtaccg tgtccggctt cagcctgacc agctacggcg tgtcatgggt
gcgccagcct 120ccaggcaagt gtctggaatg gctgggcatc atctggggcg
acggcagcac caattaccac 180agcgccctga tcagcagact gagcatctcc
aaggacaaca gcaagagcca ggtgttcctg 240aagctgaaca gcctgcagac
cgacgacacc gccacctact actgcgccaa gggcatcacc 300accgtggtgg
acgactacta cgctatggac tactggggcc agggcaccag cgtgacagtg
360tctagcggag gcggaggatc tggcggcgga ggaagtggcg gagggggatc
tgggggaggc 420ggaagcgata tccagatgac ccagagccct gccagcctgt
ctgcctctgt gggcgagaca 480gtgaccatca catgccgggc cagcgagaac
atcgacagct acctggcctg gtatcagcag 540aagcagggca agagccccca
gctgctggtg tacgccgcca cctttctggc cgacgatgtg 600cccagcagat
tcagcggcag cggaagcggc acacagtaca gcctgaagat caactccctg
660cagagcgagg
acgtggcccg gtactactgc cagcactact acagcacccc ctacaccttc
720ggctgcggca ccaagctgga aatcaaagga ggcggcggaa gtgtgcacat
gcccctgggc 780ttcctgggcc ccagacaggc cagagtcgtg aacggggggg
gcggaggcag tgggggggga 840ggatcccagg ccgtcgtgac ccaggaaccc
agcctgacag tgtctcctgg cggcaccgtg 900accctgacat gtggcagttc
tacaggcgcc gtgaccacca gcaactacgc caactgggtg 960caggaaaagc
ccggccaggc cttcagagga ctgatcggcg gcaccaacaa gagagcccct
1020ggcacccctg ccagattcag cggatctctg ctgggaggaa aggccgccct
gacactgtct 1080ggcgcccagc cagaagatga ggccgagtac tactgcgccc
tgtggtacag caacctgtgg 1140gtgttcggcg gaggcaccaa gctgacagtg
ctgagcagcg cttccaccaa gggacccagt 1200gtgttccccc tggcccccag
ctccaagtct acatccggtg gcacagctgc cctgggatgt 1260ctcgtgaagg
actactttcc tgagcctgtg acagtgtctt ggaacagcgg agccctgacc
1320agcggagtgc acacattccc tgcagtgctg cagagcagcg gcctgtatag
cctgagcagc 1380gtcgtgaccg tgccttcctc tagcctggga acacagacat
atatctgtaa tgtgaatcat 1440aagcccagta ataccaaagt ggataagaaa
gtggaaccta agagctgcga tggcggagga 1500gggtccggag gcggagggtc
ccaggtgcaa ttggttcaat ctggtgctga agtaaaaaaa 1560ccgggcgctt
ccgttaaagt gagctgcaaa gcatccggat acaccttcac ttcctattac
1620atgcactggg ttcgtcaagc cccgggccag ggtctggaat ggatgggcat
cattaaccca 1680agcggtggct ctacctccta cgcgcagaaa ttccagggtc
gcgtcacgat gacccatgac 1740actagcacct ctaccgttta tatggagctg
tccagcctgc gttctgaaga tactgcagtg 1800tactactgtg cacgctcttt
cttcactggt ttccatctgg actattgggg tcaaggcacc 1860ctcgtaacgg
tttcttctgc tagcaccaag ggcccctccg tgttccccct ggcccccagc
1920agcaagagca ccagcggcgg cacagccgct ctgggctgcc tggtcgagga
ctacttcccc 1980gagcccgtga ccgtgtcctg gaacagcgga gccctgacct
ccggcgtgca caccttcccc 2040gccgtgctgc agagttctgg cctgtatagc
ctgagcagcg tggtcaccgt gccttctagc 2100agcctgggca cccagaccta
catctgcaac gtgaaccaca agcccagcaa caccaaggtg 2160gacgagaagg
tggagcccaa gagctgcgac aaaactcaca catgcccacc gtgcccagca
2220cctgaagctg cagggggacc gtcagtcttc ctcttccccc caaaacccaa
ggacaccctc 2280atgatctccc ggacccctga ggtcacatgc gtggtggtgg
acgtgagcca cgaagaccct 2340gaggtcaagt tcaactggta cgtggacggc
gtggaggtgc ataatgccaa gacaaagccg 2400cgggaggagc agtacaacag
cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 2460gactggctga
atggcaagga gtacaagtgc aaggtctcca acaaagccct cggcgccccc
2520atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt
gtacaccctg 2580cccccatgcc gggatgagct gaccaagaac caggtcagcc
tgtggtgcct ggtcaaaggc 2640ttctatccca gcgacatcgc cgtggagtgg
gagagcaatg ggcagccgga gaacaactac 2700aagaccacgc ctcccgtgct
ggactccgac ggctccttct tcctctacag caagctcacc 2760gtggacaaga
gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct
2820ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a
28711692857DNAArtificial SequenceSynthetic Construct 169agagtccggc
cctggactgg tggcccctag ccagagcctg agcatcacct gtaccgtgtc 60cggcttcagc
ctgaccagct acggcgtgtc atgggtgcgc cagcctccag gcaagtgtct
120ggaatggctg ggcatcatct ggggcgacgg cagcaccaat taccacagcg
ccctgatcag 180cagactgagc atctccaagg acaacagcaa gagccaggtg
ttcctgaagc tgaacagcct 240gcagaccgac gacaccgcca cctactactg
cgccaagggc atcaccaccg tggtggacga 300ctactacgct atggactact
ggggccaggg caccagcgtg acagtgtcta gcggaggcgg 360aggatctggc
ggcggaggaa gtggcggagg gggatctggg ggaggcggaa gcgatatcca
420gatgacccag agccctgcca gcctgtctgc ctctgtgggc gagacagtga
ccatcacatg 480ccgggccagc gagaacatcg acagctacct ggcctggtat
cagcagaagc agggcaagag 540cccccagctg ctggtgtacg ccgccacctt
tctggccgac gatgtgccca gcagattcag 600cggcagcgga agcggcacac
agtacagcct gaagatcaac tccctgcaga gcgaggacgt 660ggcccggtac
tactgccagc actactacag caccccctac accttcggct gcggcaccaa
720gctggaaatc aaaggcgggg gaggctccgg aggcggcgga agtggaggcg
gcggaagtgg 780cggaggcgga ggggggggaa gtgggggcgg aggcagtggg
gggggaggat cccaggccgt 840cgtgacccag gaacccagcc tgacagtgtc
tcctggcggc accgtgaccc tgacatgtgg 900cagttctaca ggcgccgtga
ccaccagcaa ctacgccaac tgggtgcagg aaaagcccgg 960ccaggccttc
agaggactga tcggcggcac caacaagaga gcccctggca cccctgccag
1020attcagcgga tctctgctgg gaggaaaggc cgccctgaca ctgtctggcg
cccagccaga 1080agatgaggcc gagtactact gcgccctgtg gtacagcaac
ctgtgggtgt tcggcggagg 1140caccaagctg acagtgctga gcagcgcttc
caccaaggga cccagtgtgt tccccctggc 1200ccccagctcc aagtctacat
ccggtggcac agctgccctg ggatgtctcg tgaaggacta 1260ctttcctgag
cctgtgacag tgtcttggaa cagcggagcc ctgaccagcg gagtgcacac
1320attccctgca gtgctgcaga gcagcggcct gtatagcctg agcagcgtcg
tgaccgtgcc 1380ttcctctagc ctgggaacac agacatatat ctgtaatgtg
aatcataagc ccagtaatac 1440caaagtggat aagaaagtgg aacctaagag
ctgcgatggc ggaggagggt ctggaggcgg 1500agggtcccag gtgcaattgg
ttcaatctgg tgctgaagta aaaaaaccgg gcgcttccgt 1560taaagtgagc
tgcaaagcat ccggatacac cttcacttcc tattacatgc actgggttcg
1620tcaagccccg ggccagggtc tggaatggat gggcatcatt aacccaagcg
gtggctctac 1680ctcctacgcg cagaaattcc agggtcgcgt cacgatgacc
catgacacta gcacctctac 1740cgtttatatg gagctgtcca gcctgcgttc
tgaagatact gcagtgtact actgtgcacg 1800ctctttcttc actggtttcc
atctggacta ttggggtcaa ggcaccctcg taacggtttc 1860ttctgctagc
accaagggcc cctccgtgtt ccccctggcc cccagcagca agagcaccag
1920cggcggcaca gccgctctgg gctgcctggt cgaggactac ttccccgagc
ccgtgaccgt 1980gtcctggaac agcggagccc tgacctccgg cgtgcacacc
ttccccgccg tgctgcagag 2040ttctggcctg tatagcctga gcagcgtggt
caccgtgcct tctagcagcc tgggcaccca 2100gacctacatc tgcaacgtga
accacaagcc cagcaacacc aaggtggacg agaaggtgga 2160gcccaagagc
tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagg
2220gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac 2280ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa
gaccctgagg tcaagttcaa 2340ctggtacgtg gacggcgtgg aggtgcataa
tgccaagaca aagccgcggg aggagcagta 2400caacagcacg taccgtgtgg
tcagcgtcct caccgtcctg caccaggact ggctgaatgg 2460caaggagtac
aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccat
2520ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc
catgccggga 2580tgagctgacc aagaaccagg tcagcctgtg gtgcctggtc
aaaggcttct atcccagcga 2640catcgccgtg gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacgcctcc 2700cgtgctggac tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 2760gtggcagcag
gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
2820cacgcagaag agcctctccc tgtctccggg taaatga
28571702025DNAArtificial SequenceSynthetic Construct 170caggccgtcg
tgacccagga acccagcctg acagtgtctc ctggcggcac cgtgaccctg 60acatgtggca
gttctacagg cgccgtgacc accagcaact acgccaactg ggtgcaggaa
120aagcccggcc aggccttcag aggactgatc ggcggcacca acaagagagc
ccctggcacc 180cctgccagat tcagcggatc tctgctggga ggaaaggccg
ccctgacact gtctggcgcc 240cagccagaag atgaggccga gtactactgc
gccctgtggt acagcaacct gtgggtgttc 300ggcggaggca ccaagctgac
agtgctgagc agcgcttcca ccaagggacc cagtgtgttc 360cccctggccc
ccagctccaa gtctacatcc ggtggcacag ctgccctggg atgtctcgtg
420aaggactact ttcctgagcc tgtgacagtg tcttggaaca gcggagccct
gaccagcgga 480gtgcacacat tccctgcagt gctgcagagc agcggcctgt
atagcctgag cagcgtcgtg 540accgtgcctt cctctagcct gggaacacag
acatatatct gtaatgtgaa tcataagccc 600agtaatacca aagtggataa
gaaagtggaa cctaagagct gcgatggcgg aggagggtct 660ggaggcggag
ggtcccaggt gcaattggtt caatctggtg ctgaagtaaa aaaaccgggc
720gcttccgtta aagtgagctg caaagcatcc ggatacacct tcacttccta
ttacatgcac 780tgggttcgtc aagccccggg ccagggtctg gaatggatgg
gcatcattaa cccaagcggt 840ggctctacct cctacgcgca gaaattccag
ggtcgcgtca cgatgaccca tgacactagc 900acctctaccg tttatatgga
gctgtccagc ctgcgttctg aagatactgc agtgtactac 960tgtgcacgct
ctttcttcac tggtttccat ctggactatt ggggtcaagg caccctcgta
1020acggtttctt ctgctagcac caagggcccc tccgtgttcc ccctggcccc
cagcagcaag 1080agcaccagcg gcggcacagc cgctctgggc tgcctggtcg
aggactactt ccccgagccc 1140gtgaccgtgt cctggaacag cggagccctg
acctccggcg tgcacacctt ccccgccgtg 1200ctgcagagtt ctggcctgta
tagcctgagc agcgtggtca ccgtgccttc tagcagcctg 1260ggcacccaga
cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacgag
1320aaggtggagc ccaagagctg cgacaaaact cacacatgcc caccgtgccc
agcacctgaa 1380gctgcagggg gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc 1440tcccggaccc ctgaggtcac atgcgtggtg
gtggacgtga gccacgaaga ccctgaggtc 1500aagttcaact ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1560gagcagtaca
acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg
1620ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcggcgc
ccccatcgag 1680aaaaccatct ccaaagccaa agggcagccc cgagaaccac
aggtgtacac cctgccccca 1740tgccgggatg agctgaccaa gaaccaggtc
agcctgtggt gcctggtcaa aggcttctat 1800cccagcgaca tcgccgtgga
gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1860acgcctcccg
tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac
1920aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga
ggctctgcac 1980aaccactaca cgcagaagag cctctccctg tctccgggta aatga
2025
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