U.S. patent application number 16/891460 was filed with the patent office on 2020-12-03 for site specific her2 antibody drug conjugates.
This patent application is currently assigned to Pfizer Inc.. The applicant listed for this patent is Pfizer Inc.. Invention is credited to Edmund Idris GRAZIANI, Frank LOGANZO, Jr., Dangshe MA, Kimberly Ann MARQUETTE, Puja SAPRA, Pavel STROP.
Application Number | 20200377615 16/891460 |
Document ID | / |
Family ID | 1000004974776 |
Filed Date | 2020-12-03 |
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United States Patent
Application |
20200377615 |
Kind Code |
A1 |
MA; Dangshe ; et
al. |
December 3, 2020 |
SITE SPECIFIC HER2 ANTIBODY DRUG CONJUGATES
Abstract
The present invention provides site specific HER2 antibody drug
conjugates and methods for preparing and using the same.
Inventors: |
MA; Dangshe; (Millwood,
NY) ; LOGANZO, Jr.; Frank; (New City, NY) ;
MARQUETTE; Kimberly Ann; (Somerville, MA) ; GRAZIANI;
Edmund Idris; (Chestnut Ridge, NY) ; SAPRA; Puja;
(River Edge, NJ) ; STROP; Pavel; (San Mateo,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pfizer Inc. |
New York |
NY |
US |
|
|
Assignee: |
Pfizer Inc.
New York
NY
|
Family ID: |
1000004974776 |
Appl. No.: |
16/891460 |
Filed: |
June 3, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15356750 |
Nov 21, 2016 |
10689458 |
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16891460 |
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62409105 |
Oct 17, 2016 |
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62289727 |
Feb 1, 2016 |
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62289744 |
Feb 1, 2016 |
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62260854 |
Nov 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/565 20130101;
A61K 47/6863 20170801; A61K 47/6857 20170801; C07K 16/303 20130101;
A61K 47/6811 20170801; A61K 47/6803 20170801; C07K 2317/92
20130101; C07K 2317/73 20130101; C07K 16/32 20130101; C07K 16/3015
20130101; C07K 16/30 20130101; A61K 2039/505 20130101; C07K 16/3069
20130101; C07K 16/3023 20130101; A61K 47/6855 20170801; C07K
2317/732 20130101; C07K 2317/52 20130101; C07K 2317/94 20130101;
C07K 16/3053 20130101 |
International
Class: |
C07K 16/32 20060101
C07K016/32; A61K 47/68 20060101 A61K047/68; C07K 16/30 20060101
C07K016/30 |
Claims
1-23. (canceled)
24. A method of treating a HER2 expressing cancer in a subject,
comprising administering to the subject in need thereof a
therapeutically effective amount of a composition comprising an
antibody drug conjugate of the formula: Ab-(L-D), wherein: (a) Ab
is an antibody that binds to HER2 and comprises (1) a heavy chain
variable region comprising three CDRs comprising SEQ ID NOs:2, 3
and 4; (2) a heavy chain constant region of any of SEQ ID NOs:17,
5, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37 or 39; (3) a light chain
variable region comprising three CDRs comprising SEQ ID NOs:8, 9
and 10; (4) a light chain constant region of any of SEQ ID NOs:41,
11 or 43; and (b) L-D is a linker-drug moiety, wherein L is a
linker, and D is a drug, with the proviso that when the heavy chain
constant region is SEQ ID NO:5 the light chain constant region is
not SEQ ID NO:11.
25. The method of claim 24, wherein the cancer is a solid
tumor.
26. (canceled)
27. The method of claim 24, wherein the solid tumor is selected
from the group consisting of breast cancer, ovarian cancer, lung
cancer and gastric cancer.
28. The method of claim 27, wherein the breast cancer is estrogen
and progesterone receptor negative or triple negative breast cancer
(TNBC).
29. The method of claim 27, wherein the lung cancer is non-small
cell cancer (NSLC).
30. The method of claim 24, wherein the subject has been previously
treated with trastuzumab and/or trastuzumab emtansine either of
which alone or in combination with another therapeutic agent.
31. The method of claim 30, wherein the therapeutic agent is a
taxane.
32. The method of claim 30, wherein the cancer is resistant to,
refractory to and/or relapsed from treatment with trastuzumab
and/or trastuzumab emtansine either of which alone or in
combination with another therapeutic agent.
33. The method of claim 32, wherein the therapeutic agent is a
taxane.
34. The method of claim 24, wherein the cancer expresses HER2 at a
high level.
35. The method of claim 34, wherein the cancer expresses HER2 at a
3+ level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
.gtoreq.2.0.
36. The method of claim 24, wherein the cancer expresses HER2 at a
moderate level.
37. The method of claim 36, wherein the cancer expresses HER2 at a
2+ level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
<2.0
38. The method of claim 24, wherein the cancer expresses HER2 at a
low level.
39. The method of claim 38, wherein the cancer expresses HER2 at a
1+ level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
<2.0.
40. The method of claim 35, wherein IHC is performed using a Dako
Hercptest.TM. assay and FISH is performed using a Dako HER2 FISH
Pharm Dx' assay.
41. The method of claim 37, wherein IHC is performed using a Dako
Hercptest.TM. assay and FISH is performed using a Dako HER2 FISH
Pharm Dx' assay.
42. The method of claim 39, wherein IHC is performed using a Dako
Hercptest.TM. assay and FISH is performed using a Dako HER2 FISH
Pharm Dx' assay.
43. The method of claim 24, wherein: (a) the antibody comprises a
heavy chain comprising SEQ ID NO:18 and a light chain comprising
SEQ ID NO:42; and (b) L is a linker of vc and D is an auristatin of
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide or a pharmaceutically
acceptable salt or solvate thereof.
44. The method of claim 24, wherein: (a) the antibody comprises a
heavy chain comprising SEQ ID NO:14 and a light chain comprising
SEQ ID NO:44; and (b) L is a linker of AcLysvc and D is an
auristatin of
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1
S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-met-
hyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide or a pharmaceutically
acceptable salt or solvate thereof.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/260,854, filed Nov. 30, 2015, U.S. Provisional
Application No. 62/289,744, filed Feb. 1, 2016, U.S. Provisional
Application No. 62/289,727, filed Feb. 1, 2016, and U.S.
Provisional Application No. 62/409,105, filed Oct. 17, 2016, which
are hereby incorporated by referenced in their entireties.
SEQUENCE LISTING
[0002] This application is being filed electronically via EFS-Web
and includes an electronically submitted sequence listing in .txt
format. The .txt file contains a sequence listing entitled
"PC72091A_SequenceListing.txt" created on Oct. 18, 2016, and having
a size of 171 KB. The sequence listing contained in this .txt file
is part of the specification and is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to site specific HER2 antibody
drug conjugates. The present invention further relates to the
methods of using such antibody drug conjugates for the treatment of
cancer.
BACKGROUND OF THE INVENTION
[0004] Members of the ErbB family of transmembrane receptor
tyrosine kinases are important mediators of cell growth, cell
differentiation, cell migration, and apoptosis. The receptor family
includes four distinct members, including epidermal growth factor
receptor (EGFR or ErbB1), HER2 (ErbB2 or p185), HER3 (ErbB3) and
HER4 (ErbB4 or tyro2).
[0005] HER2 was originally identified as the product of the
transforming gene from neuroblastomas of chemically treated rats.
HER2 overexpression has been validated as tumorigenic both in vitro
(Di Fiore et al., 1987, Science 237(4811):178-82; Hudziak et al.,
1987, PNAS 84(20):7159-63; Chazin et al., 1992, Oncogene
7(9):1859-66) and in animal models (Guy et al., 1992, PNAS
89(22):10578-82). Amplification of the gene encoding HER2 with
consequent overexpression of the receptor occurs in breast and
ovarian cancers and correlates with a poor prognosis (Slamon et
al., 1987, Science 235(4785):177-82; Slamon et al., 1989, Science
244:707-12; Anbazhagan et al., 1991, Annals Oncology 2(1):47-53;
Andrulis et al., 1998, J Clinical Oncology 16(4):1340-9).
Overexpression of HER2 (frequently but not necessarily due to gene
amplification) has also been observed in other tumor types
including gastric, endometrial, non-small cell lung cancer, colon,
pancreatic, bladder, kidney, prostate and cervical (Scholl et al.,
2001, Annals Oncology 12 (Suppl. 1):581-7; Menard et al., 2001, Ann
Oncol 12(Suppl 1):515-9; Martin et al., 2014, Future Oncology
10:1469-86).
[0006] Herceptin.RTM. (trastuzumab) is a humanized monoclonal
antibody that binds to the extracellular domain of HER2 (Carter et
al. 1992, PNAS 89:4285-9 and U.S. Pat. No. 5,821,337).
Herceptin.RTM. received marketing approval from the Food and Drug
Administration on Sep. 25, 1998 for the treatment of patients with
metastatic breast cancer whose tumors overexpress the HER2 protein.
Although Herceptin.RTM. is a breakthrough in treating patients with
HER2-overexpressing breast cancers that have received extensive
prior anti-cancer therapy, segments of patients in this population
fail to respond, respond only poorly or become resistant to
Herceptin.RTM. treatment.
[0007] Kadcyla.RTM. (trastuzumab-DM1 or T-DM1) is an antibody drug
conjugate consisting of trastuzumab conjugated to the maytansinoid
agent DM1 via the stable thioether linker MCC
(4-[N-maleimidomethyl] cyclohexane-1-carboxylate) (Lewis et al.,
2008, Cancer Res. 68:9280-90; Krop et al., 2010, J Clin Oncol.
28:2698-2704; U.S. Pat. No. 8,337,856). Kadcyla.RTM. received
marketing approval from the Food and Drug Administration on Feb.
22, 2013 for the treatment of HER2 positive metastatic breast
cancer in patients who had been previously treated with
Herceptin.RTM. and a taxane drug and became Herceptin.RTM.
refractory. Like seen with Herceptin.RTM., there are segments of
the patients in the HER2-overexpressing breast cancer population
that do not experience successful long term therapy with
Kadcyla.RTM..
[0008] Therefore, there is a significant clinical need for
developing further HER2-directed cancer therapies for those
patients with HER2-overexpressing tumors or other diseases
associated with HER2 overexpression that do not respond, respond
poorly or become resistant to Herceptin.RTM. and/or Kadcyla.RTM.
treatment.
SUMMARY OF THE INVENTION
[0009] The present invention provides site specific HER2 antibody
drug conjugates (ADCs) and their use in treatment of
HER2-expressing cancers. ADCs enable targeted delivery of
therapeutics to cancer cells and offer potential for more selective
therapy while reducing known off-target toxicities.
[0010] A site specific HER2 ADC of the invention is generally of
the formula: Ab-(L-D), wherein Ab is an antibody, or
antigen-binding fragment thereof, that binds to HER2; and L-D is a
linker-drug moiety, wherein L is a linker, and D is a drug.
[0011] The antibody (Ab) of the ADCs of the invention can be any
HER2-binding antibody. In some aspects of the invention, the Ab
binds to the same epitope on HER2 as trastuzumab (Herceptin.RTM.).
In other aspects of the invention, the Ab has the same heavy chain
and light chain CDRs as trastuzumab. In specific aspects of the
invention, the Ab has the same heavy chain variable region
(V.sub.H) and the same light chain variable region (V.sub.L) as
trastuzumab.
[0012] The HER2 ADCs of the present invention are conjugated to the
drug in a site specific manner. To accommodate this type of
conjugation, the antibody must be derivatized to provide for either
a reactive cysteine residue engineered at one or more specific
sites or an acyl donor glutamine residue (either engineered at one
or more specific sites or in an attached peptide tag). Such
modifications should be at sites that do not disrupt the antigen
binding capability of the antibody. In preferred embodiments, the
one or more modifications are made in the constant region of the
heavy and/or light chains of the antibody.
[0013] In some embodiments of the present invention, the site
specific HER2 ADCs can use antibodies comprising heavy chain
variable region CDRs and light chain variable region CDRs of
trastuzumab (V.sub.H CDRs of SEQ ID NOs:2-4 and V.sub.L CDRs of SEQ
ID NOs:8-10) and any combination of heavy and light chain constant
regions disclosed in Table 1 with the proviso that when the heavy
chain constant region is SEQ ID NO:5 then the light chain constant
region is not SEQ ID NO:11. In such embodiments, the heavy chain
constant region can be selected from any of SEQ ID NOs:17, 5, 13,
21, 23, 25, 27, 29, 31, 33, 35, 37 or 39 while the light chain
constant region can be selected from any of SEQ ID NOs:41, 11 or 43
providing that the combination is not SEQ ID NO:5 and SEQ ID
NO:11.
[0014] In a specific embodiment, the antibody used to make the site
specific HER2 ADC comprises a V.sub.H domain with CDRs of SEQ ID
NOs:2-4 and a V.sub.L domain with CDRs of SEQ ID NOs:8-10 attached
to a heavy chain constant region of SEQ ID NO:17 and a light chain
constant region of SEQ ID NO:41. In another specific embodiment,
the antibody used to make the site specific HER2 ADC comprises a
V.sub.H domain with CDRs of SEQ ID NOs:2-4 and a V.sub.L domain
with CDRs of SEQ ID NOs:8-10 attached to a heavy chain constant
region of SEQ ID NO:13 and a light chain constant region of SEQ ID
NO:43.
[0015] In other embodiments, the ADCs of the invention can use
antibodies comprising of any combination of heavy and light chains
disclosed in Table 1 with the proviso that if the heavy chain is
SEQ ID NO:6 then the light chain is not SEQ ID NO:12. In such
embodiments, the heavy chain can be selected from any of SEQ ID
NOs:18, 6, 14, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 while the
light chain can be selected from any of SEQ ID NOs: 42, 12 or 44
providing that the combination is not SEQ ID NO:6 and SEQ ID
NO:12.
[0016] In a specific embodiment, the ADCs of the invention can use
an antibody comprising a heavy chain of SEQ ID NO:18 and a light
chain of SEQ ID NO:42. In another specific embodiment, the ADCs of
the invention can use an antibody comprising a heavy chain of SEQ
ID NO:14 and a light chain of SEQ ID NO:44.
[0017] Any of the site specific HER2 ADCs disclosed herein can be
prepared with a drug (D) that is a therapeutic agent useful for
treating cancer. In a specific embodiment, the therapeutic agent is
an anti-mitotic agent. In another specific embodiment, the
anti-mitotic agent drug component in the ADCs of the invention is
an auristatin (e.g., 0101, 8261, 6121, 8254, 6780 and 0131). In a
more specific embodiment, the auristatin drug component in the ADCs
of the invention is
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrroli-
din-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (also
known as 0101). Preferably, the drug component of the ADCs of the
invention is membrane permeable.
[0018] Any of the site specific HER2 ADCs disclosed herein can be
prepared with a linker (L) that is cleavable or non-cleavable.
Preferably, the linker is cleavable. Cleavable linkers include, but
are not limited to, vc, AcLysvc and m(H20)c-vc. More preferably,
the linker is vc or AcLysvc.
[0019] In a particular aspect of the invention, site specific HER2
ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:18 and a light chain of SEQ
ID NO:42; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0020] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:14 and a light chain of SEQ
ID NO:44; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is AcLysvc and wherein the drug
is 0101.
[0021] Another aspect of the invention includes methods of making,
methods of preparing, methods of synthesis, methods of conjugation
and methods of purification of the antibody drug conjugates
disclosed herein and the intermediates for the preparation,
synthesis and conjugation of the antibody drug conjugates disclosed
herein.
[0022] Further provided are pharmaceutical compositions comprising
a site specific HER2 ADC disclosed herein and a pharmaceutically
acceptable carrier.
[0023] Nucleic acids encoding the antibody portion of the site
specific HER2 ADCs are contemplated by the invention. Additional
vectors and host cells comprising the nucleic acids are also
contemplate by the invention.
[0024] The present invention also provides method of use of the
site specific HER2 ADCs in the treatment of HER2-expressing
cancers. HER2-expressing cancer to be treated with the site
specific HER2 ADCs of the invention can express HER2 at a high,
moderate or low level. In some embodiments, the cancer to be
treated is resistant to, refractory to and/or relapsed from
treatment with trastuzumab and/or trastuzumab emtansine (T-DM1)
either of which alone or in combination with a taxane. Cancers to
be treated include, but are not limited to, breast cancer, ovarian
cancer, lung cancer, gastric cancer, esophageal cancer, colorectal
cancer, urothelial cancer, pancreatic cancer, salivary gland cancer
and brain cancer or metastases of the aforementioned cancers. In a
more specific embodiment, the breast cancer is estrogen receptor
and progesterone receptor negative breast cancer or triple negative
breast cancer (TNBC). In another embodiment, the lung cancer is
non-small cell lung cancer (NSCLC).
[0025] These and other aspects of the invention will be appreciated
by a review of the application as a whole.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A-1B depict (A) T(kK183C+K290C)-vc0101 ADC and (B)
T(LCQ05+K222R)-AcLysvc0101 ADC. Each black circle represents a
linker/payload that is conjugated to the monoclonal antibody. The
structure of one such linker/payload is shown for each ADC. The
underlined entity is supplied by the amino acid residue on the
antibody through which conjugation occurs.
[0027] FIGS. 2A-2E depict spectra of selected ADCs from hydrophobic
interaction chromatography (HIC) showing changes in retention times
upon conjugation of trastuzumab derived antibodies to different
linker payloads.
[0028] FIGS. 3A-3B depict graphs of ADCs binding to HER2. (A)
direct binding to HER2 positive BT474 cells and (B) competitive
binding with PE labelled trastuzumab to BT474 cells. These results
indicate that the binding properties of antibody in these ADCs were
unaltered by the conjugation process.
[0029] FIG. 4 depicts ADCC activities of trastuzumab derived
ADCs.
[0030] FIG. 5 depicts in vitro cytotoxicity data (IC.sub.50)
reported in nM payload concentration for a number of trastuzumab
derived ADCs on a number of cell lines with different levels of
HER2 expression.
[0031] FIG. 6 depicts in vitro cytotoxicity data (IC.sub.50)
reported in ng/ml antibody concentration for a number of
trastuzumab derived ADCs on a number of cell lines with different
levels of HER2 expression.
[0032] FIGS. 7A-7I depict anti-tumor activity of nine trastuzumab
derived ADCs on N87 xenografts with tumor volume was plotted over
time. (A) T(kK183C+K290C)-vc0101; (B) T(kK183C)-vc0101; (C)
T(K290C)-vc0101; (D) T(LCQ05+K222R)-AcLysvc0101; (E)
T(K290C+K334C)-vc0101; (F) T(K334C+K392C)-vc0101; (G)
T(N297Q+K222R)-AcLysvc0101; (H) T-vc0101; (I) T-DM1. N87 gastric
cancer cells express high levels of HER2.
[0033] FIGS. 8A-8E depict anti-tumor activity of six trastuzumab
derived ADCs on HCC1954 xenografts with tumor volume plotted over
time. (A) T(LCQ05+K222R)-AcLysvc0101; (B) T(K290C+K334C)-vc0101;
(C) T(K334C+K392C)-vc0101; (D) T(N297Q+K222R)-AcLysvc0101; (E)
T-DM1. HCC1954 breast cancer cells express high levels of HER2.
[0034] FIGS. 9A-9G depict anti-tumor activity of seven trastuzumab
derived ADCs on JIMT-1 xenografts with tumor volume plotted over
time. (A) T(kK183C+K290C)-vc0101; (B) T(LCQ05+K222R)-AcLysvc0101;
(C) T(K290C+K334C)-vc0101; (D) T(K334C+K392C)-vc0101; (E)
T(N297Q+K222R)-AcLysvc0101; (F) T-vc0101; (G) T-DM1. JIMT-1 breast
cancer cells express moderate/low levels of HER2.
[0035] FIGS. 10A-10D depict anti-tumor activity of five trastuzumab
derived ADCs on MDA-MB-361(DYT2) xenografts with tumor volume
plotted over time. (A) T(LCQ05+K222R)-AcLysvc0101; (B)
T(N297Q+K222R)-AcLysvc0101; (C) T-vc0101; (D) T-DM1.
MDA-MB-361(DYT2) breast cancer cells express moderate/low levels of
HER2.
[0036] FIGS. 11A-11E depict anti-tumor activity of five trastuzumab
derived ADCs on PDX-144580 patient derived xenografts with tumor
volume plotted over time. (A) T(kK183C+K290C)-vc0101; (B)
T(LCQ05+K222R)-AcLysvc0101; (C) T(N297Q+K222R)-AcLysvc0101; (D)
T-vc0101; (E) T-DM1. PDX-144580 patient derived cells are a TNBC
PDX model.
[0037] FIGS. 12A-12D depict anti-tumor activity of four trastuzumab
derived ADCs on PDX-37622 patient derived xenografts with tumor
volume plotted over time. (A) T(kK183C+K290C)-vc0101; (B)
T(N297Q+K222R)-AcLysvc0101; (C) T(K297C+K334C)-vc0101; (D) T-DM1.
PDX-37622 patient derived cells are a NSCLC PDX model expressing
moderate levels of HER2.
[0038] FIGS. 13A-13B depict immunohistocytochemistry of N87 tumor
xenografts treated with either (A) T-DM1 or (B) T-vc0101 and
stained for phosphohistone H3 and IgG antibody. Bystander activity
is observed with T-vc0101.
[0039] FIG. 14 depicts in vitro cytotoxicity data (IC.sub.50)
reported in nM payload concentration and ng/ml antibody
concentration for a number of trastuzumab derived ADCs and free
payloads on cells made resistant to T-DM1 in vitro (N87-TM1 and
N87-TM2) or parental cells sensitive to T-DM1 (N87cells). N87
gastric cancer cells express high levels of HER2.
[0040] FIGS. 15A-15G depict anti-tumor activity of seven
trastuzumab derived ADCs on T-DM1 sensitive (N87 cells) and
resistant (N87-TM1 and N87-TM2) gastric cancer cells. (A) T-DM1;
(B) T-mc8261; (C) T(297Q+K222R)-AcLysvc0101; (D)
T(LCQ05+K222R)-AcLysvc0101; (E) T(K290C+K334C)-vc0101; (F)
T(K334C+K392C)-vc0101; (G) T(kK183C+K290C)-vc0101.
[0041] FIGS. 16A-16B depict western blots showing (A) MRP1 drug
efflux pump and (B) MDR1 drug efflux pump protein expression on
T-DM1 sensitive (N87 cells) and resistant (N87-TM1 and N87-TM2)
gastric cancer cells.
[0042] FIGS. 17A-17B depict HER2 expression and binding to
trastuzumab of T-DM1 sensitive (N87 cells) and resistant (N87-TM1
and N87-TM2) gastric cancer cells. (A) a western blot showing HER2
protein expression and (B) trastuzumab binding to cell surface
HER2.
[0043] FIGS. 18A-18D depict characterization of protein expression
levels in T-DM1 sensitive (N87 cells) and resistant (N87-TM1 and
N87-TM2) gastric cancer cells. (A) protein expression level changes
in 523 proteins; (B) western blots showing protein expression of
IGF2R, LAMP1 and CTSB; (C) western blot showing protein expression
of CAV1; (D) IHC of CAV1 protein expression in tumors generated in
vivo from implantation of N87 cells (left panel) and N87-TM2 cells
(right panel).
[0044] FIGS. 19A-19C depict sensitivity to trastuzumab and various
trastuzumab derived ADCs of tumors generated in vivo from
implantation of (A) T-DM1 sensitive N87 parental cells; (B) T-DM1
resistant N87-TM1 cells; (C) T-DM1 resistant N87-TM2 cells.
[0045] FIGS. 20A-20F depict sensitivity to trastuzumab and various
trastuzumab derived ADCs of tumors generated in vivo from
implantation of T-DM1 sensitive N87 parental cells and T-DM1
resistant N87-TM2 or N87-TM1 cells. (A) N87 tumor size was plotted
over time in the presence of trastuzumab or two trastuzumab derived
ADCs; (B) N87-TM2 tumor size was plotted over time in the presence
of trastuzumab or two trastuzumab derived ADCs; (C) time for N87
cell tumor to double in size in the presence of in the presence of
trastuzumab or two trastuzumab derived ADCs; (D) time for N87-TM2
cell tumor to double in size in the presence of trastuzumab or two
trastuzumab derived ADCs; (E) N87-TM2 tumor size was plotted over
time in the presence of seven different trastuzumab derived ADCs;
(F) N87-TM1 tumor size was plotted over time with a trastuzumab
derived ADC added at day 14.
[0046] FIGS. 21A-21E depict generation and characterization of
T-DM1 resistant cells generated in vivo. (A) N87 gastric cancer
cells were initially sensitive to T-DM1 when implanted in vivo. (B)
Over time, the implanted N87 cells became resistant to T-DM1 but
remained sensitive to (C) T-vc0101, (D) T(N297Q+K222R)-AcLysvc0101
and (E) T(kK183+K290C)-vc0101.
[0047] FIGS. 22A-22D depict in vitro cytotoxicity of four
trastuzumab derived ADCs on T-DM1 resistant cells (N87-TDM)
generated in vivo compared to T-DM1 sensitive parental N87 cells
with tumor volume plotted over time. (A) T-DM1; (B)
T(kK183+K290C)-vc0101; (C) T(LCQ05+K222R)-AcLysvc0101; (D)
T(N297Q+K222R)-AcLysvc0101.
[0048] FIGS. 23A-23B depict HER2 protein expression levels on T-DM1
resistant cells (N87-TDM1, from mice 2, 17 and 18) generated in
vivo compared to T-DM1 sensitive parental N87 cells. (A) FACS
analysis and (B) western blot analysis. No significant difference
in HER2 protein expression was observed.
[0049] FIGS. 24A-24D depict that T-DM1 resistance in N87-TDM1 (mice
2, 7 and 17) is not due to drug efflux pumps. (A) a western blot
showing MDR1 protein expression. In vitro cytotoxicity of T-DM1
resistant cells (N87-TDM1) and T-DM1 sensitive N87 parental cells
in the presence of free drug (B) 0101; (C) doxorubicin; (D)
T-DM1.
[0050] FIGS. 25A-25B depict concentration vs time profiles and
pharmacokinetics/toxicokinetics of (A) both total Ab and
trastuzumab ADC (T-vc0101) or T(kK183C+K290C) site specific ADC
after dose administration to cynomolgus monkeys and (B) the ADC
analyte of trastuzumab (T-vc0101) or various site specific ADCs
after dose administration to cynomolgus monkeys.
[0051] FIG. 26 depicts relative retention values by hydrophobic
interaction chromatography (HIC) vs exposure (AUC) in rats. The
X-axis represents Relative Retention Time by HIV; while the Y-axis
represents pharmacokinetic dose-normalized exposure in rats ("area
under curve", AUC for antibody, from 0 to 336 hours, divided by
drug dose of 10 mg/kg). Symbol shape denotes approximate drug
loading (DAR): diamond=DAR 2; circle=DAR 4. Arrow indicates
T(kK183C+K290C)-vc0101.
[0052] FIG. 27 depicts a toxicity study using T-vc0101 conventional
conjugate ADC and T(kK183C+K290C)-vc0101 site specific ADC.
T-vc0101 induced severe neutropenia at 5 mg/kg while the
T(kK183C+K290C)-vc0101 caused a minimal drop in neutrophil counts
at 9 mg/kg.
[0053] FIGS. 28A-28C depict the crystal structure of (A)
T(K290C+K334C)-vc0101; (B) T(K290C+K392C)-vc0101; and (C)
T(K334C+K392C)-vc0101.
[0054] FIG. 29 depicts in vivo efficacy on a xenograft model using
the N87 cell line. All ADCs tested showed efficacy at 3mpk.
[0055] FIG. 30 depicts anti-tumor activity of trastuzumab and two
trastuzumab derived ADCs on PDX-GA0044 patient derived xenografts
with tumor volume plotted over time. Animals were treated with
vehicle (hollow diamonds), trastuzumab (hollow triangles), T-DM1
(hollow circles), or T(kK183C+K290C)-vc0101 (solid circles and
solid squares). PDX-GA0044 patient derived cells are a Gastric PDX
model expressing moderate levels of HER2.
DETAILED DESCRIPTION OF THE INVENTION
[0056] The present invention provides site specific HER2 antibody
drug conjugates (ADCs), processes for preparing the conjugates
using HER2 antibodies, linkers, and drug payloads and nucleic acids
encoding the antibodies used in making the ADCs. The ADCs of the
invention are useful for the preparation and manufacture of
compositions, such as medicaments, that can be used in the
treatment of HER2-expressing cancers.
[0057] ADCs consist of an antibody component conjugated to a drug
payload through the use of a linker. Conventional conjugation
strategies for ADCs rely on randomly conjugating the drug payload
to the antibody through lysines or cysteines that are endogenously
on the antibody heavy and/or light chain. Accordingly, such ADCs
are a heterogeneous mixture of species showing different
drug:antibody ratios (DAR). In contrast, the ADCs disclosed herein
are site specific ADCs that conjugate the drug payload to the
antibody at particular engineered residues on the antibody heavy
and/or light chain. As such, the site specific ADCs are a
homogeneous population of ADCs comprised of a species with a
defined drug:antibody ratio (DAR). Thus, the site specific ADCs
demonstrate uniform stoichiometry resulting in improved
pharmacokinetics, biodistribution and safety profile of the
conjugate. ADCs of the invention include antibodies of the
invention conjugated to one or more linker/payload moieties.
[0058] The present invention provides antibody drug conjugates of
the formula Ab-(L-D), wherein (a) Ab is an antibody, or
antigen-binding fragment thereof, that binds to HER2, and (b) L-D
is a linker-drug moiety, wherein L is a linker, and D is a
drug.
[0059] Also encompassed by the present invention are antibody drug
conjugates of the formula Ab-(L-D).sub.p, wherein (a) Ab is an
antibody, or antigen-binding fragment thereof, that binds to HER2,
(b) L-D is a linker-drug moiety, wherein L is a linker, and D is a
drug and (c) p is the number of linker/drug moieties are attached
to the antibody. For site specific ADCs, p is a whole number due to
the homogeneous nature of the ADC. In some embodiments, p is 4. In
other embodiments, p is 3. In other embodiments, p is 2. In other
embodiments, p is 1. In other embodiments, p is greater than 4.
[0060] As used herein, the term "HER2" refers to a transmembrane
tyrosine kinase receptor that belongs to the EGFR family. HER2 is
also known as ErbB2, p185 and CD340. This family of receptors
includes four members (EGFR/HER1, HER2, HER3 and HER4) that
function by stimulating growth factor signaling pathways such as
the PI3K-AKT-mTOR pathway. Amplification and/or overexpression of
HER2 is associated with multiple human malignancies. The wild type
human HER2 protein is described, for example, in Semba et al.,
1985, PNAS 82:6497-6501 and Yamamoto et al., 1986, Nature 319:230-4
and Genbank Accession Number X03363.
[0061] As used herein, the term "Antibody (Ab)" refers to an
immunoglobulin molecule capable of recognizing and binding to a
specific target or antigen, such as a polypeptide, through at least
one antigen recognition site located in the variable region of the
immunoglobulin molecule. The term can encompass any type of
antibody, including but not limited to monoclonal antibodies,
antigen-binding fragments of intact antibodies that retain the
ability to specifically bind to a given antigen (i.e., Fab, Fab',
F(ab').sub.2, Fd, Fv, Fc, etc.) and mutants thereof.
[0062] Native or naturally occurring antibodies, and native
immunoglobulins, are typically heterotetrameric glycoproteins of
about 150,000 daltons, composed of two identical light (L) chains
and two identical heavy (H) chains. Each light chain is linked to a
heavy chain by one covalent disulfide bond, while the number of
disulfide linkages varies among the heavy chains of different
immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced intrachain disulfide bridges. Each heavy chain has
at one end a variable domain (V.sub.H) followed by a number of
constant domains. Each light chain has a variable domain at one end
(V.sub.L) and a constant domain at its other end; the constant
domain of the light chain is aligned with the first constant domain
of the heavy chain, and the light chain variable domain is aligned
with the variable domain of the heavy chain. The term "variable"
refers to the fact that certain portions of the variable domains
differ extensively in sequence among antibodies.
[0063] The antibody used in the present invention specifically
binds to HER2. In a specific embodiment, the HER2 antibody binds to
the same epitope on HER2 as trastuzumab (Herceptin.RTM.). In a more
specific embodiment, the HER2 antibody has the same variable region
CDRs as trastuzumab (Herceptin.RTM.). In yet a more specific
embodiment, the HER2 antibody has the same variable regions (i.e.,
V.sub.H and V.sub.L) as trastuzumab (Herceptin.RTM.).
[0064] As used herein, the term "Linker (L)" describes the direct
or indirect linkage of the antibody to the drug payload. Attachment
of a linker to an antibody can be accomplished in a variety of
ways, such as through surface lysines, reductive-coupling to
oxidized carbohydrates, cysteine residues liberated by reducing
interchain disulfide linkages, reactive cysteine residues
engineered at specific sites, and acyl donor glutamine-containing
tag or an endogenous glutamine made reactive by polypeptide
engineering in the presence of transglutaminase and an amine. The
present invention uses site specific methods to link the antibody
to the drug payload. In one embodiment, conjugation occurs through
cysteine residues that have been engineered into the antibody
constant region. In another embodiment, conjugation occurs through
acyl donor glutamine residues that have either been a) added to the
antibody constant region via a peptide tag, b) engineered into the
antibody constant region or c) made accessible/reactive by
engineering surrounding residues. Linkers can be cleavable (i.e.,
susceptible to cleavage under intracellular conditions) or
non-cleavable. In some embodiments, the linker is a cleavable
linker.
[0065] As used herein, the term "Drug (D)" refers to any
therapeutic agent useful in treating cancer. The drug has
biological or detectable activity, for example, cytotoxic agents,
chemotherapeutic agents, cytostatic agents, and immunomodulatory
agents. In preferred embodiments, therapeutic agents have a
cytotoxic effect on tumors including the depletion, elimination
and/or the killing of tumor cells. The terms drug, payload, and
drug payload are used interchangeably. In a specific embodiment,
the drug is an anti-mitotic agent. In a more specific embodiment,
the drug is an auristatin. In a yet more specific embodiment, the
drug is
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrroli-
din-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (also
known as 0101). In some embodiments, the drug is preferably
membrane permeable.
[0066] As used herein, the term "L-D" refers to a linker-drug
moiety resulting from a drug (D) linked to a linker (L).
[0067] Additional scientific and technical terms used in connection
with the present invention, unless indicated otherwise herein,
shall have the meanings that are commonly understood by those of
ordinary skill in the art. Further, unless otherwise required by
context, singular terms shall include pluralities and plural terms
shall include the singular. Generally, nomenclature used in
connection with, and techniques of, cell and tissue culture,
molecular biology, immunology, microbiology, genetics and protein
and nucleic acid chemistry and hybridization described herein are
those well-known and commonly used in the art.
I. HER2 Antibodies
[0068] For preparation of site specific HER2 ADCs of the invention,
the antibody can be any antibody that specifically binds to the
extracellular domain of HER2. In one embodiment, the antibody used
to make the ADC binds to the same epitope of HER2 as trastuzumab
and/or competes with trastuzumab for HER2 binding. In another
embodiment, the antibody used to make the ADC has the same heavy
chain variable region CDRs and light chain variable region CDRs as
trastuzumab. In yet another embodiment, the antibody used to make
the ADC has the same heavy chain variable region and light chain
variable region as trastuzumab.
[0069] The term "compete", as used herein with regard to an
antibody, means that a first antibody, or an antigen-binding
fragment thereof, binds to an epitope in a manner sufficiently
similar to the binding of a second antibody, or an antigen-binding
fragment thereof, such that the result of binding of the first
antibody with its cognate epitope is detectably decreased in the
presence of the second antibody compared to the binding of the
first antibody in the absence of the second antibody. The
alternative, where the binding of the second antibody to its
epitope is also detectably decreased in the presence of the first
antibody, can, but need not be the case. That is, a first antibody
can inhibit the binding of a second antibody to its epitope without
that second antibody inhibiting the binding of the first antibody
to its respective epitope. However, where each antibody detectably
inhibits the binding of the other antibody with its cognate epitope
or ligand, whether to the same, greater, or lesser extent, the
antibodies are said to "cross-compete" with each other for binding
of their respective epitope(s). Both competing and cross-competing
antibodies are encompassed by the present invention. Regardless of
the mechanism by which such competition or cross-competition occurs
(e.g., steric hindrance, conformational change, or binding to a
common epitope, or portion thereof), the skilled artisan would
appreciate, based upon the teachings provided herein, that such
competing and/or cross-competing antibodies are encompassed and can
be useful for the methods disclosed herein.
[0070] Trastuzumab (trade name Herceptin.RTM.) is a humanized
monoclonal antibody that binds to the extracellular domain of HER2.
The amino acid sequences of its variable domains are disclosed in
U.S. Pat. No. 5,821,337 (V.sub.H is SEQ ID NO:42 and V.sub.L is SEQ
ID NO:41 of U.S. Pat. No. 5,821,337) as well as in Table 1 infra
(SEQ ID NOs:1 and 7, respectively). The amino acid sequences of the
heavy chain variable region CDRs are SEQ ID NOs:2-4 while the amino
acid sequences of the light chain CDRs are SEQ ID NOs:6-10 (Table 1
infra). The amino acid sequences of the complete heavy and light
chains are SEQ ID NOs:6 and 12, respectively (Table 1 infra).
[0071] T-DM1 (trade name Kadcyla.RTM.) is an antibody drug
conjugate consisting of trastuzumab conjugated to the maytansinoid
agent DM1 via the stable thioether linker MCC
(4-[N-maleimidomethyl] cyclohexane-1-carboxylate) (U.S. Pat. No.
8,337,856). The antibody component of this ADC is identical to
trastuzumab. Payload conjugation to trastuzumab is accomplished
using conventional conjugation (rather than site specific)
techniques such that the ADC is a heterogeneous population of
species with different amounts of DM1 conjugated to each one. The
DM1 payload inhibits cell proliferation by inhibiting the formation
of microtubules during mitosis through inhibition of tubulin
polymerization (Remillard et al., 1975, Science 189:1002-5).
Kadcyla.RTM. is approved for the treatment of HER2 positive
metastatic breast cancer in patients who had been previously
treated with Herceptin.RTM. and a taxane drug and became
Herceptin.RTM. refractory. T-DM1 used in the experiments described
in the Examples Section was made internally using publically
available information.
[0072] The ADCs of the present invention are conjugated to the
payload in a site specific manner. To accommodate this type of
conjugation, the antibody must be derivatized to provide for either
a reactive cysteine residue engineered at one or more specific
sites, an acyl donor glutamine-containing tag or an endogenous
glutamine made reactive by polypeptide engineering in the presence
of transglutaminase and an amine. Amino acid modifications can be
made by any method known in the art and many such methods are well
known and routine for the skilled artisan. For example, but not by
way of limitation, amino acid substitutions, deletions and
insertions may be accomplished using any well-known PCR-based
technique. Amino acid substitutions may be made by site-directed
mutagenesis (see, for example, Zoller and Smith, 1982, Nucl. Acids
Res. 10:6487-6500; and Kunkel, 1985, PNAS 82:488).
[0073] In applications where retention of antigen binding is
required, such modifications should be at sites that do not disrupt
the antigen binding capability of the antibody. In preferred
embodiments, the one or more modifications are made in the constant
region of the heavy and/or light chains.
[0074] As used herein, the term "constant region" of an antibody
refers to the constant region of the antibody light chain or the
constant region of the antibody heavy chain, either alone or in
combination. The constant regions of the antibodies used to make
the ADCs of the invention may be derived from constant regions of
any one of IgA, IgD, IgE, IgG, IgM, or any isotypes thereof as well
as subclasses and mutated versions thereof.
[0075] The constant domains are not involved directly in binding an
antibody to an antigen, but exhibit various effector functions,
such as Fc receptor (FcR) binding, participation of the antibody in
antibody-dependent cellular toxicity (ADCC), opsonization,
initiation of complement dependent cytotoxicity, and mast cell
degranulation. As known in the art, the term "Fc region" is used to
define a C-terminal region of an immunoglobulin heavy chain. The
"Fc region" may be a native sequence Fc region or a variant Fc
region. Although the boundaries of the Fc region of an
immunoglobulin heavy chain might vary, the human IgG heavy chain Fc
region is usually defined to stretch from an amino acid residue at
position Cys226, or from Pro230, to the carboxyl-terminus thereof.
The numbering of the residues in the Fc region is that of the EU
Index of Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md., 1991). The Fc region of an
immunoglobulin generally has two constant regions, CH2 and CH3.
[0076] There are two different light chains constant regions for
use in antibodies, CL.kappa. and CL.LAMBDA.. CL.kappa. has known
polymorphic loci CL.kappa.-V/A.sup.45 and CL.kappa.-L/V.sup.83
(using the Kabat numbering system as set forth in Kabat et al.
(1991, NIH Publication 91-3242, National Technical Information
Service, Springfield, Va.), so all Kappa and Lambda positions are
numbered according to the Kabat system.) thus allowing for
polymorphisms Km(1): CL.kappa.-V.sup.48/L.sup.83; Km(1,2):
CL.kappa.-A.sup.45/L.sup.83; and Km(3): CL.kappa.-A.sup.48/V.sup.83
Polypeptides, antibodies and ADCs of the invention can have
antibody components with any of these light chain constant
regions.
[0077] For clarity, unless otherwise specified, amino acid residues
in the human IgG heavy constant domain of an antibody are numbered
according the EU index of Edelman et al., 1969, Proc. Natl. Acad.
Sci. USA 63(1):78-85 as described in Kabat et al., 1991, referred
to herein as the "EU index of Kabat". Typically, the Fc domain
comprises from about amino acid residue 236 to about 447 of the
human IgG1 constant domain. Correspondence between C numberings can
be found, e.g., at IGMT database. Amino acid residues of the light
chain constant domain are numbered according to Kabat et al., 1991.
Numbering of antibody constant domain amino acid residues is also
shown in International Patent Publication No. WO 2013/093809. The
only exception to the use of EU index of Kabat in IgG heavy
constant domain is residue A114 described in the examples. A114
refers to Kabat numbering, and the corresponding EU index number is
118. This is because the initial publication of site specific
conjugating at this site used Kabat numbering and referred this
site as A114C, and has since been widely used in the art as the
"114" site. See Junutula et al., Nature Biotechnology 26, 925-932
(2008). To be consistent with the common usage of this site in the
art, "A114," "A114C," "C114" or "114C" are used in the
examples.
[0078] Nucleic acids encoding the heavy and light chains of the
antibodies used to make the ADCs of the invention can be cloned
into a vector for expression or propagation. The sequence encoding
the antibody of interest may be maintained in vector in a host cell
and the host cell can then be expanded and frozen for future
use.
[0079] As used herein, the term "vector" refers to a construct
which is capable of delivering, and preferably, expressing, one or
more gene(s) or sequence(s) of interest in a host cell. Examples of
vectors include, but are not limited to, viral vectors, naked DNA
or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or
RNA expression vectors associated with cationic condensing agents,
DNA or RNA expression vectors encapsulated in liposomes, and
certain eukaryotic cells, such as producer cells.
[0080] As used herein, the term "host cell" includes an individual
cell or cell culture that can be or has been a recipient for
vector(s) for incorporation of polynucleotide inserts. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in genomic
DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a nucleic acids or vectors of this
invention.
[0081] Table 1 provides the amino acid (protein) sequences and
associated nucleic acid (DNA) sequences of humanized HER2
antibodies used in constructing the site specific ADCs of the
invention. The CDRs shown are defined by Kabat numbering
scheme.
[0082] The antibody heavy chains and light chains shown in Table 1
have the trastuzumab heavy chain variable region (V.sub.H) and
light chain variable region (V.sub.L). The heavy chain constant
region and light chain constant region are derivatized from
trastuzumab and contain on or more modification to allow for site
specific conjugation when making the ADCs of the invention.
[0083] Modifications to the amino acid sequences in the antibody
constant region to allow for site specific conjugation are
underlined and bolded. The nomenclature for the antibodies
derivatized from trastuzumab is T (for trastuzumab) and then in
parenthesis the position of the amino acid of modification flanked
by the single letter amino acid code for the wild type residue and
the single letter amino acid code for the residue that is now in
that position in the derivatized antibody. Two exceptions to this
nomenclature are "kK183C" which denotes that position 183 on the
light (kappa) chain has been modified from a lysine to a cysteine
and "LCQ05" which denotes an eight amino acid glutamine-containing
tag that has been attached to the C terminus of the light chain
constant region.
[0084] One of the modifications shown in Table 1 is not used for
conjugation. The residue at position 222 on the heavy chain (using
the EU Index of Kabat numbering scheme) can be altered to result in
a more homogenous antibody and payload conjugate, better
intermolecular crosslinking between the antibody and the payload
and/or significant decrease in interchain crosslinking.
TABLE-US-00001 TABLE 1 Sequences of humanized HER2 antibodies SEQ
ID NO. Description Sequence 1 Trastuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR VH protein
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG
GDGFYAMDYWGQGTLVTVSS 2 VH CDR1 DTYIH protein 3 VH CDR2
RIYPTNGYTRYADSVKG protein 4 VH CDR3 WGGDGFYAMDY protein 5
Trastuzumab ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV
heavy chain HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK
constant SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 6 Trastuzumab
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 7 Trastuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA VL protein
SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT KVEIK 8 VL CDR1
RASQDVNTAVA protein 9 VL CRD2 SASFLYS protein 10 VL CDR3 QQHYTTPPT
protein 11 Trastuzumab
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG light chain
NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS constant FNRGEC
region protein 12 Trastuzumab
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA light chain
SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT protein
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGEC 13
T(K222R) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy
chain HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK 14 T(K222R)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 T(K246C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPCPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 16 T(K246C)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPCP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 17 T(K290C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 18 T(K290C)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 19 T(N297A)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK 20 T(N297A)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 21 T(N297Q)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK 22 T(N297Q)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQ
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 23 T(K334C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 24 T(K334C)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 25 T(K392C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 26 T(K392C)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 27 T(L443C)
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV heavy chain
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK constant
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE region
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE protein
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSCSPG 28 T(L443C)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR heavy chain
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG protein
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPG 29 T(K290C +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV K334C)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 30 T(K290C +
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR K334C)
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy chain
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 31 T(K290C +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV K392C)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTCPREEQYNSTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 32 T(K290C +
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR K392C)
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy chain
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTCPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 33 T(N297A +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV K222R)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
34 T(N297A + EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR
K222R) IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy
chain GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYA
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 35 T(N297Q +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV K222R)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK 36 T(N297Q +
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR K222R)
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy chain
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQ
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 37 T(K334C +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV K392C)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIECTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPG 38 T(K334C +
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR K392C)
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy chain
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIECTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 39 T(K392C +
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV L443C)
HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK heavy chain
SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE constant
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE region
YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV protein
KGFYPSDIAVEWESNGQPENNYCTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSCSPG 40 T(K392C +
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR L443C)
IYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWG heavy chain
GDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV protein
KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ
TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYCTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSCSPG 41 T(kK183C)
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG light chain
NSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQGLSSPVTK constant SFNRGEC
region protein 42 T(kK183C)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA light chain
SFLYSGVPSRFSGSRSGTFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTK protein
VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL
QSGNSQESVTEQDSKDSTYSLSSTLTLSCADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 43
T(LCQ05) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG light
chain NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS constant
FNRGECGGLLQGPP region protein 44 T(LCQ05)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSA light chain
SFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGT protein
KVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA
LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS
PVTKSFNRGECGGLLQGPP 45 Trastuzumab
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG VH DNA
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGC 46 Trastuzumab
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 47
Trastuzumab GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCCGCCTCTGTGGGC VL DNA
GACAGAGTGACCATCACCTGTCGGGCCTCCCAGGACGTGAACACCGC
CGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA
TCTACTCCGCCTCCTTCCTGTACTCCGGCGTGCCCTCCCGGTTCTCCG
GCTCCAGATCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGCAGC
CCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCC 48 Trastuzumab
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCCGCCTCTGTGGGC light chain
GACAGAGTGACCATCACCTGTCGGGCCTCCCAGGACGTGAACACCGC DNA
CGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA
TCTACTCCGCCTCCTTCCTGTACTCCGGCGTGCCCTCCCGGTTCTCCG
GCTCCAGATCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGCAGC
CCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCC
CCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCC
GCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCC
GGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAG
GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTC
CCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCT
GTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGT
GTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCA
AGTCCTTCAACCGGGGCGAGTGC 49 T(K222R)
GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy chain
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region DNA
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACCGTACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGAAAA 50 T(K222R)
GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTTCAGCCCGGCG heavy chain
GATCACTGAGGCTCTCCTGTGCCGCCAGCGGCTTCAACATCAAGGACA DNA
CATACATCCACTGGGTTCGCCAGGCTCCTGGCAAGGGACTGGAGTGG
GTCGCTAGGATCTACCCCACCAATGGGTACACCAGGTACGCCGACTCC
GTGAAGGGGCGGTTCACAATCTCAGCCGATACTAGCAAAAATACAGCC
TACTTGCAGATGAACTCCCTGAGAGCAGAGGATACCGCCGTGTACTATT
GCTCTCGCTGGGGCGGCGACGGCTTCTACGCTATGGATTATTGGGGCC
AGGGAACCTTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACCGTACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGAAAA 51
T(K246C) GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region DNA
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CATGCCCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 52 T(K246C)
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCATGCCCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 53
T(K290C) GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region DNA
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACATGCCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 54 T(K290C)
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACATGCCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 55
T(N297A) GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region DNA
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT
CCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT
TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGAAAA 56 T(N297A)
GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTTCAGCCCGGCG heavy chain
GATCACTGAGGCTCTCCTGTGCCGCCAGCGGCTTCAACATCAAGGACA DNA
CATACATCCACTGGGTTCGCCAGGCTCCTGGCAAGGGACTGGAGTGG
GTCGCTAGGATCTACCCCACCAATGGGTACACCAGGTACGCCGACTCC
GTGAAGGGGCGGTTCACAATCTCAGCCGATACTAGCAAAAATACAGCC
TACTTGCAGATGAACTCCCTGAGAGCAGAGGATACCGCCGTGTACTATT
GCTCTCGCTGGGGCGGCGACGGCTTCTACGCTATGGATTATTGGGGCC
AGGGAACCTTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGAAAA 57
T(N297Q) GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT DNA
CCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGAAAAGCCGCCAGCGGCTT
CAACATCAAGGACACATACATCCACTGGGTTCGCCAGGCTCCTGGCAA GGG 58 T(N297Q)
GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTTCAGCCCGGCG heavy chain
GATCACTGAGGCTCTCCTGTGCCGCCAGCGGCTTCAACATCAAGGACA DNA
CATACATCCACTGGGTTCGCCAGGCTCCTGGCAAGGGACTGGAGTGG
GTCGCTAGGATCTACCCCACCAATGGGTACACCAGGTACGCCGACTCC
GTGAAGGGGCGGTTCACAATCTCAGCCGATACTAGCAAAAATACAGCC
TACTTGCAGATGAACTCCCTGAGAGCAGAGGATACCGCCGTGTACTATT
GCTCTCGCTGGGGCGGCGACGGCTTCTACGCTATGGATTATTGGGGCC
AGGGAACCTTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAAAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT
GTCTCCGGGAAAAGCCGCCAGCGGCTTCAACATCAAGGACACATACAT
CCACTGGGTTCGCCAGGCTCCTGGCAAGGG 59 T(K334C)
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy chain
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT DNA
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGTGCACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA 60 T(K334C)
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGTGCACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 61
T(K392C) GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT DNA
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACTGCACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 62 T(K392C)
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCTTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACTGCACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 63
T(L443C) GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG heavy
chain AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA constant
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT DNA
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCTGCTCCCCGGGT 64 T(L443C)
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG heavy chain
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC DNA
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC TGCTCCCCGGGT 65
T(K290C + GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG K334C)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA constant
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT region
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACATGCCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGTGCACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 66 T(K290C +
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG K334C)
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC heavy chain
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT DNA
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACATGCCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGTGCACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 67
T(K290C + GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG K392C)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA constant
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT region
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACATGCCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACTGCACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 68 T(K290C +
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG K392C)
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC heavy chain
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT DNA
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACATGCCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACTGCACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 69
T(N297A + GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG K222R)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA constant
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT region
CCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACCGTACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACGCCAGCACGTACCGTGTGGTCAGCGTCCTCACCGT
CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAA
AGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCT
TCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCC
TTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG
GGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC
TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGAAAA 70 T(N297A +
GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTTCAGCCCGGCG K222R)
GATCACTGAGGCTCTCCTGTGCCGCCAGCGGCTTCAACATCAAGGACA heavy chain
CATACATCCACTGGGTTCGCCAGGCTCCTGGCAAGGGACTGGAGTGG DNA
GTCGCTAGGATCTACCCCACCAATGGGTACACCAGGTACGCCGACTCC
GTGAAGGGGCGGTTCACAATCTCAGCCGATACTAGCAAAAATACAGCC
TACTTGCAGATGAACTCCCTGAGAGCAGAGGATACCGCCGTGTACTATT
GCTCTCGCTGGGGCGGCGACGGCTTCTACGCTATGGATTATTGGGGCC
AGGGAACCTTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACCGTACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACGCCAGCACGT
ACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATG
GCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCA
TCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG
TGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT GTCTCCGGGAAAA 71
T(N297Q + GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG K222R)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA constant
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT region
CCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACCGTACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACCAAAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCTCCGGGAAAAGCCGCCAGCGGCTT
CAACATCAAGGACACATACATCCACTGGGTTCGCCAGGCTCCTGGCAA GGG 72 T(N297Q +
GAGGTGCAGCTGGTGGAGTCCGGCGGCGGCCTGGTTCAGCCCGGCG K222R)
GATCACTGAGGCTCTCCTGTGCCGCCAGCGGCTTCAACATCAAGGACA heavy chain
CATACATCCACTGGGTTCGCCAGGCTCCTGGCAAGGGACTGGAGTGG DNA
GTCGCTAGGATCTACCCCACCAATGGGTACACCAGGTACGCCGACTCC
GTGAAGGGGCGGTTCACAATCTCAGCCGATACTAGCAAAAATACAGCC
TACTTGCAGATGAACTCCCTGAGAGCAGAGGATACCGCCGTGTACTATT
GCTCTCGCTGGGGCGGCGACGGCTTCTACGCTATGGATTATTGGGGCC
AGGGAACCTTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGG
TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG
CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT
CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCT
GTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTAGTGACCGTG
CCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCAC
AAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGT
GACCGTACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATG
ATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCA
CGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGT
GCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAAAGCACGTA
CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGG
CAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCAT
CGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT
GTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCA
GCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG
AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCT
CCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACC
GTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGT
GATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT
GTCTCCGGGAAAAGCCGCCAGCGGCTTCAACATCAAGGACACATACAT
CCACTGGGTTCGCCAGGCTCCTGGCAAGGG 73 T(K334C +
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG K39C2)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA region
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT constant
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGTGCACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACTGCACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCCTGTCCCCGGGT 74 T(K334C +
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG K392C)
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC heavy chain
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT DNA
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGTGCACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACTGCACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCCCCGGGT 75
T(K392C + GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG L443C)
AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTA heavy chain
CTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA constant
GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACT region
CCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAG DNA
ACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGAC
AAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACAT
GCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT
GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGG
GAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC
CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC
AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGA
GGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGG
AGAACAACTACTGCACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT
TCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG
GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA
CACGCAGAAGAGCCTCTCCTGCTCCCCGGGT 76 T(K392C +
GAGGTGCAGCTGGTGGAATCCGGCGGAGGCCTGGTCCAGCCTGGCGG L443C)
ATCTCTGCGGCTGTCTTGCGCCGCCTCCGGCTTCAACATCAAGGACAC heavy chain
CTACATCCACTGGGTCCGACAGGCACCTGGCAAGGGACTGGAATGGGT DNA
GGCCCGGATCTACCCCACCAACGGCTACACCAGATACGCCGACTCCGT
GAAGGGCCGGTTCACCATCTCCGCCGACACCTCCAAGAACACCGCCTA
CCTGCAGATGAACTCCCTGCGGGCCGAGGACACCGCCGTGTACTACTG
CTCCAGATGGGGAGGCGACGGCTTCTACGCCATGGACTACTGGGGCC
AGGGCACCCTGGTCACCGTGTCTAGCGCGTCGACCAAGGGCCCATCG
GTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGT
GTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG
CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCG
TGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATC
ACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTT
GTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG
GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA
TGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGC
CACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAG
GTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCAC
GTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAA
TGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA
GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGG
TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCG
TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACTGCACCACG
CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCA
CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC TGCTCCCCGGGT 77
T(kK183C) CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAG light
chain CAGCTGAAGTCCGGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTC constant
TACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCA region
GTCCGGCAACTCCCAGGAATCCGTCACCGAGCAGGACTCCAAGGACA DNA
GCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCTGCGCCGACTACG
AGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCA
GCCCCGTGACCAAGTCCTTCAACCGGGGCGAGTGC 78 T(kK183C)
GACATCCAGATGACCCAGTCCCCCTCCAGCCTGTCCGCCTCTGTGGGC light chain
GACAGAGTGACCATCACCTGTCGGGCCTCCCAGGACGTGAACACCGC DNA
CGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGA
TCTACTCCGCCTCCTTCCTGTACTCCGGCGTGCCCTCCCGGTTCTCCG
GCTCCAGATCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGCAGC
CCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCC
CCACCTTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACCGTGGCC
GCTCCCTCCGTGTTCATCTTCCCACCCTCCGACGAGCAGCTGAAGTCC
GGCACCGCCTCCGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAG
GCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTC
CCAGGAATCCGTCACCGAGCAGGACTCCAAGGACAGCACCTACTCCCT
GTCCTCCACCCTGACCCTGTCCTGCGCCGACTACGAGAAGCACAAGGT
GTACGCCTGCGAAGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCA
AGTCCTTCAACCGGGGCGAGTGC 79 T(LCQ05)
CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGC light chain
AGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT constant
CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG region
GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCAC DNA
CTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCC
CGTCACAAAGAGCTTCAACAGGGGAGAGTGT GGTGGCCTGCTTCAGGGCCCACCA 80
T(LCQ05) GATATCCAGATGACACAGTCCCCCTCCAGCCTCTCCGCTAGTGTCGGA light
chain GATAGAGTGACAATTACATGTCGGGCAAGCCAGGACGTCAATACCGCC DNA
GTGGCCTGGTATCAGCAGAAGCCAGGAAAGGCCCCAAAACTCCTGATC
TACTCCGCCTCCTTCCTGTACTCAGGGGTCCCTTCACGCTTCTCCGGTT
CCCGGAGCGGCACCGACTTCACTCTGACTATCTCAAGCTTGCAGCCCG
AGGACTTCGCCACATACTATTGCCAGCAGCACTATACCACCCCCCCTAC
CTTCGGTCAGGGAACTAAGGTGGAAATTAAACGTACGGTGGCTGCACC
ATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT
GCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAG
TACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGA
GTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCA
CCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT
GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCA
ACAGGGGAGAGTGTGGTGGCCTGCTTCAGGGCCCACCA
[0085] In some embodiments, the ADCs of the invention can use
antibodies comprising heavy chain variable region CDRs and light
chain variable region CDRs of trastuzumab (V.sub.H CDRs of SEQ ID
NOs:2-4 and V.sub.L CDRs of SEQ ID NOs:8-10) and any combination of
heavy and light chain constant regions disclosed in Table 1 with
the proviso that when the heavy chain constant region is SEQ ID
NO:5 then the light chain constant region is not SEQ ID NO:11 (due
to the fact that this combination recreates wild type trastuzumab
and would thus not allow for site specific conjugation). In such
embodiments, the heavy chain constant region can be selected from
any of SEQ ID NOs:17, 5, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37 or
39 while the light chain constant region can be selected from any
of SEQ ID NOs:41, 11 or 43 providing that the combination is not
SEQ ID NO:5 and SEQ ID NO:11 as discussed supra.
[0086] In more specific embodiments, the ADCs of the invention can
use antibodies comprising heavy chain variable region CDRs and
light chain variable region CDRs of trastuzumab (V.sub.H CDRs of
SEQ ID NOs:2-4 and V.sub.L CDRs of SEQ ID NOs:8-10) and a heavy and
light chain constant region combination selected from:
(a) a heavy chain constant region of SEQ ID NO:17 and a light chain
constant region of SEQ ID NO:41; (b) a heavy chain constant region
of SEQ ID NO:5 and a light chain constant region of SEQ ID NO:41;
(c) a heavy chain constant region of SEQ ID NO:17 and a light chain
constant region of SEQ ID NO:11; (d) a heavy chain constant region
of SEQ ID NO:21 and a light chain constant region of SEQ ID NO:11;
(e) a heavy chain constant region of SEQ ID NO:23 and a light chain
constant region of SEQ ID NO:11; (f) a heavy chain constant region
of SEQ ID NO:25 and a light chain constant region of SEQ ID NO:11;
(g) a heavy chain constant region of SEQ ID NO:27 and a light chain
constant region of SEQ ID NO:11; (h) a heavy chain constant region
of SEQ ID NO:23 and a light chain constant region of SEQ ID NO:41;
(i) a heavy chain constant region of SEQ ID NO:25 and a light chain
constant region of SEQ ID NO:41; (j) a heavy chain constant region
of SEQ ID NO:27 and a light chain constant region of SEQ ID NO:41;
(k) a heavy chain constant region of SEQ ID NO:29 and a light chain
constant region of SEQ ID NO:11; (l) a heavy chain constant region
of SEQ ID NO:31 and a light chain constant region of SEQ ID NO:11;
(m) a heavy chain constant region of SEQ ID NO:33 and a light chain
constant region of SEQ ID NO:43; (n) a heavy chain constant region
of SEQ ID NO:35 and a light chain constant region of SEQ ID NO:11;
(o) a heavy chain constant region of SEQ ID NO:37 and a light chain
constant region of SEQ ID NO:11; (p) a heavy chain constant region
of SEQ ID NO:39 and a light chain constant region of SEQ ID NO:11;
or (q) a heavy chain constant region of SEQ ID NO:13 and a light
chain constant region of SEQ ID NO:43.
[0087] In yet a more specific embodiment, an ADC of the invention
comprises an antibody with V.sub.H CDRs of SEQ ID NOs:2-4 and
V.sub.L CDRs of SEQ ID NOs:8-10 and a heavy chain constant region
of SEQ ID NO:17 and a light chain constant region of SEQ ID
NO:41.
[0088] In another more specific embodiment, an ADC of the invention
comprises an antibody with V.sub.H CDRs of SEQ ID NOs:2-4 and
V.sub.L CDRs of SEQ ID NOs:8-10 and a heavy chain constant region
of SEQ ID NO:13 and a light chain constant region of SEQ ID
NO:43.
[0089] In other embodiments, the ADCs of the invention can use
antibodies comprising any combination of heavy and light chains
disclosed in Table 1 with the proviso that if the heavy chain is
SEQ ID NO:6 then the light chain is not SEQ ID NO:12 (due to the
fact that this combination recreates wild type trastuzumab and
would thus not allow for site specific conjugation). In such
embodiments, the heavy chain can be selected from any of SEQ ID
NOs:18, 6, 14, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 while the
light chain can be selected from any of SEQ ID NOs: 42, 12 or 44
providing that the combination is not SEQ ID NO:6 and SEQ ID NO:12
as discussed supra.
[0090] In more specific embodiments, the ADCs of the invention can
use antibodies comprising a heavy chain and light chain combination
selected from:
(a) a heavy chain of SEQ ID NO:18 and a light chain of SEQ ID
NO:42; (b) a heavy chain of SEQ ID NO:6 and a light chain of SEQ ID
NO:42; (c) a heavy chain of SEQ ID NO:18 and a light chain of SEQ
ID NO:12; (d) a heavy chain of SEQ ID NO:22 and a light chain of
SEQ ID NO:12; (e) a heavy chain of SEQ ID NO:24 and a light chain
of SEQ ID NO:12; (f) a heavy chain of SEQ ID NO:26 and a light
chain of SEQ ID NO:12; (g) a heavy chain of SEQ ID NO:28 and a
light chain of SEQ ID NO:12; (h) a heavy chain of SEQ ID NO:24 and
a light chain of SEQ ID NO:42; (i) a heavy chain of SEQ ID NO:26
and a light chain of SEQ ID NO:42; (j) a heavy chain of SEQ ID
NO:28 and a light chain of SEQ ID NO:42; (k) a heavy chain of SEQ
ID NO:30 and a light chain of SEQ ID NO:12; (l) a heavy chain of
SEQ ID NO:32 and a light chain of SEQ ID NO:12; (m) a heavy chain
of SEQ ID NO:34 and a light chain of SEQ ID NO:44; (n) a heavy
chain of SEQ ID NO:36 and a light chain of SEQ ID NO:12; (o) a
heavy chain of SEQ ID NO:38 and a light chain of SEQ ID NO:12; (p)
a heavy chain of SEQ ID NO:40 and a light chain of SEQ ID NO:12; or
(q) a heavy chain of SEQ ID NO:14 and a light chain of SEQ ID
NO:44.
[0091] In yet a more specific embodiment, an ADC of the invention
comprises an antibody with a heavy chain of SEQ ID NO:18 and a
light chain of SEQ ID NO:42. Plasmids containing nucleic acids
encoding the heavy chain of SEQ ID NO:18 and the light chain of SEQ
ID NO:42 have been deposited with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209
on Nov. 17, 2015 and given Accession Nos. PTA-122672 and
PTA-122673, respectively. The deposits were made under the
provisions of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purpose of Patent
Procedure and Regulations thereunder (Budapest Treaty). This
assures maintenance of a viable culture of the deposit for 30 years
from the date of deposit. The deposit will be made available by
ATCC under the terms of the Budapest Treaty, and subject to an
agreement between Pfizer Inc. and ATCC, which assures permanent and
unrestricted availability of the progeny of the culture of the
deposit to the public upon issuance of the pertinent U.S. patent or
upon laying open to the public of any U.S. or foreign patent
application, whichever comes first, and assures availability of the
progeny to one determined by the U.S. Commissioner of Patents and
Trademarks to be entitled thereto according to 35 U.S.C. Section
122 and the Commissioner's rules pursuant thereto (including 37
C.F.R. Section 1.14 with particular reference to 886 OG 638).
[0092] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0093] In another more specific embodiment, an ADC of the invention
comprises an antibody with a heavy chain of SEQ ID NO:14 and a
light chain of SEQ ID NO:44.
[0094] In some aspects of the invention, the ADC of the invention
includes an antibody having a heavy chain and/or a light chain
comprising an amino acid sequence that is at least 90%, 95%, 98%,
or 99% identical to any of the heavy or light chains disclosed
supra. Residues that have been altered can be in the variable
region or in the constant region of the antibody. In some
embodiments, there are no more than 1, 2, 3, 4 or 5 residues that
have been altered as compared to any of the heavy or light chains
disclosed supra. In other embodiments, there are no altered
residues in any of the variable region CDRs.
[0095] The term "percent identical" (or "% identical") in the
context of amino acid sequences means the number of residues in two
sequences that are the same when aligned for maximum
correspondence. There are a number of different algorithms known in
the art which can be used to measure amino acid percent identity
(i.e., the Basic Local Alignment Tool or BLAST.RTM.). Unless
otherwise specified, default parameters for a particular program or
algorithm are used.
[0096] For use in preparation of ADCs, HER2 antibodies described
herein may be substantially pure, i.e., at least 50% pure (i.e.,
free from contaminants), more preferably, at least 90% pure, more
preferably, at least 95% pure, yet more preferably, at least 98%
pure, and most preferably, at least 99% pure.
II. Drugs
[0097] Drugs useful in preparation of the site specific HER2 ADCs
of the invention include any therapeutic agent useful in the
treatment of cancer including, but not limited to, cytotoxic
agents, cytostatic agents, immunomodulating agents and
chemotherapeutic agents. A cytotoxic effect refers to the
depletion, elimination and/or the killing of a target cell (i.e.,
tumor cell). A cytotoxic agent refers to an agent that has a
cytotoxic effect on a cell. A cytostatic effect refers to the
inhibition of cell proliferation. A cytostatic agent refers to an
agent that has a cytostatic effect on a cell, thereby inhibiting
the growth and/or expansion of a specific subset of cells (i.e.,
tumor cells). An immunomodulating agent refers to an agent that
stimulates the immune response through the production of cytokines
and/or antibodies and/or modulating T cell function thereby
inhibiting or reducing the growth of a subset of cells (i.e., tumor
cells) either directly or indirectly by allowing another agent to
be more efficacious. A chemotherapeutic agent refers to an agent
that is a chemical compound useful in the treatment of cancer. A
drug may also be a drug derivative, wherein a drug has been
functionalized to enable conjugation with an antibody of the
invention.
[0098] In some embodiments the drug is a membrane permeable drug.
In such embodiments, the payload (i.e. drug) can elicit a bystander
effect wherein cells surrounding the cell that initially
internalized the ADC are killed by the payload. This occurs when
the payload is released from the antibody (i.e., by cleaving of a
cleavable linker) and crosses the cellular membrane and, upon
diffusion, induces the killing of surrounding cells.
[0099] In accordance with the disclosed methods, the drugs are used
to prepare antibody drug conjugates of the formula Ab-(L-D),
wherein (a) Ab is an antibody that binds to HER2; and (b) L-D is a
linker-drug moiety, wherein L is a linker, and D is a drug.
[0100] The drug-to-antibody ratio (DAR) or drug loading indicates
the number of drug (D) molecules that are conjugated per antibody.
The antibody drug conjugates of the present invention use site
specific conjugation such that there is essentially a homogeneous
population of ADCs having one DAR in a composition of ADCs. In some
embodiments, the DAR is 1. In some embodiments, the DAR is 2. In
other embodiments, the DAR is 3. In other embodiments, the DAR is
4. In other embodiments, the DAR is greater than 4.
[0101] Using conventional conjugation (rather than site specific
conjugation) results in a heterogeneous population of different
species of ADCs, each of which has a different individual DAR.
Compositions of ADCs prepared in this way include a plurality of
antibodies, each antibody conjugated to a particular number of drug
molecules. As such, the compositions have an average DAR. T-DM1
(Kadcyla.RTM.) uses conventional conjugation on lysine residues and
has an average DAR of around 4 with a broad distribution which
includes ADCs loaded with 0, 1, 2, 3, 4, 5, 6, 7 or 8 drug
molecules (Kim et al., 2014, Bioconj Chem 25(7):1223-32).
[0102] Compositions, batches, and/or formulations of a plurality of
ADCs may be characterized by an average DAR. DAR and average DAR
can be determined by various conventional means such as UV
spectroscopy, mass spectroscopy, ELISA assay, radiometric methods,
hydrophobic interaction chromatography (HIC), electrophoresis and
HPLC.
[0103] In aspects of the invention, an HER2 ADC may have a DAR of
1, a DAR of 2, a DAR of 3, a DAR of 4, a DAR of 5, a DAR of 6, a
DAR of 7, a DAR of 8, a DAR of 9, a DAR of 10, a DAR of 11, a DAR
of 12 or a DAR greater than 12. In aspects of the invention, an
HER2 ADC may have one drug molecule, or 2 drug molecules, or 3 drug
molecules, or 4 drug molecules, or 5 drug molecules, or 6 drug
molecules, or 7 drug molecules, or 8 drug molecules, or 9 drug
molecules, or 10 drug molecules, or 11 drug molecules, or 12 drug
molecules or greater than 12 molecules.
[0104] In aspects of the invention, an HER2 ADC may have average
DAR in the range of about 2 to about 4, or an average DAR in the
range of about 3 to about 5, or an average DAR in the range of
about 4 to about 6, or an average DAR in the range of about 5 to
about 7, or an average DAR in the range of about 6 to about 8, or
an average DAR in the range of about 7 to about 9, or an average
DAR in the range of about 8 to about 10, or an average DAR in the
range of about 9 to about 11, or an average DAR in the range of
about 10 to about 12, etc. In some aspects the compositions,
batches and/or formulations of HER2 ADCs may have an average DAR of
about 1, or an average DAR of about 2, an average DAR of about 3,
or an average DAR of about 4, or an average DAR of about 5, or an
average DAR of about 6, or an average DAR of about 7, or an average
DAR of about 8, or an average DAR of about 9, or an average DAR of
about 10, or an average DAR of about 11, or an average DAR of about
12 or an average DAR greater than 12. As used in the foregoing
ranges of average DAR, the term "about" means +/-0.5%.
[0105] A composition, batch, and/or formulation of HER2 ADCs may be
characterized by a preferred range of average DAR, e.g., an average
DAR in the range of about 3 to about 5, an average DAR in the range
of about 3 to about 4, or an average DAR in the range of about 4 to
about 5. Further, a composition, batch, and/or formulation of HER2
ADCs may be characterized by a preferred range of average DAR,
e.g., an average DAR in the range of 3 to 5, an average DAR in the
range of 3 to 4, or an average DAR in the range of 4 to 5.
[0106] In some aspects of the invention, a composition, batch,
and/or formulation of HER2 ADCs may be characterized by an average
DAR of about 1.0, or an average DAR of 1.0, or an average DAR of
1.1, or an average DAR of 1.2, or an average DAR of 1.3, or an
average DAR of 1.4, or an average DAR of 1.5, or an average DAR of
1.6, or an average DAR of 1.7, or an average DAR of 1.8, or an
average DAR of 1.9. In another aspect, a composition, batch, and/or
formulation of HER2 ADCs may be characterized by an average DAR of
about 2.0, or an average DAR of 2.0, or an average DAR of 2.1, or
an average DAR of 2.2, or an average DAR of 2.3, or an average DAR
of 2.4, or an average DAR of 2.5, or an average DAR of 2.6, or an
average DAR of 2.7, or an average DAR of 2.8, or an average DAR of
2.9. In another aspect, a composition, batch, and/or formulation of
HER2 ADCs may be characterized by an average DAR of about 3.0, or
an average DAR of 3.0, or an average DAR of 3.1, or an average DAR
of 3.2, or an average DAR of 3.3, or an average DAR of 3.4, or an
average DAR of 3.5, or an average DAR of 3.6, or an average DAR of
3.7, or an average DAR of 3.8, or an average DAR of 3.9. In another
aspect, a composition, batch, and/or formulation of HER2 ADCs may
be characterized by an average DAR of about 4.0, or an average DAR
of 4.0, or an average DAR of 4.1, or an average DAR of 4.2, or an
average DAR of 4.3, or an average DAR of 4.4, or an average DAR of
4.5, or an average DAR of 4.6, or an average DAR of 4.7, or an
average DAR of 4.8, or an average DAR of 4.9, or an average DAR of
5.0.
[0107] In another aspect, a composition, batch, and/or formulation
of HER2 ADCs may be characterized by an average DAR of 12 or less,
an average DAR of 11 or less, an average DAR of 10 or less, an
average DAR of 9 or less, an average DAR of 8 or less, an average
DAR of 7 or less, an average DAR of 6 or less, an average DAR of 5
or less, an average DAR of 4 or less, an average DAR of 3 or less,
an average DAR of 2 or less or an average DAR of 1 or less.
[0108] In other aspects, a composition, batch, and/or formulation
of HER2 ADCs may be characterized by an average DAR of 11.5 or
less, an average DAR of 10.5 or less, an average DAR of 9.5 or
less, an average DAR of 8.5 or less, an average DAR of 7.5 or less,
an average DAR of 6.5 or less, an average DAR of 5.5 or less, an
average DAR of 4.5 or less, an average DAR of 3.5 or less, an
average DAR of 2.5 or less, an average DAR of 1.5 or less.
[0109] In some aspects of the present invention, the methods for
conventional conjugation via cysteine residues and purification
conditions disclosed herein provide a composition, batch, and/or
formulation of HER2 ADCs with an optimized average DAR in the range
of about 3 to 5, preferably about 4.
[0110] In some aspects of the present invention, the methods for
site-specific conjugation via engineered cysteine residues and
purification conditions disclosed herein provide a composition,
batch, and/or formulation of HER2 ADCs with an optimized average
DAR in the range of about 3 to 5, preferably about 4.
[0111] In some aspects of the present invention, the methods for
site-specific conjugation via transglutaminase-based conjugation
and purification conditions disclosed herein provide a composition,
batch, and/or formulation of HER2 ADCs with an optimized average
DAR in the range of about 1 to 3, preferably about 2.
[0112] Also encompassed by the present invention are antibody drug
conjugates of the formula Ab-(L-D)p, wherein (a) Ab is an antibody,
or antigen-binding fragment thereof, that binds to HER2, (b) L-D is
a linker-drug moiety, wherein L is a linker, and D is a drug and
(c) p is the number of linker/drug moieties that are attached to
the antibody. For site specific ADCs, p is a whole number due to
the homogeneous nature of the ADC. In some embodiments, p is 4. In
other embodiments, p is 3. In other embodiments, p is 2. In other
embodiments, p is 1. In other embodiments, p is greater than 4.
[0113] In one embodiment, the drug component of the ADCs of the
invention is an anti-mitotic drug. In a specific embodiment, the
anti-mitotic drug is an auristatin (e.g., 0101, 8261, 6121, 8254,
6780 and 0131; see Table 2 infra). In a more specific embodiment,
the auristatin drug is
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrroli-
din-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (also
known as 0101).
[0114] Auristatins inhibit cell proliferation by inhibiting the
formation of microtubules during mitosis through inhibition of
tubulin polymerization. PCT International Publication No. WO
2013/072813, which is incorporated by reference in its entirety,
discloses auristatins that are useful in the manufacture of the
ADCs of the invention and provides methods of producing those
auristatins.
TABLE-US-00002 TABLE 2 Drugs Name Structure IUPAC Name 0101
##STR00001## 2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-
1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-
oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-
yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-
methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide 8261 ##STR00002##
2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-
[(1R,2R)-3-{[(1S)-1-carboxy-2-
phenylethyl]amino}-1-methoxy-2-methyl-
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide 6121 ##STR00003##
2-methyl-L-prolyl-N-[(3R,4S,5S)-3-
methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-
{[(2S)-1-methoxy-1-oxo-3-phenylpropan- 2-yl]amino}-2-methyl-3-
oxopropyl]pyrrolidin-1-yl}-5-methyl-1-
oxoheptan-4-yl]-N-methyl-L-valinamide 8254 ##STR00004##
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-
1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-
methoxy-1-oxo-3-phenylpropan-2-
yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-
1-yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide 6780
##STR00005## 2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-
[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-
phenylpropan-2-yl]amino}-1-methoxy-2-
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-
methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide 0131
##STR00006## 2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-
2-[(1R,2R)-3-{[(1S)-1-carboxy-2-
phenylethyl]amino}-1-methoxy-2-methyl-
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide MMAD ##STR00007##
N-methyl-L-valyl-N-[(3R,4S,5S)-3-
methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-
methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-
thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-
1-yl}-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide MMAE
##STR00008## N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-
[(1R,2R)-3-{[(1S,2R)-1-hydroxy-1-
phenylpropan-2-yl]amino}-1-methoxy-2-
methyl-3-oxopropyl]pyrrolidin-1-yl}-3-
methoxy-5-methyl-1-oxoheptan-4-yl]-N- methyl-L-valinamide MMAF
##STR00009## N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-
[(1R,2R)-3-{[(1S)-1-carboxy-2-
phenylethyl]amino}-1-methoxy-2-methyl-
3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-
methyl-1-oxoheptan-4-yl]-N-methyl-L- valinamide
[0115] In some aspects of the invention, the cytotoxic agent can be
made using a liposome or biocompatible polymer. The HER2 antibodies
as described herein can be conjugated to the biocompatible polymer
to increase serum half-life and bioactivity, and/or to extend in
vivo half-lives. Examples of biocompatible polymers include
water-soluble polymers, such as polyethylene glycol (PEG) or
derivatives thereof and zwitterion-containing biocompatible
polymers (e.g., a phosphorylcholine containing polymer).
III. Linkers
[0116] Site specific HER2 ADCs of the invention are prepared using
a linker to link or conjugate a drug to an HER2 antibody. A linker
is a bifunctional compound which can be used to link a drug and an
antibody to form an antibody drug conjugate (ADC). Such conjugates
allow the selective delivery of drugs to tumor cells. Suitable
linkers include, for example, cleavable and non-cleavable linkers.
A cleavable linker is typically susceptible to cleavage under
intracellular conditions. Major mechanisms by which a conjugated
drug is cleaved from an antibody include hydrolysis in the acidic
pH of the lysosomes (hydrazones, acetals, and cis-aconitate-like
amides), peptide cleavage by lysosomal enzymes (the cathepsins and
other lysosomal enzymes), and reduction of disulfides. As a result
of these varying mechanisms for cleavage, mechanisms of linking the
drug to the antibody also vary widely and any suitable linker can
be used.
[0117] Suitable cleavable linkers include, but are not limited to,
a peptide linker cleavable by an intracellular protease, such as
lysosomal protease or an endosomal protease such as
maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc),
N.about.2.about.-acetyl-L-lysyl-L-valyl-L-citruline-p-aminobenzyloxycarbo-
nyl-N,N'-dimethylaminoethyl-CO-(AcLysvc) and m(H20)c-vc (Table 3
infra). In specific embodiments, the linker is a cleavable linker
such that the payload can induce a bystander effect once the linker
is cleaved. The bystander effect is when a membrane permeable drug
is released from the antibody (i.e., by cleaving of a cleavable
linker) and crosses the cellular membrane and, upon diffusion,
induces killing of cells surrounding the cell that initially
internalized the ADC.
[0118] Suitable non-cleavable linkers include, but are not limited
to, maleimidocaproyl (mc), maleimide-(polyethylene glycol).sub.6
(MalPeg6), Mal-PEG2C2, Mal-PEG3C2 and m(H20)c (Table 3 infra).
[0119] Other suitable linkers include linkers hydrolyzable at a
specific pH or a pH range, such as a hydrazone linker. Additional
suitable cleavable linkers include disulfide linkers. The linker
may be covalently bound to the antibody to such an extent that the
antibody must be degraded intracellularly in order for the drug to
be released e.g. the mc linker and the like.
[0120] In particular aspects of the invention, the linkers in the
site specific HER2 ADCs of the invention are cleavable and may be
vc or AcLysvc.
[0121] Many of the therapeutic agents (drugs) conjugated to
antibodies have little, if any, solubility in water and that can
limit drug loading on the conjugate due to aggregation of the
conjugate. One approach to overcoming this is to add solublizing
groups to the linker. Conjugates made with a linker consisting of
PEG and a dipeptide can been used, including those having a PEG
di-acid, thiol-acid, or maleimide-acid attached to the antibody, a
dipeptide spacer, and an amide bond to the amine of an
anthracycline or a duocarmycin analogue. Another example is a
conjugate prepared with a PEG-containing linker disulfide bonded to
a cytotoxic agent and amide bonded to an antibody. Approaches that
incorporate PEG groups may be beneficial in overcoming aggregation
and limits in drug loading.
TABLE-US-00003 TABLE 3 Linkers Name Structure vc ##STR00010##
AcLysvc ##STR00011## mc ##STR00012## MalPeg6 ##STR00013## m(H20)c
##STR00014## m(H20)c- vc ##STR00015## ##STR00016##
[0122] Linkers are attached to the monoclonal antibody via the left
side of the molecule and the drug via the right side of the
molecule as depicted in Table 3.
IV. Methods of Preparing Site Specific HER2 ADCs
[0123] Also provided are methods for preparing antibody drug
conjugates of the present invention. For example, a process for
producing a site specific HER2 ADC as disclosed herein can include
(a) linking the linker to the drug; (b) conjugating the linker-drug
moiety to the antibody; and (c) purifying the antibody drug
conjugate.
[0124] The HER2 ADCs of the present invention use site specific
methods to conjugate the HER2 antibody to the drug payload.
[0125] In one embodiment, the site specific conjugation occurs
through one or more cysteine residues that have been engineered
into an antibody constant region. Methods of preparing HER2
antibodies for site specific conjugation through cysteine residues
can be performed as described in PCT Publication No. WO2013/093809,
which is incorporated by reference in its entirety. One or more of
the following positions (using EU Index of Kabat numbering for the
IgG1 constant region and Kabat numbering for the Kappa chain
constant region) can be altered to be a cysteine and thus serve as
a site for conjugation: a) on the heavy chain constant region,
residues 114, 246, 249, 265, 267, 270, 276, 278, 283, 290, 292,
293, 294, 300, 302, 303, 314, 315, 318, 320, 327, 332, 333, 334,
336, 345, 347, 354, 355, 358, 360, 362, 370, 373, 375, 376, 378,
380, 382, 386, 388, 390, 392, 393, 401, 404, 411, 413, 414, 416,
418, 419, 421, 428, 431, 432, 437, 438, 439, 443, and 444 and/or b)
on the Kappa chain constant region, residues 111, 149, 183, 188,
207, and 210.
[0126] In a specific embodiment, the one or more positions (using
EU Index of Kabat numbering) that can be altered to be a cysteine
a) on the heavy chain constant region are 290, 334, 392 and/or 443
and/or b) on the light chain constant region is 183 (Kabat
numbering).
[0127] In a more specific embodiment, positions 290 on the heavy
chain constant region and position 183 on the light chain constant
region are altered to cysteine for conjugation.
[0128] In another embodiment, the site specific conjugation occurs
through one or more acyl donor glutamine residues that have been
engineered into the antibody constant region. Methods of preparing
HER2 antibodies for site specific conjugation through glutamine
residues can be performed as described in PCT Publication No.
WO2012/059882, which is incorporated by reference in its entirety.
Antibodies can be engineered to express the glutamine residue used
for site specific conjugation in three different ways.
[0129] The short peptide tag containing the glutamine residue can
be incorporated into a number of different positions of the light
and/or heavy chain (i.e., at the N-terminus, at the C-terminus,
internally). In a first embodiment, a short peptide tag containing
the glutamine residue can be attached to the C-terminus of the
heavy and/or light chain. One or more of the following glutamine
containing tags can be attached to serve as the acyl donor for drug
conjugation: GGLLQGPP (SEQ ID NO:81), GGLLQGG (SEQ ID NO:82), LLQGA
(SEQ ID NO:83), GGLLQGA (SEQ ID NO:84), LLQG (SEQ ID NO: 85),
LLQGPG (SEQ ID NO: 86), LLQGPA (SEQ ID NO: 87), LLQGP (SEQ ID NO:
88), LLQP (SEQ ID NO: 89), LLQPGK (SEQ ID NO: 90), LLQGAPGK (SEQ ID
NO: 91), LLQGAPG (SEQ ID NO: 92), LLQGAP (SEQ ID NO: 93),
LLQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5, wherein X.sub.1 is G or P,
wherein X.sub.2 is A, G, P, or absent, wherein X.sub.3 is A, G, K,
P, or absent, wherein X.sub.4 is G, K or absent, and wherein
X.sub.5 is K or absent (SEQ ID NO: 94), or
LLQX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5, wherein X.sub.1 is any
naturally occurring amino acid and wherein X.sub.2, X.sub.3,
X.sub.4, and X.sub.5 are any naturally occurring amino acids or
absent (SEQ ID NO: 95).
[0130] In a specific embodiment, GGLLQGPP (SEQ ID NO:81) is
attached to the C-terminus of the light chain.
[0131] In a second embodiment, a residue on the heavy and/or light
chain can be altered to a glutamine residue by site directed
mutagenesis. In a specific embodiment, the residue at position 297
on the heavy chain (using EU Index of Kabat numbering) can be
altered to be a glutamine (Q) and thus serve as a site for
conjugation.
[0132] In a third embodiment, a residue on the heavy chain or light
chain can be altered resulting in a glycosylation at that position
such that one or more endogenous glutamine becomes
accessible/reactive for conjugation. In a specific embodiment, the
residue at position 297 on the heavy chain (using EU Index of Kabat
numbering) is altered to an alanine (A). In such cases, the
glutamine (Q) at position 295 on the heavy chain is then capable
for use in conjugation.
[0133] Optimal reaction conditions for formation of a conjugate may
be empirically determined by variation of reaction variables such
as temperature, pH, linker-payload moiety input, and additive
concentration. Conditions suitable for conjugation of other drugs
may be determined by those skilled in the art without undue
experimentation. Site specific conjugation through engineered
cysteine residues is exemplified in Example 5A infra. Site specific
conjugation through glutamine residues is exemplified in Example 5B
infra.
[0134] To further increase the number of drug molecules per
antibody drug conjugate, the drug may be conjugated to polyethylene
glycol (PEG), including straight or branched polyethylene glycol
polymers and monomers. A PEG monomer is of the formula:
--(CH.sub.2CH.sub.2O)--. Drugs and/or peptide analogs may be bound
to PEG directly or indirectly, i.e. through appropriate spacer
groups such as sugars. A PEG-antibody drug composition may also
include additional lipophilic and/or hydrophilic moieties to
facilitate drug stability and delivery to a target site in vivo.
Representative methods for preparing PEG-containing compositions
may be found in, e.g., U.S. Pat. Nos. 6,461,603; 6,309,633; and
5,648,095.
[0135] Following conjugation, the conjugates may be separated and
purified from unconjugated reactants and/or aggregated forms of the
conjugates by conventional methods. This can include processes such
as size exclusion chromatography (SEC),
ultrafiltration/diafiltration, ion exchange chromatography (IEC),
chromatofocusing (CF) HPLC, FPLC, or Sephacryl S-200
chromatography. The separation may also be accomplished by
hydrophobic interaction chromatography (HIC). Suitable HIC media
includes Phenyl Sepharose 6 Fast Flow chromatographic medium, Butyl
Sepharose 4 Fast Flow chromatographic medium, Octyl Sepharose 4
Fast Flow chromatographic medium, Toyopearl Ether-650M
chromatographic medium, Macro-Prep methyl HIC medium or Macro-Prep
t-Butyl HIC medium.
[0136] Table 4 infra shows HER2 ADCs used to generate data in the
Examples Section set forth herein. The site specific HER2 ADCs
shown in Table 4 (in rows 1-17) are examples of site specific ADCs
of the invention.
[0137] To make a site specific HER2 ADC of the invention any HER2
antibody disclosed in Section I supra can be conjugated using site
specific techniques to any drug disclosed in Section II supra via
any linker disclosed in Section III supra. In preferred
embodiments, the linker is cleavable (e.g., vc or AcLysvc). In
other preferred embodiments, the drug is an auristatin (e.g.,
0101).
[0138] In a particular aspect of the invention, site specific HER2
ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:18 and a light chain of SEQ
ID NO:42; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101. A schematic of such an ADC is shown in FIG. 1A.
[0139] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:14 and a light chain of SEQ
ID NO:44; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is AcLysvc and wherein the drug
is 0101. A schematic of such an ADC is shown in FIG. 1B.
[0140] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:24 and a light chain of SEQ
ID NO:42; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0141] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:26 and a light chain of SEQ
ID NO:42; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0142] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:28 and a light chain of SEQ
ID NO:42; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0143] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:30 and a light chain of SEQ
ID NO:12; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0144] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:32 and a light chain of SEQ
ID NO:12; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0145] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:34 and a light chain of SEQ
ID NO:44; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is AcLysvc and wherein the drug
is 0101.
[0146] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:36 and a light chain of SEQ
ID NO:12; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is AcLysvc and wherein the drug
is 0101.
[0147] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:38 and a light chain of SEQ
ID NO:12; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
[0148] In another particular aspect of the invention, site specific
HER2 ADC of the formula Ab-(L-D) comprises (a) an antibody, Ab,
comprising a heavy chain of SEQ ID NO:40 and a light chain of SEQ
ID NO:12; and (b) a linker-drug moiety, L-D, wherein L is a linker,
and D is a drug, wherein the linker is vc and wherein the drug is
0101.
TABLE-US-00004 TABLE 4 HER2 ADCs Heavy Heavy Light Light Chain
Chain Chain Chain variable constant Heavy variable constant Light
Linker ADC region region Chain region region Chain Linker Payload
Type.sup.1 T(kK183C) - 1 5 6 7 41 42 vc 0101 C vc0101 T(K290C) - 1
17 18 7 11 12 vc 0101 C vc0101 T(N297Q) - 1 21 22 7 11 12 AcLysvc
0101 C AcLysvc0101 T(K334C) - 1 23 24 7 11 12 vc 0101 C vc0101
T(K392C) - 1 25 26 7 11 12 vc 0101 C vc0101 T(L443C) - 1 27 28 7 11
12 vc 0101 C vc0101 T(kK183C + 1 17 18 7 41 42 vc 0101 C K290C) -
vc0101 T(kK183C + 1 23 24 7 41 42 vc 0101 C K334C) - vc0101
T(kK183C + 1 25 26 7 41 42 vc 0101 C K392C) - vc0101 T(kK183C + 1
27 28 7 41 42 vc 0101 C L443C) - vc0101 T(K290C + 1 29 30 7 11 12
vc 0101 C K334C) - vc0101 T(K290C + 1 31 32 7 11 12 vc 0101 C
K392C) - vc0101 T(N297A + 1 33 34 7 43 44 AcLysvc 0101 C K222R + LC
Q05) - AcLysvc0101 T(N297Q + 1 35 36 7 11 12 AcLysvc 0101 C K222R)
- AcLysvc0101 T(K334C + 1 37 38 7 11 12 vc 0101 C K392C) - vc0101
T(K392C + 1 39 40 7 11 12 vc 0101 C L443C) - vc0101 T(LCQ05 + 1 13
14 7 43 44 AcLysvc 0101 C K222R) - AcLysvc0101 T - mc8261 1 5 6 7
11 12 mc 8261 N T - m(H20)c8261 1 5 6 7 11 12 m(H20)c 8261 N T -
MalPeg8261 1 5 6 7 11 12 MalPeg6 8261 N T - vc8261 1 5 6 7 11 12 vc
8261 C T - mc6121 1 5 6 7 11 12 mc 6121 N T - MalPeg6121 1 5 6 7 11
12 MalPeg6 6121 N T - mc0101 1 5 6 7 11 12 mc 0101 N T - vc0101 1 5
6 7 11 12 vc 0101 C T - vc8254 1 5 6 7 11 12 vc 8254 C T - vc6780 1
5 6 7 11 12 vc 6780 C T - vc0131 1 5 6 7 11 12 vc 0131 C T -
MalPegM 1 5 6 7 11 12 MalPeg6 MMAD N MAD T - vcMMAE 1 5 6 7 11 12
vc MMAE C T - DM1 1 5 6 7 11 12 mcc DM1 N .sup.1C = cleavable; N =
non-cleavable
V. Use of Site Specific HER2 Antibody Drug Conjugates
[0149] The antibody drug conjugates of the present invention are
useful in therapeutic methods to treat HER2-expressing cancer. In
some aspects of the invention, provided is a method of inhibiting
tumor growth or progression in a subject who has a HER2-expressing
tumor, including administering to the subject in need thereof an
effective amount of a composition (i.e., a pharmaceutical
composition) having one or more ADCs described herein. In other
aspects of the invention, provided is a method of inhibiting
metastasis of HER2-expressing cancer cells in a subject, including
administering to the subject in need thereof an effective amount of
a composition (i.e., a pharmaceutical composition) having one or
more ADCs described herein. In other aspects of the invention,
provided is a method of inducing regression of a HER2-expressing
tumor in a subject, including administering to the subject in need
thereof an effective amount of a composition (i.e., a
pharmaceutical composition) having one or more ADCs described
herein. In other aspects, the invention provides a pharmaceutical
composition comprising one or more ADCs described herein for use in
a method as described above. In other aspects, the invention
provides the use of one or more ADCs as described herein or a
pharmaceutical composition comprising the ADCs as described herein
in the manufacture of a medicament for use in the methods described
above.
[0150] Desired outcomes of the disclosed therapeutic methods are
generally quantifiable measures as compared to a control or
baseline measurement. As used herein, relative terms such as
"improve," "increase," or "reduce" indicate values relative to a
control, such as a measurement in the same individual prior to
initiation of treatment described herein, or a measurement in a
control individual (or multiple control individuals) in the absence
of the treatment described herein. A representative control
individual is an individual afflicted with the same form of cancer
as the individual being treated, who is about the same age as the
individual being treated (to ensure that the stages of the disorder
in the treated individual and the control individual are
comparable).
[0151] Changes or improvements in response to therapy are generally
statistically significant. As used herein, the term "significance"
or "significant" relates to a statistical analysis of the
probability that there is a non-random association between two or
more entities. To determine whether or not a relationship is
"significant" or has "significance," statistical manipulations of
the data can be "p-value." Those p-values that fall below a
user-defined cut-off point are regarded as significant. A p-value
less than or equal to 0.1, less than 0.05, less than 0.01, less
than 0.005, or less than 0.001 may be regarded as significant.
[0152] V.A. Cancers
[0153] The ADCs of the present invention are useful in treating
HER2-expressing cancers. In one embodiment, the HER2-expressing
cancer is a solid tumor. In a more specific embodiment,
HER2-expressing solid tumors include, but are not limited to,
breast cancer (e.g., estrogen and progesterone receptor negative
breast cancer, triple negative breast cancer), ovarian cancer, lung
cancer (e.g., non-small cell lung cancer (including
adenocarcinomas, squamous cell carcinomas and large cell
carcinomas) and small cell lung cancer), gastric cancer, esophageal
cancer, colorectal cancer, urothelial cancer (e.g., micropapillary
urothelial cancer and typical urothelial cancer), pancreatic
cancer, salivary gland cancer (e.g., mucoepidermoid carcinomas,
adenoid cystic carcinomas and terminal duct adenocarcinoma) and
brain cancer or metastases of the aforementioned cancers (i.e.,
lung metastasis from HER2+ breast cancer) (Martin et al., 2014,
Future Oncol. 10(8):1469-86).
[0154] In an even more specific embodiment, HER2-expressing solid
tumors include, but are not limited to, breast cancer, ovarian
cancer, lung cancer and gastric cancer.
[0155] In another embodiment, the breast cancer is estrogen
receptor and progesterone receptor negative. In a more specific
embodiment, the breast cancer is triple negative breast cancer
(TNBC).
[0156] In another embodiment, the lung cancer is non-small cell
lung cancer (NSCLC).
[0157] In one aspect of the invention, ADCs disclosed herein can be
used to treat HER2-expressing cancers that have not been previously
treated with a therapeutic agent (i.e., as a first line
treatment).
[0158] In another aspect of the invention, ADCs disclosed herein
can be used to treat HER2-expressing cancers that are resistant to,
refractory to and/or relapsed from treatment with another
therapeutic agent (i.e., as a second line treatment). In one
embodiment, the prior treatment was trastuzumab (trastuzumab or
Herceptin.RTM.) either alone or in combination with an additional
therapeutic agent (i.e., a taxane such as paclitaxel, docetaxel,
cabazitaxel, etc.). In another embodiment, the prior treatment was
trastuzumab emtansine (T-DM1 or Kadcyla.RTM.) either alone or in
combination with an additional therapeutic agent (i.e., a taxane
such as paclitaxel, docetaxel, cabazitaxel, etc.).
[0159] In another aspect of the invention, ADCs disclosed herein
can be used to treat HER2-expressing cancers that are resistant to,
refractory to and/or relapsed from treatment with more than one
other therapeutic agent (i.e., as a third line treatment or a
fourth line treatment, etc.).
[0160] ADCs of the present invention can be used to treat cancers
that express high levels of HER2 (i.e., IHC 3+), moderate levels of
HER2 (i.e., 2+ IHC or 2+/3+ IHC) or low levels of HER2 (i.e., IHC
1+, IHC 2+ or IHC 1+/2+) (see Section IVB for methods of HER2
detection). This is in contrast to trastuzumab and T-DM1 where they
are not efficacious in low or moderate HER2-expressing cancers
(Burris et al., 2011, J Clinical Oncology 29(4):398-405).
[0161] ADCs of the present invention can be used to treat cancers
that are homogeneous in nature where the majority of tumor cells
express a similar amount of HER2. Alternatively, the ADCs of the
present invention can be used to treat cancers that are
heterogeneous in nature where there are different tumor cell
populations expressing different levels of HER2.
[0162] V.B. HER2 Detection Methods
[0163] Aspects regarding the best way to assess HER2 expression
levels on a tumor have been discussed and clinical implications
have been outlined (Sauter et al., 2009, J Clin Oncol. 27:1323-33;
Wolff et al., 2007, J Clinical Oncology 25:118-45; Wolff et al.,
2013, J Clinical Oncology 31:3997-4014). Currently, HER2 status can
be assessed by immunohistochemistry (IHC), fluorescent in situ
hybridization (FISH) and chromogenic in situ hybridization
(CISH).
[0164] IHC identifies HER2 protein expression on the cell membrane.
Results are usually expressed using a semiquantitative scoring
system ranging from 0+(no expression) to 3+(high expression).
Tumors that show no (0+) or low levels (1+) of expression are
considered HER2-negative; vice-versa tumors that show high levels
(3+) of expression should be considered as HER2-positive. This
method is economically advantageous and readily available, but
suffers from low sensitivity and high interobserver variability
(Gancberg et al., 2002, Breast Cancer Res Treat. 74:113-20).
[0165] There are four FDA-approved commercial kits available for
HER2 detection using IHC: HercepTest.TM. (by Dako Denmark A/S);
Pathway (by Ventana Medical Systems, Inc.); Insite HER2/NEU kit (by
Biogenex Laboratories, Inc.) and Bond Oracle HER2 IHC System (by
Leica Biosystems). These are highly standardized, semi-quantitative
assays which stratify HER2 expression levels into; 0 (<20,000
receptors per cell, no visible expression), 1+(.about.100,000
receptors per cell, partial membrane staining, <10% of cells
overexpressing HER-2), 2+(.about.500,000 receptors per cell, light
to moderate complete membrane staining, >10% of cells
overexpressing HER-2), and 3+(.about.2,000,000 receptors per cell,
strong complete membrane staining, >10% of cells overexpressing
HER-2). The presence of cytoplasmic expression is disregarded.
[0166] FISH detects HER2 gene amplification with a DNA probe and is
more specific and sensitive than IHC (Owens et al., 2004, Clin
Breast Cancer. 5:63-69; Press et al., 2005, Clin Cancer Res.
11:6598-6607; Vogel et al., 2002, J Clinical Oncology
20(3):719-726). FISH offers quantitative results on the number of
HER2 gene copies per chromosome 17 centromeres. Results are
reported as a ratio of the number of HER2 signals to chromosome 17
centromere signals. A ratio of less than 1.8 is considered within
normal limits. A ratio of 1.8-2.0 is equivocal and requires further
testing. A ratio of greater than 2.0 is consistent with
amplification of HER2 gene sequences.
[0167] There are four FDA-approved commercial kits available for
HER2 detection using FISH: HER2 FISH Pharm Dx.TM. kit (by Dako
Denmark A/S); Pathvysion HER2 DNA Probe Kit (by Abbott Molecular
Inc.); Inform HER2/NEU and Inform HER2 Dual ISH DNA Probe Cocktail
(both by Ventana Medical Systems, Inc.).
[0168] Another method to assess HER2 gene amplification is CISH.
CISH is very similar to FISH but utilizes conventional peroxidase
or alkaline phosphatase reactions visualized under a standard
bright-field microscope. There are two FDA-approved commercial kits
available for HER2 detection using CISH: HER2 CISH PharmDx Kit (by
Dako Denmark A/S) and Spot-Light HER2 CISH Kit (by Life
Technologies, Inc.).
[0169] Both gene amplification detected by FISH or CISH and protein
expression by IHC are commonly used as initial test to assess HER2
status. There is a good correlation between the two methods (Jacobs
et al., 1999, J Clinical Oncology 17(7):1974-82). However in cases
where the tumor is scored as equivocal (i.e., IHC 2+ or FISH/CISH
ratio of 1.8-2.2 or average HER2 gene copy number of four to six
signals per nucleus), a common approach is to test the tumor with
an alternative method (Wolff et al., 2007, J Clinical Oncology
25:118-45).
[0170] Thus, HER2 expression is considered high in tumors with a 3+
level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
2.0. HER2 expression is considered moderate in tumors with a 2+
level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
<2.0. HER2 expression is considered low in tumors with a 1+
level as determined by immunohistochemistry (IHC) and/or a
fluorescence in situ hybridization (FISH) amplification ratio of
<2.0.
[0171] In one embodiment, HER2 levels are determined by IHC. In a
more specific embodiment, IHC is performed using a Dako
Hercptest.TM. assay.
[0172] In another embodiment, HER2 levels are determined by FISH.
In a more specific embodiment, FISH is performed using a Dako HER2
FISH Pharm Dx.TM. assay.
[0173] Representative tumor samples include any biological or
clinical sample which contains tumor cells, for example, a tissue
sample, a biopsy, a blood sample, a plasma sample, a saliva sample,
a urine sample, etc.
VI. Formulations
[0174] The present invention provides pharmaceutical compositions
including any of the site specific HER2 antibody drug conjugates
disclosed herein and a pharmaceutically acceptable carrier.
Further, the compositions can include more than one of the site
specific HER2 ADCs disclosed herein.
[0175] The compositions used in the present invention can further
include pharmaceutically acceptable carriers, excipients, or
stabilizers (Remington: The Science and practice of Pharmacy 21st
Ed., 2005, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in
the form of lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations, and may include
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 dextrans; 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
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
"Pharmaceutically acceptable salt" as used herein refers to
pharmaceutically acceptable organic or inorganic salts of a
molecule or macromolecule. Pharmaceutically acceptable excipients
are further described herein.
[0176] Various formulations of one or more site specific HER2 ADCs
may be used for administration including, but not limited to
formulations comprising one or more pharmaceutically acceptable
excipients. Pharmaceutically acceptable excipients are known in the
art, and are relatively inert substances that facilitate
administration of a pharmacologically effective substance. For
example, an excipient can give form or consistency, or act as a
diluent. Suitable excipients include but are not limited to
stabilizing agents, wetting and emulsifying agents, salts for
varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy 20th Ed. Mack
Publishing, 2000.
[0177] In some aspects of the invention, these agents are
formulated for administration by injection (e.g.,
intraperitoneally, intravenously, subcutaneously, intramuscularly,
etc.). Accordingly, these agents can be combined with
pharmaceutically acceptable vehicles such as saline, Ringer's
solution, dextrose solution, and the like. The particular dosage
regimen, i.e., dose, timing and repetition, will depend on the
particular individual and that individual's medical history.
[0178] Therapeutic formulations of the site specific HER2 ADCs used
in accordance with the present invention are prepared for storage
by mixing an ADC having the desired degree of purity with optional
pharmaceutically acceptable carriers, excipients or stabilizers
(Remington, The Science and Practice of Pharmacy 21st Ed. Mack
Publishing, 2005), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and may include buffers such as phosphate, citrate, and
other organic acids; salts such as sodium chloride; 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
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0179] Liposomes containing the site specific HER2 ADCs can be
prepared by methods known in the art, such as described in
Eppstein, et al., 1985, PNAS 82:3688-92; Hwang, et al., 1908, PNAS
77:4030-4; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes
with enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556. Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition including
phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter.
[0180] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacrylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nanoparticles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington, The Science and Practice of
Pharmacy 21st Ed. Mack Publishing, 2005.
[0181] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose
acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
[0182] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by, for example,
filtration through sterile filtration membranes. Therapeutic site
specific HER2 ADC compositions are generally placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0183] Suitable surface-active agents include, in particular,
non-ionic agents, such as polyoxyethylenesorbitans (e.g. TWEEN.TM.
20, 40, 60, 80 or 85) and other sorbitans (e.g. Span.TM. 20, 40,
60, 80 or 85). Compositions with a surface-active agent will
conveniently include between 0.05 and 5% surface-active agent, and
can be between 0.1 and 2.5%. It will be appreciated that other
ingredients may be added, for example mannitol or other
pharmaceutically acceptable vehicles, if necessary.
[0184] Suitable emulsions may be prepared using commercially
available fat emulsions, such as INTRALIPID.TM., LIPOSYN.TM.,
INFONUTROL.TM., LIPOFUNDIN.TM. and LIPIPHYSAN.TM.. The active
ingredient may be either dissolved in a pre-mixed emulsion
composition or alternatively it may be dissolved in an oil (e.g.
soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or
almond oil) and an emulsion formed upon mixing with a phospholipid
(e.g. egg phospholipids, soybean phospholipids or soybean lecithin)
and water. It will be appreciated that other ingredients may be
added, for example glycerol or glucose, to adjust the tonicity of
the emulsion. Suitable emulsions will typically contain up to 20%
oil, for example, between 5 and 20%. The fat emulsion can include
fat droplets between 0.1 and 1.0 .mu.m, particularly 0.1 and 0.5
.mu.m, and have a pH in the range of 5.5 to 8.0. The emulsion
compositions can be those prepared by mixing a site specific HER2
ADC with INTRALIPID.TM. or the components thereof (soybean oil, egg
phospholipids, glycerol and water).
[0185] The invention also provides kits for use in the instant
methods. Kits of the invention include one or more containers
including one or more site specific HER2 ADCs as described herein
and instructions for use in accordance with any of the methods of
the invention described herein. Generally, these instructions
include a description of administration of the site specific HER2
ADC for the above described therapeutic treatments.
[0186] The instructions relating to the use of the site specific
HER2 ADCs as described herein generally include information as to
dosage, dosing schedule, and route of administration for the
intended treatment. The containers may be unit doses, bulk packages
(e.g., multi-dose packages) or sub-unit doses. Instructions
supplied in the kits of the invention are typically written
instructions on a label or package insert (e.g., a paper sheet
included in the kit), but machine-readable instructions (e.g.,
instructions carried on a magnetic or optical storage disk) are
also acceptable.
[0187] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an infusion device such as a
minipump. A kit 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). The container
may also 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 site specific HER2 ADC. The container
may further include a second pharmaceutically active agent.
[0188] Kits may optionally provide additional components such as
buffers and interpretive information. Normally, the kit includes a
container and a label or package insert(s) on or associated with
the container.
VII. Dose and Administration
[0189] For in vivo applications, site specific HER2 ADCs are
provided or administered in an effective dosage. The phrases
"effective dosage" or "effective amount" as used herein refer to an
amount of a drug, compound or pharmaceutical composition necessary
to achieve any one or more beneficial or desired therapeutic
results either directly or indirectly. For example, when
administered to a cancer-bearing subject, an effective dosage
includes an amount sufficient to elicit anti-cancer activity,
including cancer cell cytolysis, inhibition of cancer cell
proliferation, induction of cancer cell apoptosis, reduction of
cancer cell antigens, delayed tumor growth, and/or inhibition of
metastasis. Tumor shrinkage is well accepted as a clinical
surrogate marker for efficacy. Another well accepted marker for
efficacy is progression-free survival.
[0190] An effective dosage can be administered in one or more
administrations. An effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an effective dosage may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0191] The site specific HER2 ADCs can be administered to an
individual via any suitable route. It should be understood by
persons skilled in the art that the examples described herein are
not intended to be limiting but to be illustrative of the
techniques available. Accordingly, in some aspects of the
invention, the site specific HER2 ADC is administered to an
individual in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular, intraperitoneal,
intracerebrospinal, intracranial, transdermal, subcutaneous,
intra-articular, sublingually, intrasynovial, via insufflation,
intrathecal, oral, inhalation or topical routes. Administration can
be systemic, e.g., intravenous administration, or localized.
Commercially available nebulizers for liquid formulations,
including jet nebulizers and ultrasonic nebulizers are useful for
administration. Liquid formulations can be directly nebulized and
lyophilized powder can be nebulized after reconstitution.
Alternatively, the site specific HER2 ADC can be aerosolized using
a fluorocarbon formulation and a metered dose inhaler, or inhaled
as a lyophilized and milled powder.
[0192] In some aspects of the invention, the site specific HER2 ADC
is administered via site-specific or targeted local delivery
techniques. Examples of site-specific or targeted local delivery
techniques include various implantable depot sources of site
specific HER2 ADC or local delivery catheters, such as infusion
catheters, indwelling catheters, or needle catheters, synthetic
grafts, adventitial wraps, shunts and stents or other implantable
devices, site specific carriers, direct injection, or direct
application. See, e.g. PCT International Publication No. WO
2000/53211 and U.S. Pat. No. 5,981,568.
[0193] For the purpose of the present invention, the appropriate
dosage of the site specific HER2 ADCs will depend on the particular
ADC (or compositions thereof) employed, the type and severity of
symptoms to be treated, whether the agent is administered for
therapeutic purposes, previous therapy, the patient's clinical
history and response to the agent, the patient's clearance rate for
the administered agent, and the discretion of the attending
physician. The clinician may administer a site specific HER2 ADC
until a dosage is reached that achieves the desired result and
beyond. Dose and/or frequency can vary over course of treatment,
but may stay constant as well. Empirical considerations, such as
the half-life, generally will contribute to the determination of
the dosage. For example, antibodies that are compatible with the
human immune system, such as humanized antibodies or fully human
antibodies, may be used to prolong half-life of the antibody and to
prevent the antibody being attacked by the host's immune system.
Frequency of administration may be determined and adjusted over the
course of therapy, and is generally, but not necessarily, based on
treatment and/or suppression and/or amelioration of symptoms, e.g.,
tumor growth inhibition or delay, etc. Alternatively, sustained
continuous release formulations of site specific HER2 ADCs may be
appropriate. Various formulations and devices for achieving
sustained release are known in the art.
[0194] Generally, for administration of a site specific HER2 ADC,
an initial candidate dosage can be about 2 mg/kg. For the purpose
of the present invention, a typical daily dosage might range from
about any of 3 .mu.g/kg to 30 .mu.g/kg to 300 .mu.g/kg to 3 mg/kg,
to 30 mg/kg, to 100 mg/kg or more. For example, dosage of about 1
mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25
mg/kg may be used. For repeated administrations over several days
or longer, depending on the disorder, the treatment is sustained
until a desired suppression of symptoms occurs or until sufficient
therapeutic levels are achieved, for example, to inhibit or delay
tumor growth/progression or metastases of cancer cells. An
exemplary dosing regimen includes administering an initial dose of
about 2 mg/kg, followed by a weekly maintenance dose of about 1
mg/kg of the site specific HER2 ADC, or followed by a maintenance
dose of about 1 mg/kg every other week. Other exemplary dosing
regimens include administering increasing doses (e.g., initial dose
of 1 mg/kg and gradual increase to one or more higher doses every
week or longer time period). Other dosage regimens may also be
useful, depending on the pattern of pharmacokinetic decay that the
practitioner wishes to achieve. For example, in some aspects of the
invention, dosing from one to four times a week is contemplated. In
other aspects, dosing once a month or once every other month or
every three months is contemplated, as well as weekly, bi-weekly
and every three weeks. The progress of this therapy may be easily
monitored by conventional techniques and assays. The dosing regimen
(including the particular site specific HER2 ADC used) can vary
over time.
VIII. Combination Therapies
[0195] In some aspects of the invention, the methods described
herein further include a step of treating a subject with an
additional form of therapy. In some aspects, the additional form of
therapy is an additional anti-cancer therapy including, but not
limited to, chemotherapy, radiation, surgery, hormone therapy,
and/or additional immunotherapy.
[0196] The disclosed site specific HER2 ADCs may be administered as
an initial treatment, or for treatment of cancers that are
unresponsive to conventional therapies. In addition, the site
specific HER2 ADCs may be used in combination with other therapies
(e.g., surgical excision, radiation, additional anti-cancer drugs
etc.) to thereby elicit additive or potentiated therapeutic effects
and/or reduce cytotoxicity of some anti-cancer agents. Site
specific HER2 ADCs of the invention may be co-administered or
co-formulated with additional agents, or formulated for consecutive
administration with additional agents in any order.
[0197] Site specific HER2 ADCs of the invention may be used in
combination with other therapeutic agents including, but not
limited to, therapeutic antibodies, ADCs, immunomodulating agents,
cytotoxic agents, and cytostatic agents. A cytotoxic effect refers
to the depletion, elimination and/or the killing of a target cells
(i.e., tumor cells). A cytotoxic agent refers to an agent that has
a cytotoxic and/or cytostatic effect on a cell. A cytostatic effect
refers to the inhibition of cell proliferation. A cytostatic agent
refers to an agent that has a cytostatic effect on a cell, thereby
inhibiting the growth and/or expansion of a specific subset of
cells (i.e., tumor cells). An immunomodulating agent refers to an
agent that stimulates the immune response though the production of
cytokines and/or antibodies and/or modulating T cell function
thereby inhibiting or reducing the growth of a subset of cells
(i.e., tumor cells) either directly or indirectly by allowing
another agent to be more efficacious.
[0198] For combination therapies, a site specific HER2 ADC and/or
one or more additional therapeutic agents are administered within
any time frame suitable for performance of the intended therapy.
Thus, the single agents may be administered substantially
simultaneously (i.e., as a single formulation or within minutes or
hours) or consecutively in any order. For example, single agent
treatments may be administered within about 1 year of each other,
such as within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or
1 week(s), or within about 5, 4, 3, 2 or 1 day(s).
[0199] The disclosed combination therapies may elicit a synergistic
therapeutic effect, i.e., an effect greater than the sum of their
individual effects or therapeutic outcomes. For example, a
synergistic therapeutic effect may be an effect of at least about
two-fold greater than the therapeutic effect elicited by a single
agent, or the sum of the therapeutic effects elicited by the single
agents of a given combination, or at least about five-fold greater,
or at least about ten-fold greater, or at least about twenty-fold
greater, or at least about fifty-fold greater, or at least about
one hundred-fold greater. A synergistic therapeutic effect may also
be observed as an increase in therapeutic effect of at least 10%
compared to the therapeutic effect elicited by a single agent, or
the sum of the therapeutic effects elicited by the single agents of
a given combination, or at least 20%, or at least 30%, or at least
40%, or at least 50%, or at least 60%, or at least 70%, or at least
80%, or at least 90%, or at least 100%, or more. A synergistic
effect is also an effect that permits reduced dosing of therapeutic
agents when they are used in combination.
Examples
[0200] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Example 1: Preparation of Trastuzumab Derived Antibodies for Site
Specific Conjugation
[0201] A. For Conjugation Via Cysteine
[0202] Methods of preparing trastuzumab derivatives for site
specific conjugation through cysteine residues were generally
performed as described in PCT Publication WO2013/093809 (which is
incorporated herein in its entirety). One or more residues on
either the light chain (183 using the Kabat numbering scheme) or
the heavy chain (290, 334, 392 and/or 443 using the EU index of
Kabat numbering scheme) were altered to a cysteine (C) residue by
site directed mutagenesis.
[0203] B. For Conjugation Via Transglutaminase
[0204] Methods of preparing trastuzumab derivatives for site
specific conjugation through glutamine residues were generally
performed as described in PCT Publication WO2012/059882 (which is
incorporated herein in its entirety). Trastuzumab was engineered to
express the glutamine residue used for conjugation in three
different ways.
[0205] For the first method, an 8 amino acid residue tag (LCQ05)
containing the glutamine residue was attached to the C-terminus of
the light chain (i.e., SEQ ID NO:81).
[0206] For the second method, a residue on the heavy chain
(position 297 using the EU index of Kabat numbering scheme) was
altered from an asparagine (N) to a glutamine (Q) residue by site
directed mutagenesis.
[0207] For the third method, a residue on the heavy chain (position
297 using the EU index of Kabat numbering system) was altered from
an asparagine (N) to an alanine (A). This results in aglycosylation
at position 297 and accessible/reactive endogenous glutamine at
position 295.
[0208] Additionally, some of the trastuzumab derivatives have an
alteration that is not used for conjugation. The residue at
position 222 on the heavy chain (using the EU Index of Kabat
numbering scheme) was altered from a lysine (K) to an arginine (R)
residue. The K222R substitution was found to result in more
homogenous antibody and payload conjugate, better intermolecular
crosslinking between the antibody and the payload, and/or
significant decrease in interchain crosslinking with the glutamine
tag on the C terminus of the antibody light chain.
Example 2: Production of Stably Transfected Cells Expressing
Trastuzumab Derived Antibodies
[0209] A. Cysteine Mutants
[0210] To determine that the single and double cysteine engineered
trastuzumab derived antibody variants could be stably expressed in
cells and large-scale produced, CHO cells were transfected with DNA
encoding nine trastuzumab derived antibody variants
(T(.kappa.K183C), T(K290C), T(K334C), T(K392C),
T(.kappa.K183C+K290C), T(.kappa.K183C+K392C), T(K290C+K334C),
T(K334C+K392C) and T(K290C+K392C)) and stable high production pools
were isolated using standard procedures well-known in the art. To
produce T(.kappa.K183C+K334C) for conjugation studies, HEK-293
cells (ATCC Accession # CRL-1573) were transiently co-transfected
with heavy and light chain DNA encoding this double-cysteine
engineered antibody variant using standard methods. A two-column
process, i.e. Protein-A affinity capture followed by a TMAE column
or a three-column process, i.e. Protein-A affinity capture followed
by a TMAE column and then CHA-TI column, was used to isolate these
trastuzumab variants from the concentrated CHO pool starting
material. Using these purification process, all engineered cysteine
trastuzumab derived antibody variant preparations contained >97%
peak-of-interest (POI) as determined by analytical size-exclusion
chromatography (Table 5). These results shown in Table 5
demonstrate that acceptable levels of high molecular weight (HMW)
aggregated species were detected following elution from Protein A
resin for all ten trastuzumab derived cysteine variants and that
this undesirable HMW species could be removed using size exclusion
chromatography. Additionally, the data demonstrated that the
Protein A binding site in the human IgG1 constant region was not
altered by the presence of the engineered cysteine residues.
TABLE-US-00005 TABLE 5 Production of Trastuzumab Derived Cysteine
Antibody Variants ProA Purification Eluate Yield Final Yield
Variant Process (% POI) (ProA) (% POI) (Final) T(.kappa.K183C) 2
column ND ND >99% 768 mg/L T(K290C) 2 column >99% ND >99%
100 mg/L T(K334C) 2 column >99% ND >99% 100 mg/L T(K392C 2
column >99% ND >99% 110 mg/L T(.kappa.K183C + 3 column 93%
567 mg/L >99% 248 mg/L K290C) T(K290C + 3 column 91.2% 470 mg/L
>99% 240 mg/L K334C) T(K334C + 3 column 92.4% 410 mg/L >99%
220 mg/L K392C) T(.kappa.K183C + 3 column ND ND >99% 64 mg/L
K334C) T(K290C + 2 column 93.1% 700 mg/L 97.9% 420 mg/L K392C)
T(.kappa.K183C + 2 column 91.4 ND 97.8 600 mg/L K392C) ND = Not
Determined
Example 3: Integrity of Trastuzumab Derived Antibodies
[0211] Molecular assessment of the engineered cysteine and
transglutaminase variants was performed to evaluate key biophysical
properties relative to the trastuzumab wild type antibody to ensure
the variants would be amenable to a standard antibody manufacturing
platform process.
[0212] A. Cysteine Mutants
[0213] To determine integrity of the purified engineered cysteine
antibody variant preparations produced via stable CHO expression,
the percent purity of peaks was calculated using non-reduced
capillary gel electrophoresis (Caliper LabChip GXII: Perkin Elmer
Waltham, Mass.). Results show that the engineered cysteine antibody
variants T(.kappa.K183C+K290C) and T(K290C+K334C) contained low
levels of both fragments and high molecular mass species (HMMS)
similar to the trastuzumab wild type antibody. In contrast,
T(K334C+K392C) contained high levels of fragmented antibody peaks
relative to the other double engineered cysteine variants evaluated
(Table 6). These results suggest that specific combinations of
engineered cysteines can impact integrity of the antibody intended
for site-specific conjugation.
TABLE-US-00006 TABLE 6 Percent Purity of Peaks Calculated from
Non-Reduced Electropherogram Antibody Main Peak (%) Fragments (%)
HMMS (%) trastuzumab WT 95 5 0 T(.kappa.K183C + K290C) 95.78 4.18
0.04 T(K290C + K334C) 94.6 5.2 0.2 T(K334C + K392C) 80.7 19.3 0
Example 4: Generation of Payload Drug Compounds
[0214] The auristatin drug compounds 0101, 0131, 8261, 6121, 8254
and 6780 were made according to the methods described in PCT
Publication WO2013/072813 (which is incorporated herein in its
entirety). In published application, the auristatin compounds are
indicated by the numbering system shown in Table 7.
TABLE-US-00007 TABLE 7 Auristatin Drug Compound Designation in
WO2013/072813 0101 #54 0131 #118 8261 #69 6121 #117 8254 #70 6780
#112
[0215] According to PCT Publication WO2013/072813 drug compound
0101 was made according to the following procedure.
##STR00017##
[0216] Step 1. Synthesis of
N-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-metho-
xy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-th-
iazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]--
N-methyl-L-valinamide (#53). According to general procedure D, from
#32 (2.05 g, 2.83 mmol, 1 eq.) in dichloromethane (20 mL, 0.1 M)
and N,N-dimethylformamide (3 mL), the amine #19 (2.5 g, 3.4 mmol,
1.2 eq.), HATU (1.29 g, 3.38 mmol, 1.2 eq.) and triethylamine (1.57
mL, 11.3 mmol, 4 eq.) was synthesized the crude desired material,
which was purified by silica gel chromatography (Gradient: 0% to
55% acetone in heptane), producing #53 (2.42 g, 74%) as a solid.
LC-MS: m/z 965.7 [M+H.sup.+], 987.6 [M+Na.sup.+], retention
time=1.04 minutes; HPLC (Protocol A): m/z 965.4 [M+H.sup.+],
retention time=11.344 minutes (purity >97%); .sup.1H NMR (400
MHz, DMSO-d.sub.6), presumed to be a mixture of rotamers,
characteristic signals: .delta. 7.86-7.91 (m, 2H), [7.77 (d, J=3.3
Hz) and 7.79 (d, J=3.2 Hz), total 1H], 7.67-7.74 (m, 2H), [7.63 (d,
J=3.2 Hz) and 7.65 (d, J=3.2 Hz), total 1H], 7.38-7.44 (m, 2H),
7.30-7.36 (m, 2H), 7.11-7.30 (m, 5H), [5.39 (ddd, J=11.4, 8.4, 4.1
Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz), total 1H], [4.49 (dd,
J=8.6, 7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total 1H], 3.13, 3.17,
3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s, total 3H),
1.31 and 1.36 (2 br s, total 6H), [1.05 (d, J=6.7 Hz) and 1.09 (d,
J=6.7 Hz), total 3H].
[0217] Step 2. Synthesis of
2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-met-
hyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrroli-
din-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide (#54 or
0101). According to general procedure A, from #53 (701 mg, 0.726
mmol) in dichloromethane (10 mL, 0.07 M) was synthesized the crude
desired material, which was purified by silica gel chromatography
(Gradient: 0% to 10% methanol in dichloromethane). The residue was
diluted with diethyl ether and heptane and was concentrated in
vacuo to afford #54 (or 0101) (406 mg, 75%) as a white solid.
LC-MS: m/z 743.6 [M+H.sup.+], retention time=0.70 minutes; HPLC
(Protocol A): m/z 743.4 [M+H.sup.+], retention time=6.903 minutes,
(purity >97%); .sup.1H NMR (400 MHz, DMSO-d.sub.6), presumed to
be a mixture of rotamers, characteristic signals: .delta. [8.64 (br
d, J=8.5 Hz) and 8.86 (br d, J=8.7 Hz), total 1H], [8.04 (br d,
J=9.3 Hz) and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz)
and 7.80 (d, J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d,
J=3.2 Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4
Hz) and 5.53 (ddd, J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz)
and 4.60 (dd, J=9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4
s, total 6H), 2.93 and 3.02 (2 br s, total 3H), 1.21 (s, 3H), 1.13
and 1.13 (2 s, total 3H), [1.05 (d, J=6.7 Hz) and 1.10 (d, J=6.7
Hz), total 3H], 0.73-0.80 (m, 3H).
[0218] Drug compounds MMAD, MMAE and MMAF were made in-house
according to methods disclosed in PCT Publication WO
2013/072813.
[0219] Drug compound DM1 was made in-house from purchased
maytansinol via procedures outlined in U.S. Pat. No. 5,208,020.
Example 5: Bioconjugation of Trastuzumab-Derived Antibodies
[0220] The trastuzumab-derived antibodies of the present invention
were conjugated to payload via linkers to generate ADCs. The
conjugation method used was either site specific (i.e., via
particular cysteine residues or particular glutamine residues) or
conventional conjugation.
[0221] A. Cysteine Site Specific
[0222] The ADCs of Table 8 were conjugated via cysteine site
specific methods described below.
TABLE-US-00008 TABLE 8 T(.kappa.K183C)-vc0101 T(.kappa.K183C +
K334C)-vc0101 T(K290C)-vc0101 T(.kappa.K183C + K392C)-vc0101
T(K334C)-vc0101 T(K290C + K334C)-vc0101 T(K392C)-vc0101 T(K290C +
K392C)-vc0101 T(.kappa.K183C + K290C)-vc0101 T(K334C +
K392C)-vc0101
[0223] A 500 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
solution (50 to 100 molar equivalents) was added to the antibody (5
mg) such that the final antibody concentration was 5-15 mg/mL in
PBS containing 20 mM EDTA. After allowing the reaction to stand at
37.degree. C. for 2.5 hour, the antibody was buffer exchanged into
PBS containing 5 mM EDTA using a gel filtration column (PD-10
desalting column, GE Healthcare). The resulting antibody (5-10
mg/mL) in PBS containing 5 mM EDTA was treated with a freshly
prepared 50 mM solution of DHA in 1:1 PBS/EtOH (final DHA
concentration=1 mM-4 mM) and allowed to stand at 4.degree. C.
overnight.
[0224] The antibody/DHA mixture was buffer exchanged into PBS
containing 5 mM EDTA (pH of the equilibration buffer adjusted to
.about.7.0 using phosphoric acid) and concentrated using a 50 kD MW
cutoff spin concentration device. The resulting antibody in PBS
(antibody concentration .about.5-10 mg/ml) containing 5 mM EDTA was
treated with 5-7 molar equivalents of 10 mM maleimide payload in
DMA. After standing for 1.5-2.5 hours, the material was buffer
exchanged (PD-10). Purification by SEC was performed (as needed) to
remove any aggregated material and remaining free payload.
[0225] B. Transglutaminase Site Specific
[0226] The ADCs of Table 9 were conjugated via transglutaminase
site specific methods described below.
TABLE-US-00009 TABLE 9 T(N297Q)-AcLysvc0101 T(LCQ05 +
K222R)-AcLysvc0101 T(N297Q + K222R)-AcLysvc0101 T(N297A + K222R -
LCQ05)-AcLysvc0101
[0227] In the transamidation reaction, the glutamine on the
antibody acted as an acyl donor, and the amine-containing compound
acted as an acyl acceptor (amine donor). Purified HER2 antibody in
the concentration of 33 .mu.M was incubated with a 10-25 M excess
acyl acceptor, ranging between 33-83.3 .mu.M AcLysvc-0101, in the
presence of 2% (w/v) Streptoverticillium mobaraense
transglutaminase (ACTIVA.TM., Ajinomoto, Japan) in 150-mM sodium
chloride and Tris HCl buffer at pH range 7.5-8, with 0.31 mM
reduced glutathione unless noted. The reaction conditions were
adjusted for individual acyl donors, with T(LCQ05+K222R) using 10M
excess acyl acceptor at pH 8.0 without reduced glutathione,
T(N297Q+K222R) and T(N297Q) using 20M excess acyl acceptor at pH
7.5 and T(N297A+K222R+LCQ05) using 25M excess acyl acceptor at pH
7.5. Following incubation at 37.degree. C. for 16-20 hours, the
antibody was purified on MabSelect SuReO resin or Butyl Sepharose
High Performance (GE Healthcare, Piscataway, N.J.) using standard
chromatography methods known to persons skilled in the art, such as
commercial affinity chromatography and hydrophobic interaction
chromatography from GE Healthcare.
[0228] C. Conventional Conjugation
[0229] The ADCs of Tables 10 and 11 were conjugated via
conventional conjugation methods described below.
TABLE-US-00010 TABLE 10 T-DM1 T-mc0101 T-mc8261 T-vc0101
T-MalPeg8261 T-vc8261 T-mc6121 T-vc8254 T-MalPeg6121 T-vc6780
T-MalPegMMAD T-vc0131 T-vcMMAE
TABLE-US-00011 TABLE 11 T-m(H20)c8261 T-m(H20)cvc0101
[0230] The antibody was dialyzed into Dulbecco's Phosphate Buffered
Saline (DPBS, Lonza). The dialyzed antibody was diluted to 15 mg/mL
with PBS containing 5 mM 2, 2', 2'', 2''-(ethane-1,
2-diyldinitrilo)tetraacetic acid (EDTA), pH 7. The resulting
antibody was treated with 2-3 equivalents of
tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 5 mM in
distilled water) and allowed to stand 37.degree. C. for 1-2 hours.
Upon cooling to room temperature, dimethylacetamide (DMA) was added
to achieve 10% (v/v) total organic. The mixture was treated with
8-10 equivalents of the appropriate linker-payload as a 10 mM stock
solution in DMA. The reaction was allowed to stand for 1-2 hours at
room temperature and then buffer exchanged into DPBS (pH 7. 4)
using GE Healthcare Sephadex G-25 M buffer exchange columns per
manufacturer's instructions.
[0231] Material that was to remain ring-closed (ADCs of Table 10)
was purified by size exclusion chromatography (SEC) using GE AKTA
Explorer system with GE Superdex200 column and PBS (pH 7. 4)
eluent. Final samples were concentrated to .about.5 mg/mL protein,
filter sterilized, and checked for loading using the mass
spectroscopy conditions outlined below.
[0232] Material used for succinimide ring hydrolysis (ADCs of Table
11) were immediately buffer exchanged into a 50 mM borate buffer
(pH 9.2) using an ultrafiltration device (50 kd MW cutoff). The
resulting solution was heated to 45.degree. C. for 48 h. The
resulting solution was cooled, buffer-exchanged into PBS, and
purified by SEC (as described below) in order to remove any
aggregated material. Final samples were concentrated to .about.5
mg/mL protein and filter sterilized and checked for loading using
the mass spectroscopy conditions outlined below.
[0233] D. T-DM1 Conjugation
[0234] Trastuzumab-maytansinoid conjugate (T-DM1) is structurally
similar to trastuzumab emtansine (Kadcyla.RTM.). T-DM1 is comprised
of the trastuzumab antibody covalently bound to the DM1
maytansinoid through the bifunctional linker sulfosuccinimidyl
4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC).
Sulfo-SMCC is first conjugated to the free amines on the antibody
for one hour at 25.degree. C. in 50 mM potassium phosphate, 2 mM
EDTA, pH 6.8, at a 10:1 reaction stoichiometry, and unbound linker
is then desalted from the conjugated antibody. This antibody-MCC
intermediate is then conjugated to the DM1 sulfide at the free
maleimido end on the MCC linker antibody overnight at 25.degree. C.
in 50 mM potassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.8, at a
10:1 reaction stoichiometry. Remaining unreacted maleimide is then
capped with L-cysteine, and the ADC is fractionated through a
Superdex200 column to remove non-monomeric species (Chari et al.,
1992, Cancer Res 52:127-31).
Example 6: Purification of ADCs
[0235] The ADCs were generally purified and characterized using
size-exclusion chromatography (SEC) as described below. The loading
of the drug onto the intended site of conjugation was determined
using a variety of methods including mass spectrometry (MS),
reverse phase HPLC, and hydrophobic interaction chromatography
(HIC), as more fully described below. The combination of these
three analytical methods provides a variety of ways to verify and
quantitate the loading of the payload onto the antibody thereby
providing an accurate determination of the DAR for each
conjugate.
[0236] A. Preparative SEC
[0237] ADCs were generally purified using SEC chromatography using
a Waters Superdex200 10/300GL column on an Akta Explorer FPLC
system in order to remove protein aggregate and to remove traces of
payload-linker left in the reaction mixture. On occasion, ADCs were
free of aggregate and small molecule prior to SEC purification and
were therefore not subjected to preparative SEC. The eluent used
was PBS at 1 mL/min flow. Under these conditions, aggregated
material (eluting at about 10 minutes at room temperature) was
easily separated from non-aggregated material (eluting at about 15
minutes at room temperature). Hydrophobic payload-linker
combinations frequently resulted in a "right-shift" of the SEC
peaks. Without wishing to be bound by any particular theory, this
SEC peak shift may be due to hydrophobic interactions of the
linker-payload with the stationary phase. In some cases, this
right-shift allowed for conjugated protein to be partially resolved
from non-conjugated protein.
[0238] B. Analytical SEC
[0239] Analytical SEC was carried out on an Agilent 1100 HPLC using
PBS as eluent to assess the purity and monomeric status of the
ADCs. The eluent was monitored at 220 and 280 nM. When the column
was a TSKGel G3000SW column (7.8.times.300 mm, catalog number
R874803P), the mobile phase used was PBS with a flow rate of 0.9
mL/min for 30 minutes When the column was a BiosepSEC3000 column
(7.8.times.300 mm), the mobile phase used was PBS with a flow rate
of 1.0 mL/min for 25 minutes.
Example 7: Characterization of ADCs
[0240] A. Mass Spectroscopy (MS)
[0241] Samples were prepped for LCMS analysis by combining
approximately 20 .mu.l of sample (approximately 1 mg/ml ADC in PBS)
with 20 .mu.l of 20 mM dithiothreitol (DTT). After allowing the
mixture to stand at room temperature for 5 minutes, the samples
were injected into an Agilent 110 HPLC system fitted with an
Agilent Poroshell 300SB-C8 (2.1.times.75 mm) column. The system
temperature was set to 60.degree. C. A 5 minute gradient from 20%
to 45% acetonitrile in water (with 0.1% formic acid modifier) was
utilized. The eluent was monitored by UV (220 nM) and by a Waters
Micromass ZQ mass spectrometer (ESI ionization; cone voltage: 20V;
Source temp: 120.degree. C.; Desolvation temp: 350.degree. C.). The
crude spectrum containing the multiple-charged species was
deconvoluted using MaxEnt1 within MassLynx 4.1 software package
according to the manufacturer's instructions.
[0242] B. MS Determination of Loading Per Antibody
[0243] The total loading of the payload to the antibody to make an
ADC is referred to as the Drug Antibody Ratio or DAR. The DAR was
calculated for each of the ADCs made (Table 12).
[0244] The spectra for the entire elution window (usually 5
minutes) were combined into a single summed spectrum (i.e., a mass
spectrum that represents the MS of the entire sample). MS results
for ADC samples were compared directly to the corresponding MS of
the identical non-loaded control antibody. This allowed for the
identification of loaded/nonloaded heavy chain (HC) peaks and
loaded/nonloaded light chain (LC) peaks. The ratio of the various
peaks can be used to establish loading based on the equation below
(Equation 1). Calculations are based on the assumption that loaded
and non-loaded chains ionize equally which has been determined to
be a generally valid assumption.
[0245] The following calculation was performed in order to
establish the DAR:
Loading=2*[LC1/(LC1+LC0)]+2*[HC1/(HC0+HC1+HC2)]+4*[HC2/(HC0+HC1+HC2)]
Equation 1:
Where the indicated variables are the relative abundance of:
LC0=unloaded light chain, LC1=single loaded light chain,
HC0=unloaded heavy chain, HC1=single loaded heavy chain, and
HC2=double loaded heavy chain. One of ordinary skill in the art
would appreciate that the invention encompasses expansion of this
calculation to encompass higher loaded species such as LC2, LC3,
HC3, HC4, HC5, and the like.
[0246] Equation 2, below, is used to estimate the amount of loading
onto non-engineered cysteine residues. For engineered Fc mutants,
loading onto the light chain (LC) was considered, by definition, to
be nonspecific loading. Moreover, it was assumed that loading only
the LC was the result of inadvertent reduction of the HC-LC
disulfide bridge (i.e., the antibody was "over-reduced"). Given
that a large excess of maleimide electrophile was used for the
conjugation reactions (generally approximately 5 equivalents for
single mutants and 10 equivalents for double mutants), it was
assumed that any nonspecific loading onto the light chain was
accompanied by a corresponding amount of non-specific loading onto
the heavy chain (i.e., the other "half" of the broken HC-LC
disulfide). With these assumptions in mind, the following equation
(Equation 2) was used to estimate the amount of non-specific
loading onto the protein:
Nonspecific loading=4*[LC1/(LC1+LC0)] Equation 2:
Where the indicated variables are the relative abundance of:
LC0=unloaded light chain, LC1=single loaded light chain.
TABLE-US-00012 TABLE 12 Drug Antibody Ratio (DAR) of ADCs ADC DAR
T(.kappa.K183C)-vc0101 2 T(K290C)-vc0101 2 T(K334C)-vc0101 2
T(K392C)-vc0101 2 T(.kappa.K183C + K290C)-vc0101 4 T(.kappa.K183C +
K334C)-vc0101 4 T(.kappa.K183C + K392C)-vc0101 4 T(K290C +
K334C)-vc0101 4 T(K290C + K392C)-vc0101 4 T(K334C + K392C)-vc0101 4
T(N297Q)-AcLysvc0101 4 T(N297Q + K222R)-AcLysvc0101 4 T(N297A +
K222R + LCQ05)-AcLysvc0101 4 T(LCQ05 + K222R)-AcLysvc0101 2
T-mc8261 4.2 T-m(H20)c8261 3.6 T-MalPeg8261 3.1 T-vc8261 4.3
T-mc6121 3.5 T-MalPeg6121 3.6 T-mc0101 4.8 T-vc0101 4.2 T-vc8254 4
T-vc6780 4.2 T-vc0131 4.5 T-MalPegMMAD 4.4 T-vcMMAE 3.8 T-DM1
4.2
[0247] C. Proteolysis with FabRICATOR.RTM. to Establish the Site of
Loading
[0248] For the cysteine mutant ADCs, any nonspecific loading of the
electrophillic payload onto the antibody is presumed to occur at
the "interchain" also referred to as the "internal" cysteine
residues (i.e., those that are typically part of the HC-HC or HC-LC
disulfide bridges). In order to distinguish loading of electrophile
onto the engineered cysteines in the Fc domain versus loading onto
the internal cysteine residues (otherwise typically forming the
S--S bonds between HC-HC or HC-LC), the conjugates were treated
with a protease known to cleave between the Fab domains and the Fc
domain of the antibody. One such protease is the cysteine protease
IdeS, marketed as "FabRICATOR.RTM." by Genovis, and described in
von Pawel-Rammingen et al., 2002, EMBO J. 21:1607.
[0249] Briefly, following the manufacturer's suggested conditions,
the ADC was treated with FabRICATOR.RTM. protease and the sample
was incubated at 37.degree. C. for 30 minutes. Samples were prepped
for LCMS analysis by combining approximately 20 .mu.l of sample
(approximately 1 mg/mL in PBS) with 20 .mu.l of 20 mM
dithiothreitol (DTT) and allowing the mixture to stand at room
temperature for 5 minutes. This treatment of human IgG1 resulted in
three antibody fragments, all ranging from about 23 to 26 kD in
size: the LC fragment comprising an internal cysteine which
typically forms an LC-HC interchain disulfide bond; the N-terminal
HC fragment comprising three internal cysteines (where one
typically forms an LC-HC disulfide bond and the other two cysteines
found in the hinge region of the antibody and which typically form
HC-HC disulfide bonds between the two heavy chains of the
antibody); and the C-terminal HC fragment which contains no
reactive cysteines other than those introduced by mutation in the
constructs disclosed herein. The samples were analyzed by MS as
described above. Loading calculations were performed in the same
manner as previously described (above) in order to quantitate the
loading of the LC, the N-terminal HC, and the C-terminal HC.
Loading on the C-terminal HC is considered "specific" loading while
loading onto the LC and the N-terminal HC is considered
"nonspecific" loading.
[0250] To cross-check the loading calculations, a subset of ADCs
were also assessed for loading using alternative methods (reverse
phase high performance liquid chromatography [rpHPLC]-based and
hydrophobic interaction chromatography [HIC]-based methods) as more
fully described in the sections below.
[0251] D. Reverse Phase HPLC Analysis
[0252] Samples were prepped for reverse-phase HPLC analysis by
combining approximately 20 .mu.l of sample (approximately 1 mg/mL
in PBS) with 20 .mu.l of 20 mM dithiothreitol (DTT). After allowing
the mixture to stand at room temperature for 5 minutes, the samples
were injected into an Agilent 1100 HPLC system fitted with an
Agilent Poroshell 300SB-C8 (2.1.times.75 mm) column. The system
temperature was set to 60.degree. C. and the eluent was monitored
by UV (220 nM and 280 nM). A 20-minute gradient from 20% to 45%
acetonitrile in water (with 0.1% TFA modifier) was utilized: T=0
min:25% acetonitrile; T=2 min:25% acetonitrile; T=19 min:45%
acetonitrile; and T=20 min:25% acetonitrile. Using these
conditions, the HC and LC of the antibody were baseline separated.
The results of this analysis indicate that the LC remains largely
unmodified (except for T(kK183C) and T(LCQ05) containing
antibodies) while the HC is modified (data not shown).
[0253] E. Hydrophobic Interaction Chromatography (HIC)
[0254] Compounds were prepared for HIC analysis by diluting samples
to approximately 1 mg/ml with PBS. The samples were analyzed by
auto-injection of 15 .mu.l onto an Agilent 1200 HPLC with a TSK-GEL
Butyl NPR column (4.6.times.3.5 mm, 2.5 .mu.m pore size; Tosoh
Biosciences part #14947). The system includes an auto-sampler with
a thermostat, a column heater and a UV detector.
[0255] The gradient method was used as follows:
[0256] Mobile phase A: 1.5M ammonium sulfate, 50 mM potassium
phosphate dibasic (pH7); Mobile phase B: 20% isopropyl alcohol, 50
mM potassium phosphate dibasic (pH 7); T=0 min. 100% A; T=12 min.,
0% A.
[0257] Retention times are shown in Table 13. Selected spectra are
shown in FIGS. 2A-2E. ADCs using site-specific conjugation
(T(kK183C+K290C)-vc0101, T(K334C+K392C)-vc0101 and
T(LCQ05+K222R)-AcLysvc0101) (FIGS. 1A-1C) showed primarily one peak
while ADCs using conventional conjugation (T-vc0101 and T-DM1)
(FIGS. 2D-2E) showed a mixture of differentially loaded
conjugates.
TABLE-US-00013 TABLE 13 ADC retention times by hydrophobic
interaction chromatography (HIC) ADC RT (min) RRT T-vc0101 8.8 .+-.
0.1 1.68 T(.kappa.K183C)-vc0101 7.2 .+-. 0.1 1.40 T(K334C)-vc0101
ND T(K392C)-vc0101 6.7 .+-. 0.1 1.29 T(L443C)-vc0101 10.1 .+-. 0.1
1.98 T(.kappa.K183C + K290C)-vc0101 9.0 .+-. 0.0 1.77
T(.kappa.K183C + K334C)-vc0101 ND T(.kappa.K183C + K392C)-vc0101
7.7 .+-. 0.1 1.54 T(.kappa.K183C + L443C)-vc0101 10.6 2.04 T(K290C
+ K334C)-vc0101 6.3 .+-. 0.0 1.21 T(K290C + K392C)-vc0101 7.8 .+-.
0.0 1.54 T(K334C + K392C)-vc0101 6.0 .+-. 0.3 1.18 T(K392C +
L443C)-vc0101 10.8 .+-. 0.0 2.08 T(LCQ05 + K222R)-AcLys-vc0101 6.5
1.27 T(N297A + K222R + LCQ05)-AcLys-vc0101 6.3 .+-. 0.1 1.24 ND =
not determined RT = retention time (min) on HIC RRT = mean relative
retention time, calculated by RT of ADC divided by RT of benchmark
unconjugated wild type trastuzumab having a typical retention time
of 5.0-5.2 min
[0258] F. Thermostability
[0259] Differential Scanning calorimetry (DCS) was used to
determine the thermal stability of the engineered cysteine and
transglutaminase antibody variants, and corresponding Aur-06380101
site-specific conjugates. For this analysis, samples formulated in
PBS-CMF pH 7.2 were dispensed into the sample tray of a MicroCal
VP-Capillary DSC with Autosampler (GE Healthcare Bio-Sciences,
Piscataway, N.J.), equilibrated for 5 minutes at 10.degree. C. and
then scanned up to 110.degree. C. at a rate of 100.degree. C. per
hour. A filtering period of 16 seconds was selected. Raw data was
baseline corrected and the protein concentration was normalized.
Origin Software 7.0 (OriginLab Corporation, Northampton, Mass.) was
used to fit the data to an MN2-State Model with an appropriate
number of transitions.
[0260] All single and double cysteine engineered antibody variants
as well as the engineered LCQ05 acyl donor glutamine-containing tag
antibody exhibited excellent thermal stability as determined by the
first melting transition (Tm1) >65.degree. C. (Table 14).
[0261] Trastuzumab derived monoclonal antibodies conjugated to 0101
using site specific conjugation methods were also evaluated and
shown to have exceptional thermal stability as well (Table 15).
However, the Tm1 for T(K392C+L443C)-vc0101 ADC was most impacted by
conjugation of the payload since it was -4.35.degree. C. relative
to the unconjugated antibody.
[0262] Taken together these results demonstrated that both the
engineered cysteine and acyl donor glutamine-containing tag
antibody variants were thermally stable and that site-specific
conjugation of 0101 via a vc linker yielded conjugates with
excellent thermal stability. Furthermore, the lower thermal
stability observed for T(K392C+L443C)-vc0101 relative to the
unconjugated antibody indicated that conjugation of 0101 via a vc
linker to certain combinations of engineered cysteine residues can
impact stability of the ADC.
TABLE-US-00014 TABLE 14 Thermal Stability of Engineered Trastuzumab
Derived Variants Antibody Tm1 (.degree. C.) Tm2 (.degree. C.) Tm3
(.degree. C.) T(.kappa.K183C) 72.17 .+-. 0.029 80.78 .+-. 0.37
82.81 .+-. 0.055 T(L443C) 72.02 .+-. 0.06 80.98 .+-. 1.10 82.96
.+-. 0.11 T(LCQ05) 72.22 .+-. 0.027 81.16 .+-. 0.19 82.88 .+-.
0.033 T(.kappa.K183C + K290C) 75.4 81.1 82.9 T(.kappa.K183C +
K392C) 75 81 83 T(.kappa.K183C + L443C) 72.24 .+-. 0.05 80.89 .+-.
0.89 82.87 .+-. 0.16 T(K290C + K334C) 75.0 .+-. 0.14 83.0 .+-. 0.1
81.1 .+-. 0.4 T(K334C + K392C) 75.3 .+-. 0.25 82.7 .+-. 0.53 81.0
.+-. 2.9 T(K290C + K392C) 77 81 83 T(K392C + L443C) 73.95 .+-. 0.29
80.54 .+-. 0.70 82.81 .+-. 0.17
TABLE-US-00015 TABLE 15 Thermal Stability of Site-Specific
Conjugates Conjugated to Auristatin 0101 Tm1.sub.SSC -
Site-Specific Conjugate Tm1 (.degree. C.) Tm2 (.degree. C.) Tm3
(.degree. C.) Tm1.sub.Ab T(.kappa.K183C)-vc0101 70.16 .+-. 80.45
.+-. 82.04 .+-. -2.01 0.03 0.12 0.03 T(L443C)-vc0101 72.34 .+-.
80.20 .+-. 82.44 .+-. 0.32 0.10 0.59 0.10 T(.kappa.K183C + L443C)-
70.11 .+-. 78.89 .+-. 81.38 .+-. -2.13 vc0101 0.02 0.59 0.10
T(K392C + L443C)- 69.60 .+-. 79.21 .+-. 82.10 .+-. -4.35 vc0101
0.35 0.43 0.05
Example 8: ADC Binding to HER2
[0263] A. Direct Binding
[0264] BT474 cells (HTB-20) were trypsinized, spun down and
re-suspended in fresh media. The cells were then incubated with a
serial of dilutions of either the ADCs or unconjugated trastuzumab
with starting concentration of 1 pg/ml for one hour at 4.degree. C.
The cells were then washed twice with ice cold PBS and incubated
with anti-human Alexafluor 488 secondary antibody (Cat# A-11013,
Life technologies) for 30 min. The cells were then washed twice and
then re-suspended in PBS. The mean fluorescence intensity was read
using Accuri flow cytometer (BD Biosciences San Jose, Calif.).
TABLE-US-00016 TABLE 16 ADC binding to HER2 ADC/Ab EC.sub.50
trastuzumab 0.37 T(.kappa.K183C + K392C)-vc0101 0.56 T(.kappa.K183C
+ K290C)-vc0101 0.47 T(K290C + K392C)-vc0101 0.32 T-DM1 (Kadcyla)
0.40 T(LCQ05 + K222R)-AcLysvc0101 0.37 T(N297Q + K222R)-AcLysvc0101
0.36 EC50 = the concentration of an antibody or ADC that gives
half-maximal binding.
[0265] As shown in FIG. 3A and Table 16, ADCs
T(LCQ05+K222R)-AcLysvc0101, T(N297Q+K222R)-AcLysvc0101,
T(kK183C+K290C)-vc0101, T(kK183C+K392C)-vc0101,
T(K290C+K392C)-vc0101 had similar binding affinities as T-DM1 and
trastuzumab by direct binding. This indicates that the
modifications to the antibody in the ADCs of the present invention
and the addition of the linker-payload did not significantly affect
binding.
[0266] B. Competitive Binding by FACS
[0267] BT474 cells were trypsinized, spun down and re-suspended in
fresh media. The cells were then incubated for one hour at
4.degree. C. with serial dilutions of either the ADCs or the
unconjugated trastuzumab combined with 1 .mu.g/mL of trastuzumab-PE
(custom synthesized 1:1 PE labeled trastuzumab by eBiosciences (San
Diego, Calif.)). The cells were then washed twice and then
re-suspended in PBS. The mean fluorescence intensity was read using
Accuri flow cytometer (BD Biosciences San Jose, Calif.).
[0268] As shown in FIG. 3B, ADCs T(LCQ05+K222R)-AcLysvc0101,
T(N297Q+K222R)-AcLysvc0101, T(kK183C+K290C)-vc0101,
T(kK183C+K392C)-vc0101, T(K290C+K392C)-vc0101 had similar binding
affinities as T-DM1 and trastuzumab by competition binding to PE
labeled trastuzumab. This indicates that the modifications to the
antibody in the ADCs of the present invention and the addition of
the linker-payload did not significantly affect binding.
Example 9: ADC Binding to Human FcRn
[0269] It is believed in the art that FcRn interacts with IgG
regardless of subtype in a pH dependent manner and protects the
antibody from degradation by preventing it from entering the
lysosomal compartment where it is degraded. Therefore, a
consideration for selecting positions for introduction of reactive
cysteines into the wild type IgG1-Fc region was to avoid altering
the FcRn binding properties and half-life of the antibody
comprising the engineered cysteine.
[0270] BIAcore.RTM. analysis was performed to determine the
steady-state affinity (KD) for the trastuzumab derived monoclonal
antibodies and their respective ADCs for binding to human FcRn.
BIAcore.RTM. technology utilizes changes in the refractive index at
the surface layer of a sensor upon binding of the trastuzumab
derived monoclonal antibodies or their respective ADCs to human
FcRn protein immobilized on the layer. Binding was detected by
surface plasmon resonance (SPR) of laser light refracting from the
surface. Human FcRn was specifically biotinylated through an
engineered Avi-tag using the BirA reagent (Catalog #: BIRA500,
Avidity, LLC, Aurora, Colo.) and immobilized onto a streptavidin
(SA) sensor chip to enable uniform orientation of the FcRn protein
on the sensor. Next, various concentrations of the trastuzumab
derived monoclonal antibodies or their respective ADCs or in 20 mM
MES (2-(N-morpholino)ethanesulfonic acid pH 6.0, with 150 mM NaCl,
3 mM EDTA (ethylenediaminetetraacetic acid), 0.5% Surfactant P20
(MES-EP) were injected over the chip surface. The surface was
regenerated using HBS-EP+0.05% Surfactant P20 (GE Healthcare,
Piscataway, N.J.), pH 7.4, between injection cycles. The
steady-state binding affinities were determined for the trastuzumab
derived monoclonal antibodies or their respective ADCs, and these
were compared with the wild type trastuzumab antibody (comprising
no cysteine mutations in the IgG1 Fc region, no TGase engineered
tag or site-specific conjugation of a payload).
[0271] These data demonstrated that incorporation of engineered
cysteine residues into the IgG-Fc region at the indicated positions
of the invention did not alter affinity to FcRn (Table 17).
TABLE-US-00017 TABLE 17 Steady-State Affinities of Site-Specific
Conjugates Binding Human FcRn KD [nM] KD [nM] KD [nM] Experiment 1
Experiment 2 Experiment 3 Trastuzumab WT 1050.0 705.8 859.2 T-DM1
ND 500.8 ND T(K290C + K334C) 987.0 ND ND T(K290C + K334C)-vc0101
1218.0 ND ND T(K334C + K392C) 834.1 ND ND T(K334C + K392C)-vc0101
1404.0 ND ND T(.kappa.K183C + K290C) 1173.0 ND ND T(.kappa.K183C +
K290C)-vc0101 473.8 ND ND T(.kappa.K183C + K392C) 1009.0 ND ND
T(.kappa.K183C + K392C)-vc0101 672.5 ND ND T(.kappa.K183C)-vc0101
961.5 ND ND T(LCQ05) 900.9 ND ND T(LCQ05)-vc0101 1050.0 ND ND
T(K392C) ND 468.3 ND T(K392C)-vc0101 ND 518.8 ND T(N297Q)-vc0101 ND
647.9 ND T(.kappa.K183C + K334C)-vc0101 ND 416.5 ND T(.kappa.K183C
+ K443C) ND 542.8 ND T(.kappa.K183C + K443C)-vc0101 ND 287.5 ND
T(K290C) ND ND 650.3 T(K290C)-vc0101 ND ND 874.6 T(K290C +
K392C)-vc0101 ND ND 554.7 T(K334C) ND ND 631.6 T(K334C)-vc0101 ND
ND 791.2 T(K392C + K443C) ND ND 601.7 T(K392C + K443C)-vc0101 ND ND
197.9 ND = Not Determined
Example 10: ADC Binding to Fc.gamma. Receptors
[0272] Binding of the ADCs using site-specific conjugation to human
Fc-.gamma. receptors was evaluated in order to understand if
conjugation to a payload alters binding which can impact antibody
related functionality properties such as antibody-dependent
cell-mediated cytotoxicity (ADCC). Fc.gamma.IIIa (CD16) is
expressed on NK cells and macrophages, and co-engagement of this
receptor with the target expressing cells via antibody binding
induces ADCC. BIAcore.RTM. analysis was used to examine binding of
the trastuzumab derived monoclonal antibodies and their respective
ADCs to Fc-.gamma. receptors IIa (CD32a), IIb(CD32b), IIIa (CD16)
and Fc.gamma.RI (CD64).
[0273] For this surface plasmon resonance (SPR) assay, recombinant
human epidermal growth factor receptor 2 (Her2/neu) extra-cellular
domain protein (Sino Biological Inc., Beijing, P.R. China) was
immobilized on a CM5 chip (GE Healthcare, Piscataway, N.J.) and
.about.300-400 response units (RU) of either a trastuzumab derived
monoclonal antibody or its respective ADC was captured. The T-DM1
was included in this evaluation as a positive control since it has
been shown to retain binding properties post-conjugation to
Fc.gamma. receptors comparable to the unconjugated trastuzumab
antibody. Next, various concentrations of the Fc.gamma. receptors
Fc.gamma.IIa (CD32a), Fc.gamma.IIb(CD32b), Fc.gamma.IIIa (CD16a)
and Fc.gamma.RI (CD64) were injected over the surface and binding
was determined.
[0274] Fc.gamma.Rs IIa, IIb and IIIa exhibited rapid on/off rates
and therefore the sensorgrams were fit to steady state model to
obtain Kd values. Fc.gamma.RI exhibited slower on/off rates so data
was fit to a kinetic model to obtain Kd values.
[0275] Conjugation of payload at the engineered cysteine positions
290 and 334 showed a moderate loss in Fc.gamma.R affinity,
specifically to CD16a, CD32a and CD64 compared to their
unconjugated counterpart antibodies and T-DM1 (Table 18). However,
simultaneous conjugation at sites 290, 334 and 392 resulted in a
substantial loss of affinity to CD16a, CD32a and CD32b, but not
CD64 as observed with the T(K290C+K334C)-vc0101 and
T(K334C+K392C)-vc0101 (Table 18). Interestingly,
T(.kappa.K183C+K290C)-vc0101 exhibited comparable binding to all
Fc.gamma.R evaluated in this study despite harboring drug payload
on the K290C position (Table 18). As expected the transglutaminase
mediated conjugated T(N297Q+K222R)-AcLysvc0101 did not bind to any
of the Fc.gamma. receptors evaluated since location of the acyl
donor glutamine-containing tag removes N-linked glycosylation.
Contrary, T(LCQ05+K222R)-AcLysvc0101 retained full binding to the
Fc.gamma. receptors as the glutamine-containing tag is engineered
within the human Kappa light chain constant region.
[0276] Taken together, these results suggested that location of the
conjugated payload can impact binding of the ADC to Fc.gamma.R and
may impact the antibody functionality of the conjugate.
TABLE-US-00018 TABLE 18 Binding Affinity of Site-Specific
Conjugates for Fc.gamma. Receptors binding to the CD16a, CD32a,
CD32b and CD64 K.sub.D [M] Fc.gamma.RIIIa Fc.gamma.RIIa
Fc.gamma.RIIb Fc.gamma.RI (CD16a) (CD32a) (CD32b) (CD64) [.mu.M]
[.mu.M] [.mu.M] [pM] Trastuzumab WT mAb 0.36 0.74 4.08 23 T-DM1 ADC
0.30 0.53 2.97 27 T(K290C)-vc0101 1.20 1.70 3.74 185
T(K334C)-vc0101 0.81 1.42 4.74 ND T(K290C + K334C)-vc0101 5.14 6.30
6.38 110 T(K334C + K392C)-vc0101 2.38 4.18 11.30 43 T(K392C)-vc0101
0.45 0.73 4.33 ND T(.kappa.K183C + K290C)-0101 0.47 0.70 3.63 37
T(LCQ05 + K222R)-AcLysvc0101 0.43 0.62 3.41 32 T(N297Q -
K222R)-AcLysvc0101 NB NB NB NB ND = Not Determined, NB = No
Binding
Example 11: ADCC Activities
[0277] In ADCC assays, Her2-expressing cell lines BT474 and SKBR3
were used as target cells while NK-92 cells (an interleukin-2
dependent natural killer cell line derived from peripheral blood
mononuclear cells from a 50 year old Caucasian male by Conkwest) or
human peripheral blood mononucleocytes (PBMC) isolated from the
freshly drawn blood from a healthy donor (#179) were used as
effector cells.
[0278] Target cells (BT474 or SKBR3) of 1.times.10.sup.4 cells/100
.mu.l/well were placed in 96-well plate and cultured overnight in
RPMI1640 media at 37.degree. C./5% CO.sub.2. The next day, the
media was removed and replaced with 60 .mu.l assay buffer (RPMI1640
media containing 10 mM HEPES), 20 .mu.l of 1 .mu.g/ml antibody or
ADC, followed by addition of 20 .mu.l 1.times.10.sup.5 (for SKBR3)
or 5.times.10.sup.5 (for BT474) PBMC suspension or
2.5.times.10.sup.5 NK92 cells for both cell lines to each well to
achieve effector to target ratio of 50:1 for BT474 or of 25:1 for
SKBR3 for PBMC, 10:1 for NK92. All samples were run in
triplicate.
[0279] Assay plates were incubated at 37.degree. C./5% CO.sub.2 for
6 hours and then equilibrated to room temperature. LDH release from
cell lysis was measured using CytoTox-One.TM. reagent at an
excitation wavelength of 560 nm and an emission wavelength of 590
nm. As a positive control, 8 .mu.L of Triton was added to generate
a maximum LDH release in control wells. The specific cytotoxicity
shown in FIG. 4 was calculated using the following formula:
% Specific Cytotoxicity = Experimental - effector spontaneous -
target spontaneous Target maximum - Target spontaneous .times. 100
##EQU00001##
[0280] "Experimental" corresponds to the signal measured in one of
the condition described above.
[0281] "Effector spontaneous" corresponds to the signal measured in
the presence of PBMC alone.
[0282] "Target spontaneous" corresponds to the signal measured in
the presence of target cells alone.
[0283] "Target Maximum" corresponds to the signal measured in the
presence of detergent-lysed target cells alone.
[0284] FIG. 4 shows the ADCC activities tested for trastuzumab,
T-DM1 and vc0101 ADC conjugates. The data conform the reported ADCC
activities of Trastuzumab and T-DM1. Since the mutation of N297Q is
at the glycosylation site, T(N297Q+K222R)-AcLysvc0101 was not
expected to have ADCC activities which was also confirmed in the
assays. For single mutant (K183C, K290C, K334C, K392C including
LCQ05) ADCs, ADCC activities were maintained. Surprisingly, for
double mutant (K183C+K290C, K183C+K392C, K183C+K334C K290C+K392C,
K290C+K334C, K334C+K392C) ADCs, ADCC activities were maintained in
all except two double mutant ADCs associated with K334C site
(K290C+K334C and K334C+K392C).
Example 12: In Vitro Cytotoxicity Assays
[0285] Antibody-drug conjugates were prepared as indicated in
Example 3. Cells were seeded in 96-well plates at low density, then
treated the following day with ADCs and unconjugated payloads at
3-fold serial dilutions at 10 concentrations in duplicate. Cells
were incubated for 4 days in a humidified 37.degree. C./5% CO.sub.2
incubator. The plates were harvested by incubating with
CellTiter.RTM. 96 AQueous One MTS Solution (Promega, Madison, Wis.)
for 1.5 hours and absorbance measured on a Victor plate reader
(Perkin-Elmer, Waltham, Mass.) at wavelength 490 nm. IC.sub.50
values were calculated using a four-parameter logistic model with
XLfit (IDBS, Bridgewater, N.J.) and reported as nM payload
concentration in FIG. 5 and ng/ml antibody concentration in FIG. 6.
The IC.sub.50 are shown +/-the standard deviation with the number
of independent determinations in parenthesis.
[0286] The ADCs containing vc-0101 or AcLysv-0101 linker-payloads
were highly potent against Her2-positive cell models and selective
against Her2-negative cells, compared with the benchmark ADC, T-DM1
(Kadcyla).
[0287] ADCs synthesized with site-specific conjugation to
trastuzumab showed high level potency and selectivity against Her2
cell models. Notably, several trastuzumab-vc0101 ADCs are more
potent than T-DM1 in moderate or low Her2-expressing cell models.
For example, the in vitro cytotoxicity IC.sub.50 for
T(kK183C+K290C)-vc0101 in MDA-MB-175-VII cells (with 1+Her2
expression) is 351 ng/ml, compared with 3626 ng/ml for T-DM1
(.about.10-fold lower). For cells with 2++ level Her2 expression
such as MDA-MB-361-DYT2 and MDA-MB-453 cells, the IC.sub.50 for
T(kK183C+K290C)-vc0101 is 12-20 ng/ml, compared with 38-40 ng/ml
for T-DM1.
Example 13: Xenograft Models
[0288] Trastuzumab derived ADCs of the invention tested in an N87
gastric cancer xenograft model, 37622 lung cancer xenograft model,
and a number of breast cancer xenograft models (i.e., HCC 1954,
JIMT-1, MDA-MB-361(DYT2) and 144580 (PDX) models). For each model
described below the first dose was given on Day 1. The tumors were
measured at least once a week and their volume was calculated with
the formula: tumor volume (mm.sup.3)=0.5.times.(tumor
width.sup.2)(tumor length). The mean tumor volumes (.+-.S.E.M.) for
each treatment group were calculated having a maximum of 8-10
animals and a minimum of 6-8 animals to be included.
[0289] A. N87 Gastric Xenografts
[0290] The effects of Trastuzumab derived ADCs were examined in
immunodeficient mice on the in vivo growth of human tumor
xenografts that were established from the N87 cell line (ATCC
CRL-5822) which has high level HER2 expression. To generate
xenografts, nude (Nu/Nu, Charles River Lab, Wilmington, Mass.)
female mice were implanted subcutaneously with 7.5.times.10.sup.6
N87 cells in 50% Matrigel (BD Biosciences). When the tumors reached
a volume of 250 to 450 mm.sup.3, the tumors were staged to ensure
uniformity of the tumor mass among various treatment groups. The
N87 gastric model was dosed 4 times intravenously 4 days apart
(Q4dx4) with PBS vehicle, Trastuzumab ADCs (at 0.3, 1 and 3 mg/kg)
or T-DM1 (1, 3 and 10 mg/kg) (FIG. 7).
[0291] The data demonstrates that Trastuzumab derived ADCs
inhibited growth of N87 gastric xenografts in a dose-dependent
manner (FIGS. 7A-7H).
[0292] As illustrated in FIG. 7I, T-DM1 had delayed tumor growth at
1 and 3 mg/kg and had complete regression of tumors at 10 mg/kg.
However, T(kK183C+K290C)-vc0101 provided complete regression at 1
and 3 mg/kg and partial regression at 0.3 mg/kg (FIG. 7A). The data
shows that T(kK183C+K290C)-vc0101 is significantly more potent
(.about.10 times) than T-DM1 in this model.
[0293] Similar in vivo efficacy from ADCs with DAR4 (FIGS. 6E, 6F
and 6G) were obtained compared to 183+290 (FIG. 7A). In addition,
single mutants were evaluated that are DAR2 ADCs (FIGS. 7B, 7C and
7D). In general, these ADCs are less efficacious compared to DAR4
ADCs but more efficacious than T-DM1. Among DAR2 ADCs, it appears
LCQ05 is the most potent ADC based on the in vivo efficacy
data.
[0294] B. HCC1954 Breast Xenografts
[0295] HCC1954 (ATCC# CRL-2338) is a high HER2 expression breast
cancer cell line. To generate xenografts, SHO female mice (Charles
River, Wilmington, Mass.) were implanted subcutaneously with
5.times.10.sup.6 HCC1954 cells in 50% Matrigel (BD Biosciences).
When the tumors reached a volume of 200 to 250 mm.sup.3, the tumors
were staged to ensure uniformity of the tumor mass among various
treatment groups. The HCC1954 breast model was dosed intravenously
Q4dx4 with PBS vehicle, Trastuzumab derived ADCs and negative
control ADC (FIGS. 8A-8E).
[0296] The data demonstrates that Trastuzumab ADCs inhibited growth
of HCC1954 breast xenografts in a dose-dependent manner. Comparing
the 1 mg/kg dose, vc0101 conjugates were more efficacious than
T-DM1. Comparing the 0.3 mg/kg dose, DAR4 loaded ADCs (FIGS. 8B, 8C
and 8D) are more efficacious than a DAR2 loaded ADC (FIG. 8A).
Further, the negative control ADC at 1 mg/kg had very minimal
impact on tumor growth compared to vehicle control (FIG. 8D).
However, T(N297Q+K222R)-AcLysvc0101 completely regressed the tumors
indicating the target specificity.
[0297] C. JIMT-1 Breast Xenografts
[0298] JIMT-1 is a breast cancer cell line expressing moderate/low
Her2 and is inherently resistant to trastuzumab. To generate
xenografts, nude (Nu/Nu) female mice were implanted subcutaneously
with 5.times.10.sup.6 JIMT-1 cells (DSMZ# ACC-589) in 50% Matrigel
(BD Biosciences). When the tumors reached a volume of 200 to 250
mm.sup.3, the tumors were staged to ensure uniformity of the tumor
mass among various treatment groups. The JIMT-1 breast model was
dosed intravenously Q4dx4 with PBS vehicle, T-DM1 (FIG. 9G),
trastuzumab derived ADCs using site specific conjugation (FIGS.
9A-9E), trastuzumab derived ADC using conventional conjugation
(FIG. 9F) and negative control huNeg-8.8 ADC.
[0299] The data demonstrates that all the tested vc0101 conjugates
cause tumor reduction in a dose-dependent manner. These ADCs can
cause tumor regression at 1 mg/kg. However, T-DM1 is inactive in
this moderate/low Her2 expressing model even at 6 mg/kg.
[0300] D. MDA-MB-361(DYT2) Breast Xenografts
[0301] MDA-MB-361(DYT2) is a breast cancer cell line expressing
moderate/low Her2. To generate xenografts, nude (Nu/Nu) female mice
were irradiated at 100 cGy/min for 4 minutes and three days later
implanted subcutaneously with 1.0.times.10.sup.7 MDA-MB-361(DYT2)
cells (ATCC# HTB-27) in 50% Matrigel (BD Biosciences). When the
tumors reached a volume of 300 to 400 mm.sup.3, the tumors were
staged to ensure uniformity of the tumor mass among various
treatment groups. The DYT2 breast model was dosed intravenously
Q4dx4 with PBS vehicle, trastuzumab derived ADCs using site
specific and conventional conjugation, T-DM1 and negative control
ADC (FIGS. 10A-10D).
[0302] The data demonstrates that trastuzumab ADCs inhibited growth
of DYT2 breast xenografts in a dose-dependent manner. Although DYT2
is moderate/low Her2 expression cell lines, it is more sensitive to
micro-tubule inhibitors than other Her2 low/moderate expressing
cell lines.
[0303] E. 144580 Patient-Derived Breast Cancer Xenografts
[0304] The effects of Trastuzumab derived ADCs were examined in
immunodeficient mice on the in vivo growth of human tumor
xenografts that were established from fragments of freshly resected
144580 breast tumors obtained in accordance with appropriate
consent procedures. The tumor characterization of 144580 when fresh
biopsy was taken was as a triple negative (ER-, PR-, and HER2-)
breast cancer tumor. The 144580 breast patient-derived xenografts
were subcutaneously passaged in vivo as fragments from animal to
animal in nude (Nu/Nu) female mice. When the tumors reached a
volume of 150 to 300 mm.sup.3, they were staged to ensure
uniformity of the tumor size among various treatment groups. The
144580 breast model was dosed intravenously four times every four
days (Q4dx4) with PBS vehicle, trastuzumab ADCs using site specific
conjugation, trastuzumab derived ADC using conventional conjugation
and negative control ADC (FIGS. 11A-11E).
[0305] In this HER2- (by clinical definition) PDX model, T-DM1 was
ineffective at all doses tested (1, 5, 3 and 6 mg/kg) (FIG. 10E).
For DAR4 vc0101 ADCs (FIGS. 11A, 11C and 11D), 3 mg/kg is able to
cause tumor regression (even at 1 mg/kg in FIG. 11C). The DAR2
vc0101 ADC (FIG. 11B) is less efficacious than DAR4 ADCs at 3
mg/kg. However, the DAR 2 vc0101 ADC is efficacious at 6 mg/kg
unlike T-DM1.
[0306] F. 37622 Patient-Derived Non-Small Cell Lund Cancer
Xenograft
[0307] Several ADCs were tested in patient-derived Non-Small Cell
Lung Cancer xenograft model of 37622 obtained in accordance with
appropriate consent procedures. The 37622 patient-derived
xenografts were subcutaneously passaged in vivo as fragments from
animal to animal in nude (Nu/Nu) female mice. When the tumors
reached a volume of 150 to 300 mm.sup.3, they were staged to ensure
uniformity of the tumor size among various treatment groups. The
37622 PDX model was dosed intravenously four times every four days
(Q4dx4) with PBS vehicle, trastuzumab derived ADCs using site
specific conjugation, T-DM1 and negative control ADC (FIGS.
12A-12D).
[0308] Expression of Her2 was profiled by modified Hercept test and
was classified as 2+ with more heterogeneity than seen in cell
lines. The ADCs conjugated with vc0101 as a linker-payload (FIGS.
12A-12C) were efficacious at 1 and 3 mg/kg causing tumor
regression. However, T-DM1 only provided some therapeutic benefit
at 10 mg/kg (FIG. 12D). It appears vc0101 ADCs are 10-times more
potent than T-DM1 by comparing results at 10 mg/kg from T-DM1 to 1
mg/kg from vc0101 ADCs. It is possible that the bystander effect is
important for efficacy for a heterogeneic tumor.
[0309] G. GA0044 Patient-Derived Gastric Cancer Xenograft
[0310] Trastuzumab and anti-HER2 ADCs were tested in a
patient-derived Gastric xenograft model (GA0044) obtained in
accordance with appropriate consent procedures. The GA0044
patient-derived xenografts were subcutaneously passaged in vivo as
fragments from animal to animal in nude (Nu/Nu) female mice. When
the tumors reached a volume of 150 to 300 mm.sup.3, they were
staged to ensure uniformity of the tumor size among various
treatment groups. The GA0044 PDX model was dosed intravenously four
times every four days (Q4dx4) with PBS vehicle, trastuzumab,
T-DM1or a trastuzumab derived ADC using site-specific conjugation
to vc0101 (FIG. 30).
[0311] Expression of HER2 in GA0044 was profiled by modified
Hercept test and was classified as 2+ with heterogeneous
distribution. The ADC conjugated with vc0101 as the payload
(namely, T(kK183C+K290C)-vc0101) was efficacious and resulted in
complete tumor regressions at 1 and 3 mg/kg doses. Trastuzumab and
T-DM1 showed no appreciable difference in tumor growth as compared
to vehicle treated tumors. It is possible that the bystander effect
is important for efficacy in this tumor with heterogenous target
(i.e. HER2) expression.
[0312] H. Demonstration of Bystander Effect of T-vc0101 ADC in N87
Gastric Xenograft
[0313] The released metabolite of the T-DM1 ADC has been shown to
be the lysine-capped mcc-DM1 linker payload (i.e., Lys-mcc-DM1)
which is a membrane impermeable compound (Kovtun et al., 2006,
Cancer Res 66:3214-21; Xie et al., 2004, J Pharmacol Exp Ther
310:844). However, the released metabolite from the T-vc0101 ADC is
auristatin 0101, a compound with more membrane permeability than
Lys-mcc-DM1. The ability of a released ADC payload to kill
neighboring cells is known as the bystander effect. Due to a
release of a membrane permeable payload, T-vc0101 is able to elicit
a strong bystander effect whereas T-DM1 is not. FIG. 13 shows
immunohistocytochemistry from N87 cell line xenograft tumors which
received a single dose of either T-DM1 at 6 mg/kg (FIG. 13A) or
T-vc0101 at 3 mg/kg (FIG. 13B) and then harvested and processed in
formalin fixation 96 hours later. Tumor sections were stained for
human IgG to detect ADC bound to tumor cells and phospho-histone H3
(pHH3) to detect mitotic cells as a readout of the proposed
mechanism of action for the payloads of both ADCs.
[0314] ADC is detected in the periphery of the tumors in both
cases. In T-DM1 treated tumors (FIG. 13A), the majority of pHH3
positive tumor cells are located near the ADC. However, in T-vc0101
treated tumors (FIG. 13B), the majority of pHH3 positive tumor
cells extend beyond the location of the ADC (black arrows highlight
a few examples) and are in the tumor interior. These data suggest
that an ADC with a cleavable linker and a membrane permeable
payload can elicit a strong bystander effect in vivo.
Example 14: In Vitro T-DM1 Resistance Models
[0315] A. Generation of T-DM1 Resistant Cells In Vitro
[0316] N87 cells were passaged into two separate flasks and each
flask was treated identically with respect to the
resistance-generation protocol to enable biological duplicates.
Cells were exposed to five cycles of T-DM1 conjugate at
approximately IC.sub.80 concentrations (10 nM payload
concentration) for 3 days, followed by approximately 4 to 11 days
recovery without treatment. After the five cycles at 10 nM of the
T-DM1 conjugate, the cells were exposed to six additional cycles of
100 nM T-DM1 in a similar fashion. The procedure was intended to
simulate the chronic, multi-cycle (on/off) dosing at maximally
tolerated doses typically used for cytotoxic therapeutics in the
clinic, followed by a recovery period. Parental cells derived from
N87 are referred to as N87, and cells chronically exposed to T-DM1
are referred to as N87-TM. Moderate- to high-level drug resistance
developed within 4 months for N87-TM cells. Drug selection pressure
was removed after .about.3-4 months of cycle treatments when the
level of resistance no longer increased after continued drug
exposure. Responses and phenotypes remained stable in the cultured
cell lines for approximately 3-6 months thereafter. Thereafter, a
reduction in the magnitude of the resistance phenotype as measured
by cytotoxicity assays was occasionally observed, in which case
early passage cryo-preserved T-DM1 resistant cells were thawed for
additional studies. All reported characterizations were conducted
after removal of T-DM1 selection pressure for at least 2-8 weeks to
ensure stabilization of the cells. Data were collected from various
thawed cryopreserved populations derived from a single selection,
over approximately 1-2 years after model development to ensure
consistency in the results. The gastric cancer cell line N87 was
selected for resistance to trastuzumab-maytansinoid antibody-drug
conjugate (T-DM1) by treatment cycles at doses that were
approximately the IC.sub.80 (.about.10 nM payload concentration)
for the respective cell line. Parental N87 cells were inherently
sensitive to the conjugate (IC.sub.50=1.7 nM payload concentration;
62 ng/ml antibody concentration) (FIG. 14). Two populations of
parental N87 cells were exposed to the treatment cycles and, after
only approximately four months exposure cycling at 100 nM T-DM1,
these two populations (henceforth named N87-TM-1 and N87-TM-2)
became refractory to the ADC by 114- and 146-fold, respectively,
compared with parental cells (FIG. 14 and FIG. 15A). Interestingly,
minimal cross-resistance (.about.2.2-2.5.times.) to the
corresponding unconjugated maytansinoid free drug, DM1, was
observed (FIG. 14).
[0317] B. Cytotoxicity Studies
[0318] ADCs were prepared as indicated in Example 3. Unconjugated
maytansine analog (DM1) and auristatin analogs were prepared by
Pfizer Worldwide Medicinal Chemistry (Groton, Conn.). Other
standard-of-care chemotherapeutics were purchased from Sigma (St.
Louis, Mo.). Cells were seeded in 96-well plates at low density,
then treated the following day with ADCs and unconjugated payloads
at 3-fold serial dilutions at 10 concentrations in duplicate. Cells
were incubated for 4 days in a humidified 37.degree. C./5% CO.sub.2
incubator. The plates were harvested by incubating with
CellTiter.RTM. 96 AQueous One MTS Solution (Promega, Madison, Wis.)
for 1.5 hours and absorbance measured on a Victor plate reader
(Perkin-Elmer, Waltham, Mass.) at wavelength 490 nm. IC.sub.50
values were calculated using a four-parameter logistic model with
XLfit (IDBS, Bridgewater, N.J.).
[0319] The cross-resistance profile to other trastuzumab derived
ADCs was determined. Significant cross-resistance to many
trastuzumab derived ADCs composed of non-cleavable linkers and
delivering payloads with anti-tubulin mechanisms of action was
observed (FIG. 14). For example, in N87-TM vs. N87-parental cells,
>330- and >272-fold reduced potency was observed to T-mc8261
(FIG. 14 and FIG. 15B) and T-MalPeg8261 (FIG. 14), which represent
an auristatin-based payload linked to trastuzumab via non-cleavable
maleimidocaproyl or Mal-PEG linkers, respectively. Over 235-fold
resistance was observed in N87-TM cells against T-mcMalPegMMAD,
another trastuzumab ADC with a different non-cleavable linker
delivering monomethyl dolastatin (MMAD) (FIG. 14).
[0320] Remarkably, it was observed that the N87-TM cell line
retained sensitivity to payloads when delivered via a cleavable
linker, even though these drugs functionally inhibit similar
targets (i.e., microtubule depolymerization). Examples of ADCs
which overcome resistance include, but are not limited to,
T(N297Q+K222R)-AcLysvc0101 (FIG. 14 and FIG. 15C),
T(LCQ05+K222R)-AcLysvc0101 (FIG. 14 and FIG. 15D),
T(K290C+K334C)-vc0101 (FIG. 10 and FIG. 11E), T(K334C+K392C)-vc0101
(FIG. 14 and FIG. 15F) and T(kK183C+K290C)-vc0101 (FIG. 14 and FIG.
15G). These represent trastuzumab-based ADCs delivering the
auristatin analog 0101, but where the payloads are released
intracellularly by proteolytic cleavage of the vc linker.
[0321] In order to determine whether these ADC-resistant cancer
cells were broadly resistant to other therapies, the N87-TM cell
models were treated with a panel of standard-of-care
chemotherapeutics with various mechanisms of action. In general,
small molecule inhibitors of microtubule and DNA function remained
effective against the N87-TM resistant cell lines (FIG. 14). While
these cells were made resistant against an ADC delivering an analog
of the microtubule depolymerizing agent, maytansine, minimal or no
cross-resistance was observed to several tubulin depolymerizing or
polymerizing agents. Similarly, both cell lines retained
sensitivity to agents which interfere with DNA function, including
topoisomerase inhibitors, anti-metabolites, and
alklyating/cross-linking agents. In general, the N87-TM cells were
not refractory to a broad range of cytotoxics, ruling out generic
growth or cell cycle defects which might mimic drug resistance.
[0322] Both N87-TM populations also retained sensitivity to the
corresponding unconjugated drugs (i.e., DM1 and 0101; FIG. 14).
Hence, N87-TM cells made refractory to a trastuzumab-maytansinoid
conjugate displayed cross-resistance to other microtubule-based
ADCs when delivered via non-cleavable linkers, but remained
sensitive to unconjugated microtubule inhibitors and other
chemotherapeutics.
[0323] To determine the molecular mechanism of resistance to T-DM1
in the N87-TM cells protein expression levels of MDR1 and MRP1 drug
efflux pumps were determined. This was because small molecule
tubulin inhibitors are known substrates of the MDR1 and MRP1 drug
efflux pumps (Thomas and Coley, 2003, Cancer Control
10(2):159-165). The protein expression levels of these two proteins
from total cell lysates of the parental N87 and N87-TM resistant
cells was determined (FIG. 16). Immunoblot analysis showed that the
N87-TM resistant cells do not significantly overexpress the MRP1
(FIG. 16A) or MDR1 (FIG. 16B) proteins. Taken together, these data
combined with the lack of cross-resistance to known substrates of
drug efflux pumps (e.g. paclitaxel, doxorubicin) in the N87-TM
cells suggests that drug efflux pump overexpression is not the
molecular mechanism of T-DM1 resistance in N87-TM cells.
[0324] Since the mechanism of action for ADCs requires binding to a
specific antigen, antigen depletion or reduced antibody binding may
account for T-DM1 resistance in N87-TM cells. To determine if the
antigen for T-DM1 had been significantly depleted in N87-TM cells,
HER2 protein expression levels from total cell lysates of the
parental N87 and N87-TM resistant cells were compared (FIG. 17A).
Immunoblot analysis showed that the N87-TM cells did not have a
markedly reduced amount of HER2 protein expression compared with
the parental N87 cells.
[0325] The amount of antibody binding to cell surface HER2 antigens
of the N87-TM cells was determined. In a cell surface binding study
using fluorescence activated cell sorting, the N87-TM cells did
have .about.50% decrease in trastuzumab binding to cell surface
antigens (FIG. 17B). Since N87 cells are high expressers of HER2
protein among cancer cell lines (Fujimoto-Ouchi et al., 2007,
Cancer Chemother Pharmacol 59(6):795-805), a .about.50% reduction
in HER2 antibody binding in these cells probably does not represent
the driving mechanism of resistance to T-DM1 in N87-TM cells.
Evidence supporting this interpretation is that the N87-TM
resistant cells remain sensitive to other HER2 binding trastuzumab
derived ADCs with different linkers and payloads (FIG. 14).
[0326] In order to determine potential mechanisms of T-DM1
resistance in an unbiased approach, the parental N87 and N87-TM
resistant cell models were profiled via a proteomic approach in
order to globally identify changes in membrane protein expression
levels that may be responsible for T-DM1 resistance. Significant
expression level changes in 523 proteins between both cell line
models was observed (FIG. 18A). To validate a selection of these
predicted protein changes, immunoblots on N87 and N87-TM whole cell
lysates were performed for proteins predicted to be under-expressed
(IGF2R, LAMP1, CTSB) (FIG. 18B) and over-expressed (CAV1) (FIG.
18C) in the N87-TM cells relative to the N87 cells. In vivo tumors
were generated by subcutaneous implantation of the N87 and N87-TM-2
cells into NSG mice to assess if protein changes observed in vivo
mimic those seen in vitro. N87-TM-2 tumors retained over-expression
of the CAV1 protein compared with the N87 tumors (FIG. 18D). While
CAV1 staining in the mouse stroma in both models is expected,
epithelial CAV1 staining was only seen in the N87-TM-2 model.
[0327] C. In Vivo Efficacy Studies
[0328] In order to determine if the resistance observed in cell
culture was recapitulated in vivo, parental N87 cells and N87-TM-2
cells were expanded and injected into the flanks of Female NOD scid
gamma (NSG) immunodeficient mice (NOD.Cg-Prkdcscid II2rgtm1Wjl/SzJ)
obtained from The Jackson Laboratory (Bar Harbor, Me.). Mice were
injected subcutaneously in the right flank with suspensions of
either N87 or N87-TM cells (7.5.times.10.sup.6 cells per injection,
with 50% Matrigel). Mice were randomized into study groups when
tumors reached .about.0.3 g (.about.250 mm.sup.3). T-DM1 conjugate
or vehicle, were administered intravenously in saline on day 0 and
repeated for a total of four doses, four days apart (Q4Dx4). Tumors
were measured weekly and mass calculated as
volume=(width.times.width.times.length)/2. Time-to-event analysis
(tumor doubling) was conducted and significance evaluated by
Log-rank (Mantel-Cox) test. No weight loss was observed in mice in
all treatment groups in these studies.
[0329] Mice were treated with the following agents: (1) vehicle
control PBS, (2) trastuzumab antibody at 13 mg/kg, followed by 4.5
mg/kg; (3) T-DM1 at 6 mg/kg; (4) T-DM1 at 10 mg/kg; (5) T-DM1 at 10
mg/kg, then T(N297Q+K222R)-AcLysvc0101 at 3 mg/kg; (6)
T(N297Q+K222R)-AcLysvc0101 at 3 mg/kg. Tumor sizes were monitored
and results are indicated in FIG. 20. The N87 (FIG. 19 and FIG.
20A) and N87-TM-2 (FIG. 19 and FIG. 20B) tumors showed an ADC
efficacy profile similar to that seen in the in vitro cytotoxicity
assays (FIGS. 19 and 20B), wherein the N87-TM drug resistant cells
were refractory to T-DM1 but still responded to trastuzumab derived
ADCs with cleavable linkers. In fact, tumors that were refractory
to T-DM1 and grew to about 1 gram were switched to therapy with
T(N297Q+K222R)-AcLysvc0101 and effectively regressed (FIG. 20B). In
a time-to-event analysis of this study, T-DM1 at 6 and 10 mg/kg
prevented tumor doubling in >50% of mice for at least 60 days in
the N87 model, but T-DM1 failed to do so in the N87-TM-2 model
(FIGS. 20C and 20D). T(N297Q+K222R)-AcLysvc0101 dosed at 3 mg/kg
prevented any tumor doubling of both N87 and N87-TM tumors in the
mice for the duration of the study (.about.80 days) (FIGS. 20C and
20D).
[0330] In another study, all cleavable linked ADCs that overcame
T-DM1 resistance in vitro remained effective in this N87-TM2 tumor
model that was non-responsive to T-DM1 (FIG. 19 and FIG. 20E).
[0331] It was then assessed whether T(kK183+K290C)-vc0101 ADC could
inhibit the growth of tumors which were refractory to TDM1. N87-TM
tumors treated with either vehicle or T-DM1 grew through these
treatments, however tumors switched to T(kK183C+K290C)-vc0101
therapy at day 14 immediately regressed (FIG. 20F).
Example 15: In Vivo T-DM1 Resistant Models
[0332] A. Generation of T-DM1 Resistant Cells In Vivo
[0333] All animal studies were approved by the Pfizer Pearl River
Institutional Animal Care and Use Committee according to
established guidelines. To generate xenografts, nude (Nu/Nu) female
mice were implanted subcutaneously with 7.5.times.10.sup.6 N87
cells in 50% Matrigel (BD Biosciences). The animals were randomized
when average tumor volume reach .about.300 mm.sup.3 into two
groups: 1) vehicle control (n=10) and 2) T-DM1 treated (n=20).
T-DM1 ADC (6.5 mg/kg) or vehicle (PBS) were administered
intravenously in saline on day 0 and then the animals were dosed
weekly with 6.5 mg/kg for up to 30 weeks. Tumors were measured
twice per week or weekly and mass calculated as
volume=(width.times.width.times.length)/2. No weight loss was
observed in mice in all treatment groups in these studies.
[0334] Animals were considered refractory or relapsed under T-DM1
treatment when the individual tumor volume reached .about.600
mm.sup.3 (doubled original size of tumor at randomization).
Compared to control group, most tumors initially responded to T-DM1
treatment as shown in FIG. 21A. More specifically, 17 out of 20
mice responded to initial T-DM1 treatment but significant number of
tumors (13 out of 20) relapsed under T-DM1 treatment. Over time the
implanted N87 tumor cells became resistant to T-DM1 (FIG. 21B).
Three tumors that did not initially responded to T-DM1 treatment
were harvested for Her2 expression determination by IHC indicating
no HER2 expression changes. The remaining 10 relapsed tumors are
described below.
[0335] Four tumors which initially responded to T-DM1 treatment and
then relapsed were switched to T-vc0101 treatment weekly at 2.6
mg/kg on day 77 (mice 1 and 16), 91 (mouse 19), 140 (mouse 6). As
shown in FIG. 19C, T-DM1 resistant tumors generated in vivo
responded to T-vc0101 indicating acquired T-DM1 resistant tumors
are sensitive to vc0101 ADC treatment.
[0336] Another three tumors initially responded to T-DM1 treatment
and then relapsed were switched to T(N297Q+K222R)-AcLysvc0101
treatment weekly at 2.6 mg/kg on day 110 (mice 4, 13, and 18). As
shown in FIG. 21D, T-DM1 resistant tumors generated in vivo also
responded to T(N297Q+K222R)-AcLysvc0101. A follow-on experiment was
performed to evaluate T(kK183C+K290C)-vc0101, similar results were
obtained indicating that T-DM1 resistant tumors generated in vivo
were sensitive to T(kK183C+K290C)-vc0101 treatment as shown in FIG.
21E.
[0337] In summary, all T-DM1 refractory tumors having follow-on
treatment were sensitive to the vc0101 ADC treatment (7 of 7)
indicating that in vivo resistant T-DM1 tumors can be treated with
cleavable vc0101 conjugates.
[0338] Additional three tumors (mouse 7, 17 and 2 as shown in FIG.
21B) initially responded to T-DM1 and then relapsed were excised
for in vitro characterization. After 2-5 months of culturing the
excised tumors in vitro these cells were evaluated for resistance
to T-DM1 and characterized in vitro (see Sections B and C of this
Example below).
[0339] B. Cytotoxicity Studies
[0340] Cells relapsed from T-DM1 treatment and cultured in vitro
(as described in Section A of this Example) were seeded in 96-well
plates and dosed the following day with 4-fold serial dilutions of
the ADCs or unconjugated payloads. Cells were incubated for 96
hours in a humidified 37.degree. C./5% CO.sub.2 incubator.
CellTiter Glo Solution (Promega, Madison, Wis.) was added to the
plates and absorbance measured on a Victor plate reader
(Perkin-Elmer, Waltham, Mass.) at wavelength 490 nm. IC.sub.50
values were calculated using a four-parameter logistic model with
XLfit (IDBS, Bridgewater, N.J.).
[0341] Cytotoxicity screening results are summarized in Tables 19
and 20. The cells were resistant to T-DM1 (FIG. 22A) when compared
to the parental but sensitive to cleavable vc0101 conjugates
T-vc0101 (data not shown), T(kK183C+K290C)-vc0101 (FIG. 22B),
T(LCQ05+K222R)-AcLysvc0101 (FIG. 22C) and
T(N297Q+K222R)-AcLysvc0101 (FIG. 22D) (Table 19). The T-DM1
resistant cells were surprisingly sensitive to the parent payload
DM1 as well as the 0101 payload (Table 20).
TABLE-US-00019 TABLE 19 Resistant Cell Sensitivity to ADCs N87-T-
N87-T- N87-T- N87 DM1 DM1 DM1 Fold ADC parental Mouse #7 Mouse #17
Mouse #2 Resistance T-DM1 16 1388 944 3700 ~60-230 T(.kappa.K183C +
5 -- -- 5 1 K290C)-vc0101 T(LCQ05 + 25 9 10 18 ~1 K222R)-
AcLysvc0101 T(N297Q + 9 7 13 16 ~1 K222R)- AcLysvc0101 T(K334C + 6
-- 11 4 ~1 K392C)-vc0101 T(K290C + 6 -- 16 4 ~1-2 K334C)-vc0101
IC.sub.50 values are shown for each of the cell lines
TABLE-US-00020 TABLE 20 Resistant Cell Line Sensitivity to Free
Payload Cell Line DMI-Sme Aur-0101 Doxorubicin N87 10 0.5 48
N87-T-DM1_Ms2 23 0.40 46 N87-T-DM1_Ms7 20 0.60 79 N87-T-DM1_Ms17 27
0.28 34
[0342] C. Her2 Expression by FACS and Western Blot
[0343] Her2 expression was characterized on cells relapsed from
T-DM1 treatment and cultured in vitro (as described in Section A of
this Example). For FACS analysis, cells were trypsinized, spun down
and resuspended in fresh media. The cells were then incubated for
one hour at 4.degree. C. with 5 .mu.g/mL of Trastuzumab-PE (custom
synthesized 1:1 PE labeled Trastuzumab by eBiosciences (San Diego,
Calif.)). The cells were then washed twice and then resuspended in
PBS. The mean fluorescence intensity was read using Accuri flow
cytometer (BD Biosciences San Jose, Calif.).
[0344] For western blot analysis, the cells were lysed using RIPA
lysis buffer (with protease inhibitors and phosphatase inhibitor)
on ice for 15 minutes then vortexed and spun down at maximum speed
in a microcentrifuge at 4.degree. C. The supernatant was collected
and 4.times. sample buffer and reducing agent were added to the
samples normalizing for total protein in each sample. The samples
were run on a 4-12% Bis tris gel and transferred on to
nitrocellulose membrane. The membranes were blocked for an hour and
incubated with HER2 antibody (Cell Signalling, 1:1000) over night
at 4.degree. C. The membranes were then washed 3 times in
1.times.TBST and incubated with an anti-mouse HRP antibody (Cell
Signalling, 1:5000) for 1 hour washed 3 times and probed.
[0345] The HER2 expression levels of the T-DM1 relapsed tumors were
similar to the control tumors (without T-DM1 treatment) as
evaluated by FACS (FIG. 23A) and western blot (FIG. 23B).
[0346] D. T-DM1 Resistance is not Due to Expression of Drug Efflux
Pumps
[0347] The cell lines do not express MDR1 by western blot (FIG.
24A) and cells are not resistant to MDR-1 substrate free drug 0101
(FIG. 24B). No resistance to doxorubicin (FIG. 24C) was observed
indicating that resistant mechanism is not through MRP1. However,
the cells are still resistant to free DM1 (FIG. 24D).
Example 16: Pharmacokinetics (PK)
[0348] Exposure of conventional or site specific vc0101 antibody
drug conjugates were determined after an IV bolus dose
administration of either 5 or 6 mg/kg to cynomolgus monkeys.
Concentrations of total antibody (total Ab; measurement of both
conjugated mAb and unconjugated mAb) and ADC (mAb that is
conjugated to at least one drug molecule) was measured using ligand
binding assays (LBA). The ADC in was made using vc0101 in all cases
except for T(LCQ05) were AcLysvc0101 was used. Conventional
conjugation (not site specific conjugation) was used to make the
ADC from trastuzumab.
[0349] Concentration vs time profiles and
pharmacokinetics/toxicokinetics of both total Ab and trastuzumab
ADC (T-vc0101) (5 mg/kg) or T(kK183C+K290C) site specific ADC (6
mg/kg) after dose administration to cynomolgus monkeys (FIG. 25A
and Table 21). Exposure of the T(kK183C+K290C) site specific ADC
has both increased exposure and stability when compared to the
conventional conjugate.
[0350] Concentration vs time profiles and
pharmacokinetics/toxicokinetics of the ADC analyte of trastuzumab
(T-vc0101) (5 mg/kg) or T(kK183C+K290C), T(LCQ05), T(K334C+K392C),
T(K290C+K334C), T(K290C+K392C) and T(kK183C+K392C) site specific
ADC (6 mg/kg) after dose administration to cynomolgus monkeys (FIG.
25B and Table 21). Exposure several site specific ADC (T(LCQ05),
T(kK183C+K290C), T(K290C+K392C) and T(kK183C+K392C)) are higher
compared to that of the trastuzumab ADC using conventional
conjugation. However, exposure of two other site specific ADC
(T(K290C+K334C) and T(K334C+K392C)) do not have higher exposure
than the trastuzumab ADC indicating that not all site specific ADCs
will have pharmacokinetic properties better than the trastuzumab
ADC made using conventional conjugation.
TABLE-US-00021 TABLE 21 Pharmacokinetics Dose Cmax AUC( 0-336 h)
mAb/ADC (mg/kg) Analyte (.mu.g/mL) (.mu.g h/mL) trastuzumab 5 Total
Ab 157 11100 ADC 154 7660 T(K290C + K334C) 6 Total Ab 165 5770 ADC
163 5060 T(K334C + K392C) 6 Total Ab 159 5320 ADC 157 4770 T(LCQ05)
5 Total Ab 165 .+-. 19 16400 .+-. 1020 ADC 164 .+-. 22 16300 .+-.
989 T(.kappa.K183C + K290C) 6 Total Ab 187 16800 ADC 181 15300
T(K183C + K392C) 6 Total Ab 195 18500 ADC 196 16900 T(K290C +
K392C) 6 Total Ab 205 13300 ADC 208 12300
Example 17: Relative Retention Values by Hydrophobic Interaction
Chromatography Vs. Exposure (AUC) in Rats
[0351] Hydrophobicity is a physical property of a protein that can
be assessed by hydrophobicity interaction chromatography (HIC), and
the retention times of protein samples differ based on their
relative hydrophobicity. ADCs can be compared with their respective
antibody by calculating a relative retention time (RRT), which is
the ratio of the HIC retention time of the ADC divided by the HIC
retention time of the respective antibody. Highly hydrophobic ADCs
have higher RRT, and it is possible that these ADCs may also have
more pharmacokinetic liability, specifically lower
area-under-the-curve (AUC, or exposure). When the HIC values of
ADCs with various site mutations were compared with their measured
AUC in rats, the distribution in FIG. 26 was observed.
[0352] ADCs with RRT 1.9 showed lower AUC values, while ADCs with
lower RRT tended to have higher AUC, although the relationship was
not direct. The ADC T(kK183C+K290C)-vc0101 was observed to have a
relatively higher RRT (mean value of 1.77) and therefore was
expected to have a relatively lower AUC. Surprisingly, the observed
AUC was relatively high, hence it was not obvious to predict the
exposure of this ADC from the hydrophobicity data.
Example 18:Toxicity Studies
[0353] In two independent exploratory toxicity studies, a total of
ten male and female cynomolgus monkeys were divided into 5 dosage
groups (1/gender/dosage) and dosed IV once every 3 weeks (study
days 1, 22 and 43). On study day 46 (3 days after the 3.sup.rd dose
administration) animals were euthanized and protocol specified
blood and tissue samples were collected. Clinical observations,
clinical pathology, macroscopic and microscopic pathology
evaluations were conducted in life and post necropsy. For anatomic
pathology evaluation, severity of histopathology findings was
recorded on a subjective, relative, study specific basis.
[0354] In cynomolgus monkey exploratory toxicity studies at 3 and 5
mg/kg, T-vc0101 caused transient but marked (390/.mu.l) to severe
(40/.mu.l to non-detectable) neutropenia at Day 11 post the first
dose. In contrast at 9 mg/kg, all cynomolgus monkeys dosed with
T(kK183C+K290C)-vc0101 had none to minimal neutropenia with
neutrophil counts well above 500/.mu.l at any time-points tested
(FIG. 27). In fact, T(kK183C+K290C)-vc0101 dosed animals showed
average neutrophil counts (>1000 .mu.L) at day 11 and 14 as
compared to vehicle controls.
[0355] Microscopically in the bone marrow at 3 and 5 mg/kg, the
cynomolgus monkey dosed with T-vc0101 had compound-related
increased M/E ratio. Increased myeloid/erythroid (M/E) ratio
consisted of decreased erythroid precursors combined with an
increase of primarily mature granulocytes. In contrast, at 6 and 9
mg/kg, only the male dosed with T(kK183C+K290C)-vc0101 at 6
mg/kg/dose had minimal to mild increased cellularity of mature
granulocytes (data not shown).
[0356] Therefore, the hematologic and microscopic data clearly
indicated that the ADC conjugate based on site-specific-mutation
technology, T(kK183C+K290C)-vc010 clearly improved the T-vc010
induced bone marrow toxicity and neutropenia.
Example 19: ADC Crystal Structure
[0357] The crystal structures were obtained for
T(K290C+K334C)-vc0101, T(K290C+K392C)-vc0101 and
T(K334C+K392C)-vc0101. These particular ADCs were chosen for
crystallography since conjugation to the K290C+K334C and
K334C+K392C double cysteine-variants, but not the K290C+K392C,
abolished ADCC activity.
[0358] The conjugated Fc regions were prepared for crystallography
using papain cleavage of the ADCs. Crystals of the same morphology
were obtained for the three conjugated IgG1-Fc regions using the
same conditions: 100 mM NaCitrate pH 5.0+100 mM MgCl.sub.2+15% PEG
4K.
[0359] Wild type human IgG1-Fc structures deposited in the PDB are
relatively similar showing that the CH2-CH2 domains contact each
other through Asn297-linked glycans (carbohydrate or glycan
antennas) and that the CH3-CH3 domains form a stable interface that
is relatively constant between structures. Fc structures exist in
either a "closed" or "open" confirmation and the deglycosylated Fc
structure adopts the "open" structure conformation thus
demonstrating that the glycan antennas hold the CH2 regions
together. Additionally, a published structure of an unconjugated
Phe241Ala-IgG1 Fc mutant (Yu et al. "Engineering Hydrophobic
Protein-Carbohydrate interactions to fine-tune monoclonal
antibodies". JACS 2013) shows one partially disordered CH2 domain
since this mutation leads to destabilization of CH2-glycan
interface and CH2-CH2 interface since aromatic Phe residue cannot
stabilize the carbohydrate.
[0360] The "CH2 domain" of a human IgG Fc region (also referred to
as "Cy2" domain) usually extends from about amino acid 231 to about
amino acid 340. The CH2 domain is unique in that it is not closely
paired with another domain. Rather, two N-linked branched
carbohydrate chains are interposed between the two CH2 domains of
an intact native IgG molecule. It has been speculated that the
carbohydrate may provide a substitute for the domain-domain pairing
and help stabilize the CH2 domain (Burton et al., 1985, Molec.
Immunol. 22: 161-206).
[0361] The "CH3 domain" comprises the stretch of residues
C-terminal to a CH2 domain in an Fc region (i.e. from about amino
acid residue 341 to about amino acid residue 447 of an IgG).
[0362] The solved structures for both T(K290C+K334C)-vc0101 and
T(K290C+K392C)-vc0101 Fc regions were similar showing that the Fc
dimer contained one CH2 and both CH3s that were highly ordered
(like wild type Fc). However, they also contain a disordered CH2
with glycan attached (FIG. 28A and FIG. 28B). The higher degree of
destabilization of one CH2 domain was attributed to the close
proximity of conjugation sites to glycan antennas. Considering 0101
payload geometry, conjugation at any of K290, K334, K392 sites
could perturb the overall trajectory of the glycan away from the
CH2 surface destabilizing the glycan and the CH2 structure itself
and as a result the CH2-CH2 interface (FIG. 28C). A higher degree
of heterogeneity is available to these 0101 site-specifically
conjugated double cysteine-Fc-variants relative to WT-Fc,
Phe241Ala-Fc or deglycosylated-Fc. When engineered cysteine-variant
positions were mapped on the structure of WT-Fc in complex with
Fc.gamma.R type IIb, it showed that conjugation at C334 could
directly interfere with binding to Fc.gamma.RIIb (FIG. 28C). This
heterogeneity in CH2 positioning caused by mutation or conjugation
could result in significant decrease in FcRIIb binding. Therefore
these results suggested that either conformation heterogeneity or
conjugation of 0101 to certain combinations of engineered cysteines
within the IgG1-Fc could affect ADCC activity for the double
cysteine variants containing the K334C site, or perhaps both.
Example 20: Different Conjugation Sites Results in Different ADC
Properties
[0363] A. General Procedure for the Synthesis of Cys-Mutant
ADCs
[0364] A solution of trastuzumab incorporating one or more
engineered cyststeine residues (as shown in the Table 22) was
prepared in 50 mM phosphate buffer, pH 7.4. PBS, EDTA (0.5 M
stock), and TCEP (0.5 M stock) were added such that the final
protein concentration was 10 mg/mL, the final EDTA concentration
was 20 mM, and the final TCEP concentration was approximately 6.6
mM (100 molar eq.). The reaction was allowed to stand at rt for 48
h then buffer exchanged into PBS using GE PD-10 Sephadex G25
columns per the manufacturer's instructions. The resulting solution
was treated with approximately 50 equivalents of dehydroascorbate
(50 mM stock in 1:1 EtOH/water). The antibody was allowed to stand
at 4.degree. C. overnight and subsequently buffer exchanged into
PBS using GE PD-10 Sephadex G25 columns per the manufacturer's
instructions. Slight variations of the above procedure were
employed on some mutants.
[0365] The antibody thus prepared was diluted to .about.2.5 mg/mL
in PBS containing 10% DMA (vol/vol) and treated with vc0101 (10
molar eq.) as a 10 mM stock solution in DMA. After 2 h at rt, the
mixture was buffer exchanged into PBS (per above) and purified by
size-exclusion chromatography on a Superdex200 column. The
monomeric fractions were concentrated and filter sterilized to give
the final ADC. See Table 22 below for product characterization.
TABLE-US-00022 TABLE 22 Summary of ADC properties HIC HIC LCMS LCMS
RT relative LCMS Observed Expected % Main retention DAR HIC DAR
Mass Mass mono Peak time ADC (mol/mol) (mol/mol) Shift Shift mer
(min) (RRT) T(A114C) - vc0101 1.9 1.74 1342 1341 94% 7.15 1.40
T(kK183C) - vc0101 2 2 1341 1341 99% 7.05 1.38 T(K290C) - vc0101
2.1 2.1 1341 1341 99% 7.85 1.53 T(K334C) - vc0101 2.1 2.1 1341 1341
99% 5.90 1.15 T(Q347C) - vc0101 1.9 NA 1341 1341 99% 8.41 1.64
T(S375C) - vc0101 2 NA 1340 1341 99% 6.23 1.22 T(E380C) - vc0101 2
1.9 1341 1341 99% 7.93 1.55 T(K388C) - vc0101 1.9 NA 1340 1341 97%
8.75 1.71 T(K392C) - vc0101 2.1 2.1 1341 1341 98% 6.60 1.29
T(N421C) - vc0101 1.9 NA 1342 1341 93% 8.20 1.60 T(L443C) - vc0101
2 2 1344 1341 90% 9.10 1.78 T(kK183C + K334C) - vc0101 3.7 NA 1341
1341 95% 7.00 1.37 T(kK183C + K392C) - vc0101 4 4 1342 1341 97%
7.70 1.50 T(K290C + K334C) - vc0101 4 4 1342 1341 97% 6.03 1.18
T(K334C + K392C) - vc0101 4 4 1343 1341 97% 5.91 1.15 T(K392C +
L443C) - vc0101 3.2 NA 1340 1341.68 97% 10.85 2.12 Trastuzumab mAb
5.12 1.00
[0366] B. General Analytical Methods for Conjugation Examples
[0367] LCMS: Column=Waters BEH300-C4, 2.1.times.100 mm
(P/N=186004496); Instrument=Acquity UPLC with an SQD2 mass spec
detector; Flow rate=0.7 mL/min; Temperature=80.degree. C.; Buffer
A=water+0.1% formic acid; Buffer B=acetonitrile+0.1% formic acid.
The gradient ran from 3% B to 95% B over 2 minutes, holds at 95% B
for 0.75 min, and then re-equilibrates at 3% B. The sample was
reduced with TCEP or DTT immediately prior to injection. The eluate
was monitored by LCMS (400-2000 daltons) and the protein peak was
deconvoluted using MaxEnt1. DAR was reported as a weight average
loading.
[0368] SEC: Column: Superdex200 (5/150 GL); Mobile phase: Phosphate
buffered saline containing 2% acetonitrile, pH 7.4; Flow rate=0.25
mL/min; Temperature=ambient; Instrument: Agilent 1100 HPLC.
[0369] HIC:_Column: TSKGel Butyl NPR, 4.6 mm.times.3.5 cm
(P/N=S0557-835); Buffer A=1.5 M ammonium sulfate containing 10 mM
phosphate, pH 7; Buffer B=10 mM phosphate, pH 7+20% isopropyl
alcohol; Flow rate=0.8 mL/min; Temperature=ambient; Gradient=0% B
to 100% B over 12 minutes, hold at 100% B for 2 minutes, then
re-equilibrate at 100% A; Instrument: Agilent 1100 HPLC.
[0370] C. Determination of Hydrophobicity of Site Specific vc0101
Conjugates
[0371] ADCs of Table 22 were evaluated by hydrophobic interaction
chromatography (method above) in order to determine the relative
hydrophobicity of the various conjugates. ADC hydrophobicity has
been reported to correlate with total antibody exposure.
[0372] Conjugates to sites 334, 375, and 392 exhibited to smallest
shift in retention time as compared to the unmodified antibody
while conjugates to sites 421, 443, and 347 showed the largest
shift in retention time. The relative hydrophobicity of each ADC
was calculated by dividing the retention time of the ADC by the
retention time of the unmodified antibody, thus resulting in a
"relative retention time" or "RRT". An RRT of .about.1 indicates
that the ADC has approximately the same hydrophobicity as the
unmodified antibody. The RRT for each ADC is shown in Table 22.
[0373] D. ADC Plasma Stability of Site Specific vc0101
Conjugates
[0374] ADC samples (.about.1.5 mg/mL) were diluted into mouse, rat
or human plasma to yield a final solution of 50 .mu.g/mL ADC in
plasma. Samples were incubated at 37.degree. C. under 5% CO.sub.2,
and aliquots were taken at three time points (0, 24 h, and 72 h).
Each time point of ADC samples from the plasma incubation (25
.mu.L) was deglycosylated with IgG0 at 37.degree. C. for 1 h.
Following the deglycosylation, a capture antibody (biotinylated
goat anti-human IgG1 Fc.gamma. fragment specific at 1 mg/mL for
mouse and rat plasma, or biotinylated anti-trastuzumab antibody at
1 mg/mL for human plasma) was added and the mixture was heated at
37.degree. C. for 1 h followed by gentle shaking at room
temperature for a second hour. Dynabead MyOne Streptavidin T1
magnetic beads were added to the samples and incubated at room
temperature for 1 h with gentle shaking. The sample plate was then
washed with 200 .mu.L PBS+0.05% Tween-20, 200 .mu.L PBS and HPLC
grade water. The bound ADC was eluted with 55 .mu.L of 2% of formic
acid (FA) (v/v). 50 .mu.L aliquot of each sample were transferred
into a new plate followed by an additional 5 .mu.L of 200 mM
TCEP.
[0375] The intact protein analysis was carried out with Xevo G2
Q-TOF mass spectrometer coupled with nanoAcquity UPLC (Waters)
using BEH300 C4, 1.7 .mu.m, 0.3.times.100 mm iKey column. The
mobile phase A (MPA) consisted of 0.1% FA in water (v/v) and the
mobile phase B (MPB) consisted of 0.1% FA in acetonitrile (v/v).
The chromatographic separation was achieved at a flow rate of 0.3
.mu.L/min using a linear gradient of MPB from 5% to 90% over 7 min.
The LC column temperature was set at 85.degree. C. Data acquisition
was conducted with MassLynx software version 4.1. The mass
acquisition range was from 700 Da to 2400 Da. Data analysis
including deconvolution was performed using Biopharmalynx version
1.33.
[0376] Loading and succinimide ring opening (a +18 dalton peak) was
monitored over time. The loading data is reported as % DAR loss
compared to 0 h DAR. The ring-opening data is reported as the % of
ring-opened species as compared to total species present at 72 h.
Several site mutants resulted in very stable ADCs (334C, 421C, and
443C) while some sites lost significant amounts of linker-payload
(380C and 114C). The rate of ring-opening varied considerably
between the sites. Several sites such as 392C, 183C, and 334C
resulted in very little ring opening while other sites such as
421C, 388C, and 347C resulted in rapid and spontaneous ring
opening.
[0377] Sites that result in rapid and spontaneous ring opening may
be useful for the generation of conjugates that have reduced
hydrophobicity and/or increased PK exposure. This finding runs
counter to the prevailing understanding that ring stability
correlates with plasma stability. In some aspects therefore,
conjugation at one or more of sites 421C, 388C, and 347C can be
particularly advantageous when using a linker-payload with a high
hydrophobicity. In some aspects, high hydrophobicity is a relative
retention time (RRT) value (as measured by HIC) of 1.5 or more. In
some aspects, high hydrophobicity is a RRT value of 1.7 or more. In
some aspects, high hydrophobicity is a RRT value of 1.8 or more. In
some aspects, high hydrophobicity is a RRT value of 1.9 or more. In
some aspects, high hydrophobicity is a RRT value of 2.0 or
more.
TABLE-US-00023 TABLE 23 Plasma stability of various ADCs %
Succinamide ADC % DAR Loss @ 72-h hydrolysis @ 72-h T(K334C)-vc0101
0% 18% T(N421C)-vc0101 0% 100 T(L443C)-vc0101 0% 40%
T(K388C)-vc0101 -1.3% 100% T(K392C)-vc0101 3.0% 0% T(K290C)-vc0101
9.5% 21% T(Q347C)-vc0101 10% 66% T(.kappa.K183C)-vc0101 11% 29%
T(S375C)-vc0101 12% 46% T(A114C)-vc0101 20% 33% T(E380C)-vc0101 49%
29%
[0378] E. Glutathione Stability of Site Specific vc0101
Conjugates
[0379] The ADC samples were diluted into aqueous glutathione to
yield a final GSH concentration of 0.5 mM and final protein
concentration of .about.0.1 mg/mL in a phosphate buffer, pH 7.4.
The samples were then incubated at 37.degree. C. and aliquots were
removed at three time points to determine the DAR (T-0, T-3 day,
T-6 day). The aliquot from each time point was treated with TCEP
and analyzed by LC-MS per the method described in Example 20.A.
[0380] Loading and succinimide ring opening (a +18 dalton peak) was
monitored over time. The loading data is reported as % DAR loss
compared to 0 h DAR. (Table 24) The ring-opening data is reported
as the % of ring-opened species as compared to total species
present at 72 h. Several site mutants resulted in very stable ADCs
(334C, 421C, and 443C) while some sites lost significant amounts of
linker-payload (380C and 114C). The rate of ring-opening varied
considerably between the sites. Several sites such as 392C, 183C,
and 334C resulted in very little ring opening while other sites
such as 421C, 388C, and 347C resulted in considerable ring-opening.
The results of this assay correlates quite well with the plasma
stability results (Example 20.D) suggesting that thiol-mediated
deconjugation is the major pathway of payload loss in plasma.
Combined, these results suggest that particular sites such as 334,
443, 290, and 392 may be especially useful for the conjugation of
payload-linkers that are readily lost through a thiol-mediated
deconjugation. Such payload-linkers include those that utilize the
common mc and vc linkages (e.g. vc-101, vc-MMAE, mc-MMAF etc).
TABLE-US-00024 TABLE 24 Glutathione stability of various vc0101
site specific conjugates % Succinamide ADC % DAR Loss @ 72-h
hydrolysis @ 72-h T(A114C)-vc0101 12% 41% T(.kappa.K183C)-vc0101 7%
17% T(K334C)-vc0101 4% 26% T(Q347C)-vc0101 10% 71% T(S375C)-vc0101
18% 47% T(E380C)-vc0101 79% 50% T(K388C)-vc0101 19% 100%
T(K392C)-vc0101 0% 17% T(N421C)-vc0101 0% 80% T(L443C)-vc0101 12%
41% T(K290C)-vc0101 17% 33%
[0381] F. Pharmacokinetic evaluation of select site specific vc0101
conjugates in mice Non-tumor bearing athymic female nu/nu (nude)
mice (6-8 weeks of age) were obtained from Charles River
Laboratories. All procedures using mice were approved by the
Institutional Animal Care and Use Committee according to
established guidelines. Mice (n=3 or 4) were administered a single
intravenous dose of an ADC at 3 mg/kg based on the antibody
component. Blood samples were collected from each mouse via the
tail vein at 0.083, 6, 24, 48, 96, 168 and 336 hours post-dose. The
total antibody (T.sub.ab) and ADC concentrations were determined by
a LBA where a sheep anti-human IgG antibody was used for capture, a
goat anti-human IgG antibody was used for detection of T.sub.ab or
an anti-payload antibody was used for detection of ADC. Plasma
concentration data for each animal was analyzed using Watson LIMS
version 7.4 (Thermo). Exposure varied based on site. The ADCs made
from the 290C and 443C mutants exhibited the lowest exposure, while
ADCs made from the 183C and 392C sites exhibited the highest
exposure. For many applications, sites with a high exposure may be
preferred, as this will lead to increased duration of therapeutic
agent. However, for certain applications, it may be preferable to
use a conjugate with a lower exposure (such as 290C and 443C). In
particular, applications where a lower exposure (i.e. lower PK) may
include, but are not limited to, use in the brain, the CNS, and the
eye. Indications include cancer, especially of the brain, CNS
and/or eye.
TABLE-US-00025 TABLE 25 PK exposure of various site-specific vc0101
ADCs tAb AUC (0-last) ADC AUC (0-last) ADC (mg * h/mL) (mg * h/mL)
T(.kappa.K183C)-vc0101 7150 5980 T(K290C)-vc0101 4240 3480
T(K334C)-vc0101 5130 4500 T(Q347C)-vc0101 5080 4070 T(K388C)-vc0101
6100 3680 T(K392C)-vc0101 6400 6010 T(L443C)-vc0101 4430 4500
[0382] G. Cathepsin Cleavage of Site Specific vc0101 Conjugates
[0383] Cathepsin B was activated using 6 mM dithiothreitol (DTT) in
150 mM sodium acetate, pH 5.2 for 15 min at 37.degree. C. 50 ng of
the activated cathepsin-B was then mixed with 20 uL of 1 mg/mL of
ADC at a final concentration of 2 mM DTT, 50 mM sodium acetate, pH
5.2. Reactions were quenched using 10 uM E-64 cysteine protease
inhibitor in 250 mM borate buffer, pH 8.5 following incubation at
37.degree. C. for 20 min, 1 h, 2 h and 4 h. After the assay, the
samples were reduced using TCEP and analyzed by LC/MS using the
conditions described in Example 21.A. The data showed that the rate
of linker cleavage depends heavily on the site of conjugation.
Particular sites are cleaved very quickly, such as 443C, 388C, and
290C while other sites are cleaved very slowly, such as 334C, 375C,
and 392C. In some aspects, it may be advantageous to conjugate to
sites that lend themselves to slow cleavage. In other aspects,
quick cleavage is preferred. For example, it may be preferable to
release the payload quickly to reduce time spent in the endosome.
In further aspects rapid payload cleavage can be advantageously
permit penetration of the payload where the conjugated molecule may
not be able to do so, such as certain solid tumors. In further
aspects, rapid cleavage can permit the payload to be delivered to
adjacent cells that do not express the antibody's antigen, thus
permitting treatment of a heterogenous tumor, for example.
TABLE-US-00026 TABLE 26 Linker cleavage kinetics of various
site-specific vc0101 ADCs % Linker % Linker % Linker % Linker
cleavage cleavage cleavage cleavage ADC @ 20 min @ 1 h @ 2 h @ 4 h
T(A114C)-vc0101 29% 71% 100% 100% T(.kappa.K183C)-vc0101 31% 95%
100% 100% T(K290C)-vc0101 54% 100% 100% 100% T(K334C)-vc0101 0% 0%
0% 13% T(Q347C)-vc0101 42% 89% 100% 100% T(S375C)-vc0101 0% 0% 0%
5% T(E380C)-vc0101 15% 48% 83% 92% T(K388C)-vc0101 82% 100% 100%
100% T(K392C)-vc0101 0% 0% 0% 0% T(N421C)-vc0101 31% 61% 73% 100%
T(L443C)-vc0101 100% 100% 100% 100%
[0384] H. Thermal Stability of Site Specific vc0101 Conjugates
[0385] The ADC was diluted to 0.2 mg/mL in PBS (pH 7.4) containing
10 mM EDTA. The ADCs were placed in a sealed vial and heated to
45.degree. C. An aliquot (10 .mu.L) was removed at 1-week
increments to evaluate the level of high molecular weight species
(HMWS) and low molecular weight species (LMWS) that formed over
time by size exclusion chromatography (SEC). The SEC conditions are
outlined in Example 21.A. Under these conditions, the monomer
eluted at approximately 3.6 minutes. Any protein material eluting
to the left of the monomer peak was counted as HMWS and any protein
material eluting to the right of the monomer peak was counted as
LMWS. Results are shown in Table 27 below. Select ADCs showed
excellent thermal stability, such as 183C, 375C, and 334C, while
other ADCs showed significant decomposition, such as 443C and
392C+443C.
TABLE-US-00027 TABLE 27 Thermal stability of various site-specific
vc0101 ADCs Day 1 Day 1 Day 1 Day 21 Day 21 Day 21 ADC (HMWS)
(LMWS) (Monomer) (HMWS) (LMWS) (Monomer) T(A114C) - vc0101 3.31%
3.00% 93.60% 1.70% 5.30% 93.80% T(kK183C) - vc0101 0.40% 0.60%
99.00% 0.40% 1.30% 98.30% T(K290C) - vc0101 0.90% 0.30% 98.70%
2.00% 2.80% 95.20% T(K334C) - vc0101 0.80% 0.40% 98.80% 1.10% 2.60%
96.30% T(Q347C) - vc0101 1.10% 0.40% 98.50% 1.20% 1.50% 97.30%
T(S375C) - vc0101 0.70% 0.60% 98.70% 0.80% 2.10% 97.20% T(E380C) -
vc0101 0.90% 0.30% 98.80% 1.60% 1.70% 96.60% T(K388C) - vc0101
1.90% 0.70% 97.40% 1.20% 2.10% 96.70% T(K392C) - vc0101 1.20% 0.50%
98.30% 1.40% 2.40% 96.10% T(N421C) - vc0101 2.60% 4.30% 93.00%
2.60% 6.10% 91.30% T(L443C) - vc0101 5.20% 4.60% 90.10% 5.80% 6.30%
87.40% T(kK183C + K334C) - vc0101 4.60% 0.50% 94.90% 5.70% 1.90%
92.40% T(kK183C + K392C) - vc0101 2.10% 0.70% 97.10% 2.10% 1.60%
96.30% T(K290C + K334C) - vc0101 2.80% 0.60% 96.60% 4.30% 1.90%
93.70% T(K334C + K392C) - vc0101 1.90% 0.70% 97.40% 2.70% 2.40%
94.90% T(K392C + L443C) - vc0101 2.80% 0.60% 96.60% 8.80% 2.90%
88.30%
[0386] I. Efficacy of Various vc0101 Site-Mutants
[0387] In vivo efficacy studies of antibody-drug conjugates were
performed in a target-expressing xenograft model using the N87 cell
line. Approximately 7.5 million tumor cells in 50% matrigel were
implanted subcutaneously into 6-8 weeks old nude mice until the
tumor sizes reach between 250 and 350 mm.sup.3. The drug was dosed
through bolus tail vein injection. Animals were injected with 10,
3, or 1 mg/kg of antibody drug conjugate a total of four times,
once every 4 days (on days 1, 5, 9, and 13). All experimental
animals are monitored for body weight changes weekly. Tumor volume
is measured twice a week for the first 50 days and once weekly
thereafter by a Caliper device and calculated with the following
formula: Tumor volume=(length.times.width.sup.2)/2. Animals are
humanely sacrificed before their tumor volumes reach 2500 mm.sup.3.
The tumor size is generally observed to decrease after the first
week of treatment. Animals were monitored continuously for tumor
re-growth after the treatment has discontinued (up to 100 days
post-treatment). Data from the 3mpk dosing group is shown in FIG.
29. ADCs generated from the 388C and 347C mutants exhibited
slightly lower potency than ADCs from the 334C, 183C, 392C and 443C
mutants.
Sequence CWU 1
1
951120PRTArtificial SequenceSynthetic peptide sequence 1Glu 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 Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
12025PRTArtificial SequenceSynthetic peptide sequence 2Asp Thr Tyr
Ile His1 5317PRTArtificial SequenceSynthetic peptide sequence 3Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys1 5 10
15Gly411PRTArtificial SequenceSynthetic peptide sequence 4Trp Gly
Gly Asp Gly Phe Tyr Ala Met Asp Tyr1 5 105329PRTArtificial
SequenceSynthetic peptide sequence 5Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230
235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly 3256449PRTArtificial SequenceSynthetic peptide
sequence 6Glu 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 Asp Gly
Phe Tyr Ala Met 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 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 Glu Glu Met 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 445Gly7107PRTArtificial
SequenceSynthetic peptide sequence 7Asp 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 Asp Val Asn 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
Leu Tyr Ser 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 105811PRTArtificial
SequenceSynthetic peptide sequence 8Arg Ala Ser Gln Asp Val Asn Thr
Ala Val Ala1 5 1097PRTArtificial SequenceSynthetic peptide sequence
9Ser Ala Ser Phe Leu Tyr Ser1 5109PRTArtificial SequenceSynthetic
peptide sequence 10Gln Gln His Tyr Thr Thr Pro Pro Thr1
511107PRTArtificial SequenceSynthetic peptide sequence 11Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25
30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
100 10512214PRTArtificial SequenceSynthetic peptide sequence 12Asp
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 Asp Val Asn 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 Leu Tyr Ser 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 Glu Gln 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 21013330PRTArtificial
SequenceSynthetic peptide sequence 13Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val Glu Pro Lys Ser Cys Asp Arg Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33014450PRTArtificial
SequenceSynthetic peptide sequence 14Glu 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 Asp Gly Phe Tyr Ala Met 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
220Arg 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 Glu Glu Met 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 45015329PRTArtificial SequenceSynthetic peptide sequence 15Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Cys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32516449PRTArtificial
SequenceSynthetic peptide sequence 16Glu 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 Asp Gly Phe Tyr Ala Met 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 Leu Leu Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Cys 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 Glu Glu Met 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
445Gly17329PRTArtificial SequenceSynthetic peptide sequence 17Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32518449PRTArtificial SequenceSynthetic peptide sequence 18Glu 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 Asp Gly Phe Tyr Ala Met
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 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 Cys 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 Glu Glu
Met 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 445Gly19330PRTArtificial SequenceSynthetic peptide
sequence 19Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 33020450PRTArtificial SequenceSynthetic peptide sequence
20Glu 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 Asp Gly Phe Tyr
Ala Met 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 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 Ala 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 Glu
Glu Met 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 45021330PRTArtificial
SequenceSynthetic peptide sequence 21Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Gln Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33022450PRTArtificial SequenceSynthetic peptide sequence 22Glu 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 Asp Gly Phe Tyr Ala Met
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 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 Gln 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 Glu Glu
Met 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 45023329PRTArtificial SequenceSynthetic
peptide sequence 23Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly 32524449PRTArtificial SequenceSynthetic peptide sequence
24Glu 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 Asp Gly Phe Tyr
Ala Met 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 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 335Cys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu
Glu Met 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 445Gly25329PRTArtificial SequenceSynthetic
peptide sequence 25Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly 32526449PRTArtificial SequenceSynthetic peptide sequence
26Glu 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 Asp Gly Phe Tyr
Ala Met 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 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 Glu
Glu Met 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 Cys 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 445Gly27329PRTArtificial SequenceSynthetic
peptide sequence 27Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Cys Ser
Pro Gly 32528449PRTArtificial SequenceSynthetic peptide sequence
28Glu 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 Asp Gly Phe Tyr Ala Met 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 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 Glu Glu Met 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 Cys Ser Pro 435 440
445Gly29329PRTArtificial SequenceSynthetic peptide sequence 29Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Cys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32530449PRTArtificial SequenceSynthetic peptide sequence 30Glu 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 Asp Gly Phe Tyr Ala Met
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 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 Cys 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 335Cys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu Glu
Met 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 445Gly31329PRTArtificial SequenceSynthetic peptide
sequence 31Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Cys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32532449PRTArtificial SequenceSynthetic peptide sequence 32Glu 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 Asp Gly Phe Tyr Ala Met
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 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 Cys 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 Glu Glu
Met 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 Cys 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 445Gly33330PRTArtificial SequenceSynthetic peptide
sequence 33Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys
Asp Arg Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 33034450PRTArtificial SequenceSynthetic peptide sequence
34Glu 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 Asp Gly Phe Tyr
Ala Met 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 220Arg 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 Ala 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 Glu Glu Met 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 45035330PRTArtificial SequenceSynthetic peptide sequence
35Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1
5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu
Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Arg
Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155
160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280
285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33036450PRTArtificial SequenceSynthetic peptide sequence 36Glu 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 Asp Gly Phe Tyr Ala Met
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 220Arg 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 Gln 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 Glu Glu
Met 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 45037329PRTArtificial SequenceSynthetic
peptide sequence 37Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Cys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser
Pro Gly 32538449PRTArtificial SequenceSynthetic peptide sequence
38Glu 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 Asp Gly Phe Tyr
Ala Met 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 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 335Cys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Glu
Glu Met 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 Cys 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 445Gly39329PRTArtificial SequenceSynthetic
peptide sequence 39Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250
255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270Asn Tyr Cys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Cys Ser
Pro Gly 32540449PRTArtificial SequenceSynthetic peptide sequence
40Glu 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 Asp Gly Phe Tyr
Ala Met 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 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 Glu Glu Met 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 Cys 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 Cys Ser Pro 435 440
445Gly41107PRTArtificial SequenceSynthetic peptide sequence 41Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Cys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10542213PRTArtificial SequenceSynthetic peptide sequence
42Asp 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 Asp Val Asn 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 Leu Tyr Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Arg Ser Gly Thr Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His
Tyr Thr Thr Pro Pro Thr 85 90 95Phe Gly Gln Gly Thr Lys Val 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 Cys 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
21043115PRTArtificial SequenceSynthetic peptide sequence 43Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25
30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp
Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
Gly Gly Leu Leu Gln 100 105 110Gly Pro Pro 11544222PRTArtificial
SequenceSynthetic peptide sequence 44Asp 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 Asp Val Asn 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 Leu Tyr Ser 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 Glu Gln 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 Gly Gly Leu Leu Gln Gly Pro Pro 210 215
22045360DNAArtificial SequenceSynthetic nucleotide sequence
45gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360461347DNAArtificial
SequenceSynthetic nucleotide sequence 46gaggtgcagc tggtggaatc
cggcggaggc ctggtccagc ctggcggatc tctgcggctg 60tcttgcgccg cctccggctt
caacatcaag gacacctaca tccactgggt ccgacaggca 120cctggcaagg
gactggaatg ggtggcccgg atctacccca ccaacggcta caccagatac
180gccgactccg tgaagggccg gttcaccatc tccgccgaca cctccaagaa
caccgcctac 240ctgcagatga actccctgcg ggccgaggac accgccgtgt
actactgctc cagatgggga 300ggcgacggct tctacgccat ggactactgg
ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 540ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 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 ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc cccgggt 134747321DNAArtificial SequenceSynthetic
nucleotide sequence 47gacatccaga tgacccagtc cccctccagc ctgtccgcct
ctgtgggcga cagagtgacc 60atcacctgtc gggcctccca ggacgtgaac accgccgtgg
cctggtatca gcagaagccc 120ggcaaggccc ccaagctgct gatctactcc
gcctccttcc tgtactccgg cgtgccctcc 180cggttctccg gctccagatc
tggcaccgac tttaccctga ccatctccag cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacacca ccccccccac ctttggccag
300ggcaccaagg tggaaatcaa g 32148642DNAArtificial SequenceSynthetic
nucleotide sequence 48gacatccaga tgacccagtc cccctccagc ctgtccgcct
ctgtgggcga cagagtgacc 60atcacctgtc gggcctccca ggacgtgaac accgccgtgg
cctggtatca gcagaagccc 120ggcaaggccc ccaagctgct gatctactcc
gcctccttcc tgtactccgg cgtgccctcc 180cggttctccg gctccagatc
tggcaccgac tttaccctga ccatctccag cctgcagccc 240gaggacttcg
ccacctacta ctgccagcag cactacacca ccccccccac ctttggccag
300ggcaccaagg tggaaatcaa gcggaccgtg gccgctccct ccgtgttcat
cttcccaccc 360tccgacgagc agctgaagtc cggcaccgcc tccgtcgtgt
gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagtccgg caactcccag 480gaatccgtca ccgagcagga
ctccaaggac agcacctact ccctgtcctc caccctgacc 540ctgtccaagg
ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc
600ctgtccagcc ccgtgaccaa gtccttcaac cggggcgagt gc
64249990DNAArtificial SequenceSynthetic nucleotide sequence
49gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtagtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg accgtactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc tccgggaaaa 990501350DNAArtificial SequenceSynthetic
nucleotide sequence 50gaggtgcagc tggtggagtc cggcggcggc ctggttcagc
ccggcggatc actgaggctc 60tcctgtgccg ccagcggctt caacatcaag gacacataca
tccactgggt tcgccaggct 120cctggcaagg gactggagtg ggtcgctagg
atctacccca ccaatgggta caccaggtac 180gccgactccg tgaaggggcg
gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240ttgcagatga
actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc
300ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac
cgtctcctca 360gcctccacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg accgtactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 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 ctcccagccc
ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggaaaa
135051987DNAArtificial SequenceSynthetic nucleotide sequence
51gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccatgcccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc cccgggt 987521347DNAArtificial SequenceSynthetic
nucleotide sequence 52gaggtgcagc tggtggaatc cggcggaggc ctggtccagc
ctggcggatc tctgcggctg 60tcttgcgccg cctccggctt caacatcaag gacacctaca
tccactgggt ccgacaggca 120cctggcaagg gactggaatg ggtggcccgg
atctacccca ccaacggcta caccagatac 180gccgactccg tgaagggccg
gttcaccatc tccgccgaca cctccaagaa caccgcctac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga
300ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac
cgtgtctagc 360gcgtcgacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccatgcccc
aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc cccgggt
134753987DNAArtificial SequenceSynthetic nucleotide sequence
53gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacatgcc
cgcgggagga gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc cccgggt 987541347DNAArtificial SequenceSynthetic
nucleotide sequence 54gaggtgcagc tggtggaatc cggcggaggc ctggtccagc
ctggcggatc tctgcggctg 60tcttgcgccg cctccggctt caacatcaag gacacctaca
tccactgggt ccgacaggca 120cctggcaagg gactggaatg ggtggcccgg
atctacccca ccaacggcta caccagatac 180gccgactccg tgaagggccg
gttcaccatc tccgccgaca cctccaagaa caccgcctac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga
300ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac
cgtgtctagc 360gcgtcgacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacatgcc
cgcgggagga gcagtacaac 900agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc cccgggt 134755990DNAArtificial SequenceSynthetic
nucleotide sequence 55gcctccacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 180ggactctact ccctcagcag
cgtagtgacc gtgccctcca gcagcttggg cacccagacc 240tacatctgca
acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc
300aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact
cctgggggga 360ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc
tcatgatctc ccggacccct 420gaggtcacat gcgtggtggt ggacgtgagc
cacgaagacc ctgaggtcaa gttcaactgg 480tacgtggacg gcgtggaggt
gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 540agcacgtacc
gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag
600gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa
aaccatctcc 660aaagccaaag ggcagccccg agaaccacag gtgtacaccc
tgcccccatc ccgggaggag 720atgaccaaga accaggtcag cctgacctgc
ctggtcaaag gcttctatcc cagcgacatc 780gccgtggagt gggagagcaa
tgggcagccg gagaacaact acaagaccac gcctcccgtg 840ctggactccg
acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg
900cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 960cagaagagcc tctccctgtc tccgggaaaa
990561350DNAArtificial SequenceSynthetic nucleotide sequence
56gaggtgcagc tggtggagtc cggcggcggc ctggttcagc ccggcggatc actgaggctc
60tcctgtgccg ccagcggctt caacatcaag gacacataca tccactgggt tcgccaggct
120cctggcaagg gactggagtg ggtcgctagg atctacccca ccaatgggta
caccaggtac 180gccgactccg tgaaggggcg gttcacaatc tcagccgata
ctagcaaaaa tacagcctac 240ttgcagatga actccctgag agcagaggat
accgccgtgt actattgctc tcgctggggc 300ggcgacggct tctacgctat
ggattattgg ggccagggaa ccttggtcac cgtctcctca 360gcctccacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtagtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
720ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacgcc 900agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc tccgggaaaa 1350571055DNAArtificial
SequenceSynthetic nucleotide sequence 57gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtagtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtaccaa 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc
tccgggaaaa gccgccagcg gcttcaacat caaggacaca 1020tacatccact
gggttcgcca ggctcctggc aaggg 1055581415DNAArtificial
SequenceSynthetic nucleotide sequence 58gaggtgcagc tggtggagtc
cggcggcggc ctggttcagc ccggcggatc actgaggctc 60tcctgtgccg ccagcggctt
caacatcaag gacacataca tccactgggt tcgccaggct 120cctggcaagg
gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac
180gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa
tacagcctac 240ttgcagatga actccctgag agcagaggat accgccgtgt
actattgctc tcgctggggc 300ggcgacggct tctacgctat ggattattgg
ggccagggaa ccttggtcac cgtctcctca 360gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 540ggactctact ccctcagcag cgtagtgacc gtgccctcca
gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtaccaa 900agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca
1380tacatccact gggttcgcca ggctcctggc aaggg 141559987DNAArtificial
SequenceSynthetic nucleotide sequence 59gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagtg caccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc cccgggt
987601347DNAArtificial SequenceSynthetic nucleotide sequence
60gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
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 ctcccagccc ccatcgagtg caccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc cccgggt 134761987DNAArtificial
SequenceSynthetic nucleotide sequence 61gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc cccgggt
987621347DNAArtificial SequenceSynthetic nucleotide sequence
62gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccttggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
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 ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact actgcaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc cccgggt 134763987DNAArtificial
SequenceSynthetic nucleotide sequence 63gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctcctgctc cccgggt
987641347DNAArtificial SequenceSynthetic nucleotide sequence
64gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
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 ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact acaagaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctcctgctc cccgggt 134765987DNAArtificial
SequenceSynthetic nucleotide sequence 65gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacatgcc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagtg caccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc cccgggt
987661347DNAArtificial SequenceSynthetic nucleotide sequence
66gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac
900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagtg caccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc cccgggt
134767987DNAArtificial SequenceSynthetic nucleotide sequence
67gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacatgcc
cgcgggagga gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
actgcaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc cccgggt 987681347DNAArtificial SequenceSynthetic
nucleotide sequence 68gaggtgcagc tggtggaatc cggcggaggc ctggtccagc
ctggcggatc tctgcggctg 60tcttgcgccg cctccggctt caacatcaag gacacctaca
tccactgggt ccgacaggca 120cctggcaagg gactggaatg ggtggcccgg
atctacccca ccaacggcta caccagatac 180gccgactccg tgaagggccg
gttcaccatc tccgccgaca cctccaagaa caccgcctac 240ctgcagatga
actccctgcg ggccgaggac accgccgtgt actactgctc cagatgggga
300ggcgacggct tctacgccat ggactactgg ggccagggca ccctggtcac
cgtgtctagc 360gcgtcgacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacatgcc cgcgggagga gcagtacaac
900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact actgcaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc cccgggt
134769990DNAArtificial SequenceSynthetic nucleotide sequence
69gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtagtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg accgtactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtacgcc 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc tccgggaaaa 990701350DNAArtificial SequenceSynthetic
nucleotide sequence 70gaggtgcagc tggtggagtc cggcggcggc ctggttcagc
ccggcggatc actgaggctc 60tcctgtgccg ccagcggctt caacatcaag gacacataca
tccactgggt tcgccaggct 120cctggcaagg gactggagtg ggtcgctagg
atctacccca ccaatgggta caccaggtac 180gccgactccg tgaaggggcg
gttcacaatc tcagccgata ctagcaaaaa tacagcctac 240ttgcagatga
actccctgag agcagaggat accgccgtgt actattgctc tcgctggggc
300ggcgacggct tctacgctat ggattattgg ggccagggaa ccttggtcac
cgtctcctca 360gcctccacca agggcccatc ggtcttcccc ctggcaccct
cctccaagag cacctctggg 420ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 480tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540ggactctact
ccctcagcag cgtagtgacc gtgccctcca gcagcttggg cacccagacc
600tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 660aaatcttgtg accgtactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 720ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 780gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc
900agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 960gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 1020aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggaggag 1080atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
1200ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa
gagcaggtgg 1260cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 1320cagaagagcc tctccctgtc tccgggaaaa
1350711055DNAArtificial SequenceSynthetic nucleotide sequence
71gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
60ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
120tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180ggactctact ccctcagcag cgtagtgacc gtgccctcca
gcagcttggg cacccagacc 240tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300aaatcttgtg accgtactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
420gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtaccaa 540agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 600gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
720atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc
tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca
1020tacatccact gggttcgcca ggctcctggc aaggg 1055721415DNAArtificial
SequenceSynthetic nucleotide sequence 72gaggtgcagc tggtggagtc
cggcggcggc ctggttcagc ccggcggatc actgaggctc 60tcctgtgccg ccagcggctt
caacatcaag gacacataca tccactgggt tcgccaggct 120cctggcaagg
gactggagtg ggtcgctagg atctacccca ccaatgggta caccaggtac
180gccgactccg tgaaggggcg gttcacaatc tcagccgata ctagcaaaaa
tacagcctac 240ttgcagatga actccctgag agcagaggat accgccgtgt
actattgctc tcgctggggc 300ggcgacggct tctacgctat ggattattgg
ggccagggaa ccttggtcac cgtctcctca 360gcctccacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 420ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg
480tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 540ggactctact ccctcagcag cgtagtgacc gtgccctcca
gcagcttggg cacccagacc 600tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg accgtactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 720ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct
780gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa
gttcaactgg 840tacgtggacg gcgtggaggt gcataatgcc aagacaaagc
cgcgggagga gcagtaccaa 900agcacgtacc gtgtggtcag cgtcctcacc
gtcctgcacc aggactggct gaatggcaag 960gagtacaagt gcaaggtctc
caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020aaagccaaag
ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag
1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 1200ctggactccg acggctcctt cttcctctat
agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320cagaagagcc
tctccctgtc tccgggaaaa gccgccagcg gcttcaacat caaggacaca
1380tacatccact gggttcgcca ggctcctggc aaggg 141573987DNAArtificial
SequenceSynthetic nucleotide sequence 73gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagtg caccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc cccgggt
987741347DNAArtificial SequenceSynthetic nucleotide sequence
74gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
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 ctcccagccc ccatcgagtg caccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact actgcaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctccctgtc cccgggt 134775987DNAArtificial
SequenceSynthetic nucleotide sequence 75gcgtcgacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg
cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag
gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720atgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc
780gccgtggagt gggagagcaa tgggcagccg gagaacaact actgcaccac
gcctcccgtg 840ctggactccg acggctcctt cttcctctat agcaagctca
ccgtggacaa gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg
atgcatgagg ctctgcacaa ccactacacg 960cagaagagcc tctcctgctc cccgggt
987761347DNAArtificial SequenceSynthetic nucleotide sequence
76gaggtgcagc tggtggaatc cggcggaggc ctggtccagc ctggcggatc tctgcggctg
60tcttgcgccg cctccggctt caacatcaag gacacctaca tccactgggt ccgacaggca
120cctggcaagg gactggaatg ggtggcccgg atctacccca ccaacggcta
caccagatac 180gccgactccg tgaagggccg gttcaccatc tccgccgaca
cctccaagaa caccgcctac 240ctgcagatga actccctgcg ggccgaggac
accgccgtgt actactgctc cagatgggga 300ggcgacggct tctacgccat
ggactactgg ggccagggca ccctggtcac cgtgtctagc 360gcgtcgacca
agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg
420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt
gacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcc
cggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgacc
gtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatca
caagcccagc aacaccaagg tggacaagaa agttgagccc 660aaatcttgtg
acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
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 ctcccagccc ccatcgagaa aaccatctcc
1020aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc
ccgggaggag 1080atgaccaaga accaggtcag cctgacctgc ctggtcaaag
gcttctatcc cagcgacatc 1140gccgtggagt gggagagcaa tgggcagccg
gagaacaact actgcaccac gcctcccgtg 1200ctggactccg acggctcctt
cttcctctat agcaagctca ccgtggacaa gagcaggtgg 1260cagcagggga
acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg
1320cagaagagcc tctcctgctc cccgggt 134777321DNAArtificial
SequenceSynthetic nucleotide sequence 77cggaccgtgg ccgctccctc
cgtgttcatc ttcccaccct ccgacgagca gctgaagtcc 60ggcaccgcct ccgtcgtgtg
cctgctgaac aacttctacc cccgcgaggc caaggtgcag 120tggaaggtgg
acaacgccct gcagtccggc aactcccagg aatccgtcac cgagcaggac
180tccaaggaca gcacctactc cctgtcctcc accctgaccc tgtcctgcgc
cgactacgag 240aagcacaagg tgtacgcctg cgaagtgacc caccagggcc
tgtccagccc cgtgaccaag 300tccttcaacc ggggcgagtg c
32178642DNAArtificial SequenceSynthetic nucleotide sequence
78gacatccaga tgacccagtc cccctccagc ctgtccgcct ctgtgggcga cagagtgacc
60atcacctgtc gggcctccca ggacgtgaac accgccgtgg cctggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctactcc gcctccttcc tgtactccgg
cgtgccctcc 180cggttctccg gctccagatc tggcaccgac tttaccctga
ccatctccag cctgcagccc 240gaggacttcg ccacctacta ctgccagcag
cactacacca ccccccccac ctttggccag 300ggcaccaagg tggaaatcaa
gcggaccgtg gccgctccct ccgtgttcat cttcccaccc 360tccgacgagc
agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac
420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg
caactcccag 480gaatccgtca ccgagcagga ctccaaggac agcacctact
ccctgtcctc caccctgacc 540ctgtcctgcg ccgactacga gaagcacaag
gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa
gtccttcaac cggggcgagt gc 64279321DNAArtificial SequenceSynthetic
nucleotide sequence 79cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat
ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg cctgctgaat aacttctatc
ccagagaggc caaagtacag 120tggaaggtgg ataacgccct ccaatcgggt
aactcccagg agagtgtcac agagcaggac 180agcaaggaca gcacctacag
cctcagcagc accctgacgc tgagcaaagc agactacgag 240aaacacaaag
tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag
300agcttcaaca
ggggagagtg t 32180666DNAArtificial SequenceSynthetic nucleotide
sequence 80gatatccaga tgacacagtc cccctccagc ctctccgcta gtgtcggaga
tagagtgaca 60attacatgtc gggcaagcca ggacgtcaat accgccgtgg cctggtatca
gcagaagcca 120ggaaaggccc caaaactcct gatctactcc gcctccttcc
tgtactcagg ggtcccttca 180cgcttctccg gttcccggag cggcaccgac
ttcactctga ctatctcaag cttgcagccc 240gaggacttcg ccacatacta
ttgccagcag cactatacca ccccccctac cttcggtcag 300ggaactaagg
tggaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca
360tctgatgagc 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 gtggtggcct gcttcagggc 660ccacca
666818PRTArtificial SequenceSynthetic peptide sequence 81Gly Gly
Leu Leu Gln Gly Pro Pro1 5827PRTArtificial SequenceSynthetic
peptide sequence 82Gly Gly Leu Leu Gln Gly Gly1 5835PRTArtificial
SequenceSynthetic peptide sequence 83Leu Leu Gln Gly Ala1
5847PRTArtificial SequenceSynthetic peptide sequence 84Gly Gly Leu
Leu Gln Gly Ala1 5854PRTArtificial SequenceSynthetic peptide
sequence 85Leu Leu Gln Gly1866PRTArtificial SequenceSynthetic
peptide sequence 86Leu Leu Gln Gly Pro Gly1 5876PRTArtificial
SequenceSynthetic peptide sequence 87Leu Leu Gln Gly Pro Ala1
5885PRTArtificial SequenceSynthetic peptide sequence 88Leu Leu Gln
Gly Pro1 5894PRTArtificial SequenceSynthetic peptide sequence 89Leu
Leu Gln Pro1906PRTArtificial SequenceSynthetic peptide sequence
90Leu Leu Gln Pro Gly Lys1 5918PRTArtificial SequenceSynthetic
peptide sequence 91Leu Leu Gln Gly Ala Pro Gly Lys1
5927PRTArtificial SequenceSynthetic peptide sequence 92Leu Leu Gln
Gly Ala Pro Gly1 5936PRTArtificial SequenceSynthetic peptide
sequence 93Leu Leu Gln Gly Ala Pro1 5948PRTArtificial
SequenceSynthetic peptide sequenceMISC_FEATURE(4)..(4)Xaa is G or
PMISC_FEATURE(5)..(5)Xaa is A, G, P, or
absentMISC_FEATURE(6)..(6)Xaa is A, G, K, P, or
absentMISC_FEATURE(7)..(7)Xaa is G, K or
absentMISC_FEATURE(8)..(8)Xaa is K or absent 94Leu Leu Gln Xaa Xaa
Xaa Xaa Xaa1 5958PRTArtificial SequenceSynthetic peptide
sequenceMISC_FEATURE(4)..(4)Xaa is any naturally occurring amino
acidMISC_FEATURE(5)..(5)Xaa is any naturally occurring amino acid
or absentMISC_FEATURE(6)..(6)Xaa is any naturally occurring amino
acid or absentMISC_FEATURE(7)..(7)Xaa is any naturally occurring
amino acid or absentMISC_FEATURE(8)..(8)Xaa is any naturally
occurring amino acid or absent 95Leu Leu Gln Xaa Xaa Xaa Xaa Xaa1
5
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