U.S. patent application number 17/395223 was filed with the patent office on 2022-06-16 for antibody for anti-claudin 18a2 and use thereof.
The applicant listed for this patent is CAFA THERAPEUTICS LIMITED. Invention is credited to Hua JIANG, Zonghai LI, Zhimin SHI, Huamao WANG, Peng WANG, Linlin YANG.
Application Number | 20220185880 17/395223 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220185880 |
Kind Code |
A1 |
WANG; Peng ; et al. |
June 16, 2022 |
ANTIBODY FOR ANTI-CLAUDIN 18A2 AND USE THEREOF
Abstract
Provided in the present invention is an antibody for
anti-claudin 18A2 and an immune effector cell targeting claudin
18A2. Also provided are methods for inducing cell death and
treating tumours.
Inventors: |
WANG; Peng; (Shanghai,
CN) ; JIANG; Hua; (Shanghai, CN) ; YANG;
Linlin; (Shanghai, CN) ; SHI; Zhimin;
(Shanghai, CN) ; WANG; Huamao; (Shanghai, CN)
; LI; Zonghai; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CAFA THERAPEUTICS LIMITED |
Dublin |
|
IE |
|
|
Appl. No.: |
17/395223 |
Filed: |
August 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16316331 |
Jan 8, 2019 |
11111295 |
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PCT/CN2017/092381 |
Jul 10, 2017 |
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17395223 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; C12N 15/85 20060101 C12N015/85; A61P 35/00 20060101
A61P035/00; C12N 5/10 20060101 C12N005/10; A61K 39/395 20060101
A61K039/395; C07K 16/30 20060101 C07K016/30; C07K 19/00 20060101
C07K019/00; A61K 47/68 20060101 A61K047/68; A61K 9/00 20060101
A61K009/00; A61K 31/282 20060101 A61K031/282; A61K 31/704 20060101
A61K031/704; A61K 31/7115 20060101 A61K031/7115; A61K 35/17
20060101 A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2016 |
CN |
201610536449.9 |
Apr 26, 2017 |
CN |
PCT/CN2017/082024 |
Claims
1. An antibody or antigen binding unit thereof that specifically
binds to claudin 18A2, wherein the antibody or antigen binding unit
comprises a variable heavy chain comprising at least one CDR
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:31, 32, 33, 37, 38, 39, 43, 44, 45, 49, 50,
51, 83, 84, 85, and an amino acid sequence which is at least 80%
identical thereto and/or a variable light chain comprising at least
one CDR comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:34, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53,
54, and an amino acid sequence which is at least 80% identical
thereto.
2. The antibody or antigen binding unit of claim 1, wherein (a) the
antibody or antigen binding unit does not significantly bind to a
claudin 18A1 peptide, or (b) the antibody or antigen binding unit
exhibits less non-specific binding to a claudin 18A1 peptide when
compared with a reference antigen binding unit.
3. The antibody or antigen binding unit of claim 2, wherein, (a)
HCDR1, HCDR2 and HCDR3 comprise the amino acid sequences of,
respectively: i) SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; ii)
SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39; iii) SEQ ID NO:43,
SEQ ID NO:44, and SEQ ID NO:45; iv) SEQ ID NO:49, SEQ ID NO:50, and
SEQ ID NO:51; v) SEQ ID NO:31, SEQ ID NO:83, and SEQ ID NO:33; vi)
SEQ ID NO:31, SEQ ID NO:84, SEQ ID NO:33; or vii) SEQ ID NO:49, SEQ
ID NO:85, SEQ ID NO:51, and/or (b) LCDR1, LCDR2 and LCDR3 comprise
the amino acid sequences of, respectively: i) SEQ ID NO:34, SEQ ID
NO:35, and SEQ ID NO:36; ii) SEQ ID NO:40, SEQ ID NO:41, and SEQ ID
NO:42; iii) SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48; or iv)
SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54.
4. The antibody or antigen binding unit of claim 1, comprising a
heavy chain variable region (VH) and a light chain variable region
(VL), wherein VH comprises an amino acid sequence of SEQ ID NO:3,
SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:29; and has VL
comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:5, SEQ
ID NO:9, SEQ ID NO:13, SEQ ID NO:21 or SEQ ID NO:25.
5. The antibody or antigen binding unit of claim 4, wherein: (a) VH
comprises an amino acid sequence of SEQ ID NO:3 and VL comprises an
amino acid sequence of SEQ ID NO:1; (b) VH comprises an amino acid
sequence of SEQ ID NO:7 and VL comprises an amino acid sequence of
SEQ ID NO:5; (c) VH comprises an amino acid sequence of SEQ ID
NO:11 and VL comprises an amino acid sequence of SEQ ID NO:9; (d)
VH comprises an amino acid sequence of SEQ ID NO:15 and VL
comprises an amino acid sequence of SEQ ID NO:13; (e) VH comprises
an amino acid sequence of SEQ ID NO:17 and VL comprises an amino
acid sequence of SEQ ID NO:1; (f) VH comprises an amino acid
sequence of SEQ ID NO:19 and VL comprises an amino acid sequence of
SEQ ID NO:1; (g) VH comprises an amino acid sequence of SEQ ID
NO:23 and VL comprises an amino acid sequence of SEQ ID NO:21; (h)
VH comprises an amino acid sequence of SEQ ID NO:27 and VL
comprises an amino acid sequence of SEQ ID NO:25; or (i) VH
comprises an amino acid sequence of SEQ ID NO:29 and VL comprises
an amino acid sequence of SEQ ID NO:25.
6. The antibody of claim 1, having (a) a heavy chain comprising an
amino acid sequence of SEQ ID NO:63 and a light chain comprising an
amino acid sequence of SEQ ID NO:65; (b) a heavy chain comprising
an amino acid sequence of SEQ ID NO:59 and a light chain comprising
an amino acid sequence of SEQ ID NO:61; or (c) a heavy chain
comprising an amino acid sequence of SEQ ID NO:67 and a light chain
comprising an amino acid sequence of SEQ ID NO:65.
7-9. (canceled)
10. A nucleic acid encoding the antibody or antigen binding
fragment of claim 1.
11. An expression vector comprising the nucleic acid of claim
10.
12. A host cell comprising the expression vector of claim 11.
13. A method for preparing an anti-claudin 18A2 targeting drug, an
anti-claudin 18A2 antibody-drug conjugate, a multifunctional
anti-claudin 18A2 antibody, a reagent for diagnosing a tumor
expressing claudin 18A2, or an anti-claudin 18A2 chimeric antigen
receptor modified immune cell, wherein the method comprises
providing the antibody or antigen binding unit of claim 1 and
incorporating said antibody or antigen binding unit into the
claudin 18A2 targeting drug, the anti-claudin 18A2 antibody-drug
conjugate, the multifunctional anti-claudin 18A2 antibody, the
reagent for diagnosing a tumor expressing claudin 18A2, or the anti
claudin 18A2 chimeric antigen receptor modified immune cell.
14. (canceled)
15. A chimeric antigen receptor comprising the following
sequentially linked components: the antibody or antigen binding
unit of claim 1, a transmembrane region and an intracellular signal
region.
16. The chimeric antigen receptor of claim 15, wherein the
intracellular signal region is selected from: an intracellular
signal region sequence of CD3.zeta., Fc.epsilon.RI.gamma., CD27,
CD28, CD137, CD134, MyD88, CD40, or a combination thereof; and/or
the transmembrane region comprises a transmembrane region of CD8 or
CD28.
17. (canceled)
18. A nucleic acid encoding the chimeric antigen receptor claim
16.
19. An expression vector comprising the nucleic acid of claim
18.
20. A virus comprising the vector of claim 19.
21-25. (canceled)
26. A multifunctional immunoconjugate comprising: an antibody or
antigen binding unit of claim 1; and a functional molecule linked
thereto, wherein the functional molecule is selected from the group
consisting of: a molecule targeting a surface marker on a tumor, a
tumor-inhibiting molecule, a molecule targeting a surface marker of
an immune cell, and a detectable label.
27. The multifunctional immunoconjugate of claim 26, wherein the
molecule targeting a surface marker of an immune cell is an
antibody binding to a T cell surface marker, which forms a
bifunctional antibody in which T cell is involved.
28. A nucleic acid encoding the multifunctional immunoconjugate of
claim 25.
29-30. (canceled)
31. A kit comprising: a container, and the antibody or antigen
binding unit of claim 1.
32-74. (canceled)
75. A method for inducing the death of a cell comprising a claudin
18A2 peptide, comprising contacting the cell with the antibody or
antigen binding unit of claim 1.
76-80. (canceled)
81. A method for treating a tumor in an individual in need thereof,
comprising administering to the individual an effective amount of
the antibody or antigen binding unit of claim 1.
82. The method of claim 81, wherein the tumor is a solid tumor.
83. The method of claim 81, wherein the tumor is gastric cancer,
esophageal cancer, intestinal cancer, pancreatic cancer,
nephroblastoma, lung cancer, ovarian cancer, colon cancer, rectal
cancer, liver cancer, head and neck cancer, chronic myelogenous
leukemia or gallbladder cancer.
84. The method of claim 81, further comprising administering to the
individual an additional therapeutic agent.
85. The method of claim 84, wherein the additional therapeutic
agent is at least one selected from the group consisting of
epirubicin, oxaliplatin and 5-fluorouracil.
86. The antibody or antigen binding unit of claim 2, wherein the
claudin 18A2 peptide comprises an amino acid sequence of SEQ ID
NO:55 and/or the claudin 18A1 peptide comprises an amino acid
sequence of SEQ ID NO:57.
87. The antibody or antigen binding unit of claim 1, comprising (a)
a heavy chain variable region comprising three CDRs, namely HCDR1,
HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence
selected from SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID
NO:49, or an amino acid sequence which is at least 80% identical
thereto; HCDR2 comprises an amino acid sequence selected from SEQ
ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:83,
SEQ ID NO:84, SEQ ID NO:85, or an amino acid sequence which is at
least 80% identical thereto; and HCDR3 comprises an amino acid
sequence selected from SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45,
SEQ ID NO:51, or an amino acid sequence which is at least 80%
identical thereto; and/or (b) a light chain variable region
comprising three CDRs, namely LCDR1, LCDR2, and LCDR3, wherein
LCDR1 comprises an amino acid sequence selected from SEQ ID NO:34,
SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:52, or an amino acid sequence
which is at least 80% identical thereto; LCDR2 comprises an amino
acid sequence selected from SEQ ID NO:35, SEQ ID NO:41, SEQ ID
NO:47, SEQ ID NO:53, or an amino acid sequence which is at least
80% identical thereto; and LCDR3 comprises an amino acid sequence
selected from SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID
NO:54, or an amino acid sequence which is at least 80% identical
thereto.
88. The antibody or antigen binding unit of claim 87, wherein, (a)
HCDR1 is SEQ ID NO:31, HCDR2 is SEQ ID NO:32, HCDR3 is SEQ ID
NO:33, LCDR1 is SEQ ID NO: 34, LCDR2 is SEQ ID NO: 35, and LCDR3 is
SEQ ID NO: 36; (b) HCDR1 is SEQ ID NO:37, HCDR2 is SEQ ID NO:38,
HCDR3 is SEQ ID NO:39, LCDR1 is SEQ ID NO: 40, LCDR2 is SEQ ID NO:
41, and LCDR3 is SEQ ID NO:42; (c) HCDR1 is SEQ ID NO:43, HCDR2 is
SEQ ID NO:44, HCDR3 is SEQ ID NO:45, LCDR1 is SEQ ID NO:46, LCDR2
is SEQ ID NO:47, and LCDR3 is SEQ ID NO:48; (d) HCDR1 is SEQ ID
NO:49, HCDR2 is SEQ ID NO:50, HCDR3 is SEQ ID NO:51, LCDR1 is SEQ
ID NO:52, LCDR2 is SEQ ID NO:53, and LCDR3 is SEQ ID NO:54; (e)
HCDR1 is SEQ ID NO:31, HCDR2 is SEQ ID NO:83, HCDR3 is SEQ ID
NO:33, LCDR1 is SEQ ID NO:34, LCDR2 is SEQ ID NO:35, and LCDR3 is
SEQ ID NO:36; (f) HCDR1 is SEQ ID NO:31, HCDR2 is SEQ ID NO:84,
HCDR3 is SEQ ID NO:33, LCDR1 is SEQ ID NO:34, LCDR2 is SEQ ID
NO:35, and LCDR3 is SEQ ID NO: 36; or (g) HCDR1 is SEQ ID NO:49,
HCDR2 is SEQ ID NO:85, HCDR3 is SEQ ID NO:51, LCDR1 is SEQ ID
NO:52, LCDR2 is SEQ ID NO:53, and LCDR3 is SEQ ID NO: 54.
89. The antibody or antigen binding unit of claim 1, which is a
humanized antibody, a chimeric antibody, a fully human antibody, a
monoclonal antibody, a single chain antibody, a domain antibody, a
multivalent antibody, a chimeric antigen receptor, an scFv, an Fv,
a Fab, or a (Fab)2.
90. A pharmaceutical composition comprising the antibody or antigen
binding unit of claim 1, and a pharmaceutically acceptable
carrier.
91. A method for producing an antibody or antigen binding unit that
specifically binds to claudin 18A2, comprising culturing the host
cell of claim 12 under suitable conditions, and obtaining the
product expressed by the host cell.
92. A chimeric antigen receptor-modified immune cell having the
chimeric antigen receptor of claim 15.
93. The chimeric antigen receptor-modified immune cell of claim 92,
wherein the immune cell is selected from a T lymphocyte, NK cell,
or NKT lymphocyte.
94. The immune cell of claim 93, further comprising: (a) an
encoding sequence for an exogenous cytokine; (b) another expressed
chimeric antigen receptor which does not contain CD3.zeta. but
contains an intracellular signal domain of CD28, an intracellular
signal domain of CD137, or a combination of the both; (c) an
expressed chemokine receptor; or (d) an expressed siRNA which can
reduce expression of PD-1; (e) an expressed protein which can block
PD-L1; (f) a knock-out of endogenous PD-1 in the immune cell by
gene editing techniques; or (g) an expressed safety switch.
95. The composition of claim 90, further comprising a Type I
interferon.
Description
CROSS-REFERENCE
[0001] The present application claims the benefit of
CN201610536449.9 filed on Jul. 8, 2016 and PCT International
Application No. PCT/CN2017/082024 filed on Apr. 26, 2017, the
entire disclosures of which are hereby incorporated by
reference.
TECHNICAL FIELD
[0002] The present invention belongs to the field of immunology,
and in particular, the invention relates to antibodies against
claudin 18A2 and uses thereof.
BACKGROUND
[0003] Chimeric antigen receptor (CAR) is an artificial recombinant
receptor that usually contains the antigen recognition domain of a
monoclonal antibody located in extracellular region, a
transmembrane region and an intracellular activation signal domain
of an immune response cell.
[0004] Gastric cancer is one of the cancers with the highest
incidence rate worldwide. According to the statistics from WHO
Cancer Control Project, there are 7 million patients who die of
cancer every year in the world, and 700,000 of them die of gastric
cancer. Compared with conventional gastric cancer treatment
regimens, antibody-based treatment regimens have far-reaching
application prospects due to high specificity and low side
effects.
[0005] Claudin 18 (CLD18) is an intrinsic membrane protein located
in the tight junction of the epithelium and endothelium with a
molecular weight of approximately 27.9 KD. The GenBank accession
number is splice variant 1 (CLD18A1, CLD18.1): NP_057453, NM016369,
and splice variant 2 (CLD18A2, CLD18.2): NM_001002026,
NP_001002026. FIG. 1A shows a comparison of identity between
claudin 18A2 (SEQ ID NO:55) and claudin 18A1 (SEQ ID NO:57). In
normal cells, CLD18A1 is selectively expressed in the epithelium of
the lung and stomach, while CLD18A2 is slightly expressed in normal
gastric epithelial short-lived cells. However, in tumor cells,
CLD18A2 is strongly expressed in various types of cancer. For
example, 75% of patients with gastric cancer have high expression
of CLD18A2, 50% of patients with pancreatic cancer have high
expression of CLD18A2, and 30% of patients with esophageal cancer
have high expression of CLD18A2, which is also highly expressed in
lung cancer. Therefore, it is of great significance for the
treatment and detection of cancer to find antibodies binding
CLD18A2 with higher specificity without binding to CLD18A1.
[0006] Type I interferons contain IFN.alpha. protein (a class of
identical proteins encoded by 13 human genes from IFNA1 to IFNA13),
IFN.beta. (encoded by a single human and mouse gene IFNB1), and
other less studied interferons. Studies have shown that type I
interferons have anticancer effects on some tumors, probably due to
their immune stimulating function. However, systemic administration
of type I interferons may have immunosuppressive effects (Lotrich,
F E Major depression during interferon-.alpha. treatment:
vulnerability and prevention. Dialogues Clin. Neurosci. 11, 417-425
(2009)) with major undesirable events, the most common of which are
fatigue, anorexia, hepatotoxicity, flu-like symptoms and severe
depression (Kreutzer, K., Bonnekoh, B., Franke, I., Ulrich, J.
& Gollnick, H. Sarcoidosis, myasthenia gravis And anterior
ischaemic optic neuropathy: severe side effects of adjuvant
interferon-.alpha. therapy in malignant melanoma?. J. Dtsch.
Dermatol. Ges. 2,689-694 (in German) (2004)), and such severe side
effects severely limit its application.
SUMMARY OF THE INVENTION
[0007] The present invention overcomes the aforementioned problems
and has additional advantages.
[0008] According to an aspect of the present invention, the present
invention provides an antibody specifically binding to claudin
18A2, characterized in that the antibody comprises a heavy chain
CDR comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:31, 32, 33, 37, 38, 39, 43, 44, 45, 49,
50, 51, 83, 84, 85 or a variant thereof and/or a light chain CDR
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:34, 35, 36, 40, 41, 42, 46, 47, 48, 52,
53, 54 or a variant thereof.
[0009] In some embodiments, the antibody of the invention is
selected from the group consisting of (a) an antibody comprising a
heavy chain variable region, wherein the heavy chain variable
region has CDR1 comprising an amino acid sequence of SEQ ID NO:31,
SEQ ID NO:37, SEQ ID NO:43 or SEQ ID NO:49, CDR2 comprising an
amino acid sequence of SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44,
SEQ ID NO:50, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85, CDR3
comprising an amino acid sequence of SEQ ID NO:33, SEQ ID NO:39,
SEQ ID NO:45 or SEQ ID NO:51; (b) an antibody comprising a light
chain variable region, wherein the light chain variable region has
CDR1 comprising an amino acid sequence of SEQ ID NO:34, SEQ ID
NO:40, SEQ ID NO:46 or SEQ ID NO:52, CDR2 comprising an amino acid
sequence of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47 or SEQ ID
NO:53, CDR3 comprising an amino acid sequence of SEQ ID NO:36, SEQ
ID NO:42, SEQ ID NO:48 or SEQ ID NO:54; (c) an antibody comprising
(a) a heavy chain variable region of said antibody and (b) a light
chain variable region of said antibody; (d) an antibody,
recognizing the same antigenic determinant site as that of the
antibody of any one of (a) to (c).
[0010] In some embodiments, the CDR1, CDR2 and CDR3 regions of the
heavy chain variable region of the antibody are SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33; or SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39; or SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45; or SEQ ID
NO:49, SEQ ID NO:50, SEQ ID NO:51; or SEQ ID NO:31, SEQ ID NO:83,
SEQ ID NO:33; or SEQ ID NO:31, SEQ ID NO:84, SEQ ID NO:33; or SEQ
ID NO:49, SEQ ID NO 85, SEQ ID NO:51, respectively; and/or the
CDR1, CDR2 and CDR3 regions of the light chain variable region of
the antibody are SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36; or SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42; or SEQ ID NO:46, SEQ ID
NO:47, SEQ ID NO:48; or SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54,
respectively.
[0011] In some embodiments, the antibody comprises a heavy chain
variable region and a light chain variable region, wherein the
heavy chain variable region has an amino acid sequence of SEQ ID
NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:23, SEQ ID NO:27 or SEQ ID NO:29; and the light
chain variable region has an amino acid sequence of SEQ ID NO:1,
SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:21 or SEQ ID
NO:25. In some embodiments, the antibody is an antibody having a
heavy chain variable region of SEQ ID NO:3 and a light chain
variable region of SEQ ID NO:1; an antibody having a heavy chain
variable region of SEQ ID NO:7 and a light chain variable region of
SEQ ID NO:5; an antibody having a heavy chain variable region of
SEQ ID NO:11 and a light chain variable region of SEQ ID NO:9; an
antibody having a heavy chain variable region of SEQ ID NO:15 and a
light chain variable region of SEQ ID NO:13; an antibody having a
heavy chain variable region of SEQ ID NO:17 and a light chain
variable region of SEQ ID NO:1; an antibody having a heavy chain
variable region of SEQ ID NO:19 and a light chain variable region
of SEQ ID NO:1; an antibody having a heavy chain variable region of
SEQ ID NO:23 and a light chain variable region of SEQ ID NO:21; an
antibody having a heavy chain variable region of SEQ ID NO:27 and a
light chain variable region of SEQ ID NO:25; or an antibody having
a heavy chain variable region of SEQ ID NO:29 and a light chain
variable region of SEQ ID NO:25. In some embodiments, the antibody
is an antibody having a heavy chain variable region of SEQ ID NO:3
and a light chain variable region of SEQ ID NO:1; an antibody
having a heavy chain variable region of SEQ ID NO:17 and a light
chain variable region of SEQ ID NO:1; an antibody having a heavy
chain variable region of SEQ ID NO:19 and a light chain variable
region of SEQ ID NO:1; an antibody having a heavy chain variable
region of SEQ ID NO:23 and a light chain variable region of SEQ ID
NO:21; an antibody having a heavy chain variable region of SEQ ID
NO:27 and a light chain variable region of SEQ ID NO:25; or an
antibody having a heavy chain variable region of SEQ ID NO:29 and a
light chain variable region of SEQ ID NO:25. In some embodiments,
the antibody is a humanized antibody, a chimeric antibody or a
fully humanized antibody; or the antibody is a monoclonal antibody;
or the antibody is a single chain antibody or a domain antibody. In
some embodiments, the antibody is a humanized antibody selected
from the group consisting of an antibody having a heavy chain
variable region of SEQ ID NO:27 and a light chain variable region
of SEQ ID NO:25; an antibody having a heavy chain variable region
of SEQ ID NO:23 and a light chain variable region of SEQ ID NO:21;
an antibody having a heavy chain variable region of SEQ ID NO:29
and a light chain variable region of SEQ ID NO:25. In some
embodiments, the antibody is selected from the group consisting of
an antibody having a heavy chain of SEQ ID NO:63 and a light chain
of SEQ ID NO:65; an antibody having a light chain of SEQ ID NO:61
and a heavy chain of SEQ ID NO:59; and an antibody having a heavy
chain of SEQ ID NO:67 and a light chain of SEQ ID NO:65.
[0012] According to one aspect of the invention, the invention
provides a nucleic acid encoding the antibody as mentioned above.
According to another aspect of the invention, the invention
provides an expression vector comprising the nucleic acid.
According to another aspect of the present invention, the present
invention provides a host cell comprising the expression vector of
the present invention or having the nucleic acid of the present
invention integrated into its genome.
[0013] According to one aspect of the invention, the invention
provides the use of an antibody according to the invention for
preparing a targeting drug, antibody-drug conjugate or
multifunctional antibody specifically targeting tumor cells
expressing claudin 18A2; or for preparing a reagent for diagnosing
a tumor expressing claudin 18A2; or for preparing a chimeric
antigen receptor modified immune cell. In some embodiments, the
tumor expressing claudin 18A2 includes: gastric cancer, pancreatic
cancer, esophageal cancer, lung cancer.
[0014] According to one aspect of the invention, the invention
provides a chimeric antigen receptor comprising an antibody of the
invention, wherein the chimeric antigen receptor comprises
following sequentially linked components: an antibody of the
invention, a transmembrane region and an intracellular signal
region. In some embodiments, the intracellular signal region is
selected from the group consisting of: an intracellular signal
region sequence of CD3.zeta., FccRI.gamma., CD27, CD28, CD137,
CD134, MyD88, CD40, or a combination thereof; or the transmembrane
region comprises a transmembrane region of CD8 or CD28. In some
embodiments, the chimeric antigen receptor comprises sequentially
linked an antibody, a transmembrane region and an intracellular
signaling region: an antibody of the invention, CD8 and CD3.zeta.;
an antibody of the invention, CD8, CD137 and CD3.zeta.; or an
antibody of the invention, a transmembrane region of CD28 molecule,
an intracellular signal region of CD28 molecule, and CD3.zeta.; or
an antibody of the invention, a transmembrane region of CD28
molecule, an intracellular signal region of CD28 molecule, CD137
and CD3.zeta..
[0015] According to another aspect of the invention, the invention
provides a nucleic acid encoding the chimeric antigen receptor.
According to another aspect of the invention, the invention
provides an expression vector comprising the nucleic acid of the
invention. According to another aspect of the invention, the
invention provides a virus comprising the vector of the
invention.
[0016] According to an aspect of the present invention, the present
invention provides uses of a chimeric antigen receptor, nucleic
acid, expression vector or virus of the present invention for
preparing chimeric antigen receptor-modified immune cells targeting
tumor cells expressing claudin 18A2. In some embodiments, the tumor
expressing claudin 18A2 includes: gastric cancer, pancreatic
cancer, esophageal cancer, lung cancer.
[0017] According to one aspect of the present invention, the
present invention provides a chimeric antigen receptor-modified
immune cell transduced with a nucleic acid, expression vector or
virus of the present invention; or having a chimeric antigen
receptor of the present invention expressed on the surface. In some
embodiments, the immune cell is: a T lymphocyte, NK cell or NKT
lymphocyte. In some embodiments, the immune cell further carries an
encoding sequence for an exogenous cytokine; or further expresses
another chimeric antigen receptor which does not contain CD3.zeta.
but contains an intracellular signal domain of CD28, an
intracellular signal domain of CD137, or a combination of the both;
or further expresses a chemokine receptor (preferably, said
chemokine receptor comprises: CCR); or further expresses an siRNA
which can reduce expression of PD-1 or a protein which can block
PD-L1; or endogenous PD-1 in the immune cell is knocked out by gene
editing techniques; or further expresses a safety switch.
[0018] According to one aspect of the present invention, the
present invention provides uses of the chimeric antigen
receptor-modified immune cell for producing a tumor-inhibiting
drug, wherein the tumor is a tumor expressing claudin 18A2;
preferably, the tumor includes: gastric cancer, pancreatic cancer,
esophageal cancer, and lung cancer.
[0019] According to one aspect of the present invention, the
present invention provides a multifunctional immunoconjugate
comprising an antibody of the present invention; and a functional
molecule linked thereto; and the functional molecule is selected
from the group consisting of: a molecule targeting a surface marker
on a tumor, a tumor-inhibiting molecule, a molecule targeting a
surface marker of an immune cell or a detectable label. In some
embodiments, the molecule targeting a surface marker of an immune
cell is an antibody binding to a T cell surface marker, which forms
a bifunctional antibody with the antibody of the invention in which
T cell is involved. According to another aspect of the invention,
the invention provides a nucleic acid encoding said multifunctional
immunoconjugate and uses thereof for preparing anti-tumor drugs. In
some embodiments, the nucleic acid encoding the multifunctional
immunoconjugate is used to prepare a reagent for diagnosing a tumor
expressing claudin 18A2. In some embodiments, the nucleic acid
encoding the multifunctional immunoconjugate is used to prepare
chimeric antigen receptor modified immune cells. In some
embodiments, the immune cells includes: a T lymphocyte, NK cell or
NKT lymphocyte.
[0020] According to one aspect of the invention, the invention
provides a pharmaceutical composition comprising an antibody of the
invention or a nucleic acid encoding the antibody. According to one
aspect of the invention, the invention provides a pharmaceutical
composition comprising an immunoconjugate of the invention or a
nucleic acid encoding the conjugate. According to one aspect of the
invention, the invention provides a pharmaceutical composition
comprising a chimeric antigen receptor of the invention or a
nucleic acid encoding the chimeric antigen receptor. According to
one aspect of the invention, the invention provides a
pharmaceutical composition comprising a chimeric antigen receptor
modified immune cell of the invention. In some embodiments, the
pharmaceutical composition comprises a pharmaceutically acceptable
carrier or excipient.
[0021] According to an aspect of the invention, a kit is provided
in the invention comprising a container, and a pharmaceutical
composition of the invention in the container; or a container, and
an antibody of the invention or a nucleic acid encoding the
antibody in the container; or the immunoconjugate of the present
invention or a nucleic acid encoding the conjugate; or the chimeric
antigen receptor of the present invention or a nucleic acid
encoding the chimeric antigen receptor; or the chimeric antigen
receptor modified immune cell of the invention.
[0022] According to one aspect of the present invention, the
present invention provides an antigen binding unit comprising a
light chain CDR and a heavy chain CDR, wherein the antigen binding
unit specifically binds to a claudin 18A2 peptide; and the antigen
binding unit does not significantly bind to a claudin 18A1 peptide.
According to another aspect of the present invention, the present
invention provides an antigen binding unit comprising a light chain
CDR and a heavy chain CDR, wherein the antigen binding unit
specifically binds to a claudin 18A2 peptide; and the antigen
binding unit, compared with a reference antigen binding unit,
exhibits less non-specific binding to the claudin 18A1 peptide. In
some embodiments, the reference antigen binding unit comprises a
light chain amino acid sequence of SEQ ID NO:86 or SEQ ID NO:88
and/or a heavy chain amino acid sequence of SEQ ID NO:87 or SEQ ID
NO:89. In some embodiments, the claudin 18A2 peptide comprises an
amino acid sequence of SEQ ID NO:55. In some embodiments, the
claudin 18A1 peptide comprises an amino acid sequence of SEQ ID
NO:57. In some embodiments, the non-specific binding of the antigen
binding unit to the claudin 18A1 peptide does not exceed 20% of the
specific binding to the claudin 18A2 peptide. In some embodiments,
the binding specificity is determined by flow cytometry. In some
embodiments, the binding specificity is determined by FACS. In some
embodiments, the binding specificity is determined by ELISA. In
some embodiments, the antigen binding unit binds to the claudin
18A2 peptide with an EC50 of less than about 100 nM. In some
embodiments, the antigen binding unit is a monoclonal antibody, a
humanized antibody, a chimeric antibody, a multivalent antibody or
a chimeric antigen receptor. In some embodiments, the light chain
CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR
comprises HCDR1, HCDR2 and HCDR3; wherein the LCDR1, LCDR2 and
LCDR3 respectively have an amino acid sequence selected from the
group consisting of: SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47,
SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54; and the
HCDR1, HCDR2 and HCDR3 respectively have an amino acid sequence
selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32,
SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:50, SEQ
ID NO:51, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85. In some
embodiments, the LCDR1 comprises an amino acid sequence selected
from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID
NO:46 and SEQ ID NO:52. In some embodiments, the LCDR2 comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:34, SEQ ID NO:41, SEQ ID NO:47 and SEQ ID NO:53. In some
embodiments, the LCDR3 comprises an amino acid sequence selected
from the group consisting of SEQ ID NO:35, SEQ ID NO:42, SEQ ID
NO:48 and SEQ ID NO:54. In some embodiments, the HCDR1 comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:31, SEQ ID NO:37, SEQ ID NO:43 and SEQ ID NO:49. In some
embodiments, the HCDR2 comprises an amino acid sequence selected
from the group consisting of SEQ ID NO:32, SEQ ID NO:38, SEQ ID
NO:44, SEQ ID NO:50, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85.
In some embodiments, the HCDR3 comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39,
SEQ ID NO:45 and SEQ ID NO:51. In some embodiments, the antigen
binding unit is scFv, Fv, Fab or (Fab)2.
[0023] According to one aspect of the invention, the invention
provides an antigen binding unit comprising a light chain CDR and a
heavy chain CDR, wherein said light chain CDR comprises LCDR1,
LCDR2 and LCDR3; said heavy chain CDR comprises HCDR1, HCDR2 and
HCDR3; the LCDR1, LCDR2 and LCDR3 respectively comprise an amino
acid sequence which is at least 80% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53
and SEQ ID NO:54; and the HCDR1, HCDR2 and HCDR3 respectively
comprise an amino acid sequence which is at least 80% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID
NO:85. In some embodiments, the light chain CDR comprises LCDR1,
LCDR2 and LCDR3; the heavy chain CDR comprises HCDR1, HCDR2 and
HCDR3; the LCDR1, LCDR2 and LCDR3 respectively have an amino acid
sequence selected from the group consisting of: SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 SEQ ID NO:52, SEQ ID NO:53
and SEQ ID NO:54; and the HCDR1, HCDR2 and HCDR3 respectively have
an amino acid sequence selected from the group consisting of SEQ ID
NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO: 39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID
NO:85. In some embodiments, the LCDR1 comprises an amino acid
sequence which is at least 80% identical to an amino acid sequence
selected from the group consisting of: SEQ ID NO:34, SEQ ID NO:40,
SEQ ID NO:46 and SEQ ID NO:52. In some embodiments, the LCDR2
comprises an amino acid sequence which is at least 80% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:34, SEQ ID NO:41, SEQ ID NO:47 and SEQ ID NO:53. In some
embodiments, the LCDR3 comprises an amino acid sequence which is at
least 80% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:48 and
SEQ ID NO:54. In some embodiments, the HCDR1 comprises an amino
acid sequence which is at least 80% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:31, SEQ ID
NO:37, SEQ ID NO:43 and SEQ ID NO:49. In some embodiments, the
HCDR2 comprises an amino acid sequence which is at least 80%
identical to an amino acid sequence selected from the group
consisting of: SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID
NO:50 SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85. In some
embodiments, the HCDR3 comprises an amino acid sequence which is at
least 80% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45 and
SEQ ID NO:51. In some embodiments, the antigen binding unit is a
monoclonal antibody, a humanized antibody, a chimeric antibody, a
multivalent antibody or a chimeric antigen receptor. In some
embodiments, the antigen binding unit is scFv, Fv, Fab or
(Fab)2.
[0024] According to one aspect of the invention, the invention
provides a chimeric antigen receptor comprising an extracellular
antigen binding unit, a transmembrane domain and an intracellular
domain, wherein the extracellular antigen binding unit comprises an
antigen binding unit of the invention. According to one aspect of
the invention, the invention provides a composition comprising an
antigen binding unit or a chimeric antigen receptor of the
invention. In some embodiments, the composition comprises a Type I
interferon. According to one aspect of the invention, the invention
provides an isolated nucleic acid encoding an antigen binding unit
or chimeric antigen receptor of the invention, and optionally a
type I interferon. According to one aspect of the invention, the
invention provides a vector comprising a nucleic acid of the
invention.
[0025] According to one aspect of the invention, the invention
provides a host cell which expresses an antigen binding unit or
chimeric antigen receptor of the invention, and optionally a type I
interferon. According to one aspect of the invention, the invention
provides a host cell comprising a nucleic acid encoding the antigen
binding unit or chimeric antigen receptor of the invention, and
optionally a type I interferon. In some embodiments, the host cell
is an immune response cell. In some embodiments, the host cell is a
T cell, natural killer cell, cytotoxic T lymphocyte, natural killer
T cell, DNT cell, and/or regulatory T cell. In some embodiments,
the host cell is an NK92 cell.
[0026] In some embodiments, the host cell is cytotoxic to a cell
comprising a claudin 18A2 peptide comprising the amino acid
sequence of SEQ ID NO:55. In some embodiments, the host cell does
not have significant cytotoxicity to a cell comprising a claudin
18A1 peptide while not comprising a claudin 18A2 peptide, and the
claudin 18A1 peptide comprises an amino acid sequence of SEQ ID
NO:57, and the claudin 18A2 peptide comprises the amino acid
sequence of SEQ ID NO:55.
[0027] According to one aspect of the invention, the invention
provides a method for producing an antigen binding unit or chimeric
antigen receptor or composition of the invention, including:
culturing a host cell of the invention under suitable conditions,
and obtaining the product expressed by the host cell.
[0028] According to one aspect of the invention, the invention
provides a method for inducing the death of a cell comprising a
claudin 18A2 peptide, including contacting the cell with an antigen
binding unit, chimeric antigen receptor, composition or host cell
of the invention. In some embodiments, the cell is contacted with
the antigen binding unit, the chimeric antigen receptor, the
composition or host cell in vitro. In some embodiments, the cell is
contacted with the antigen binding unit, the chimeric antigen
receptor, the composition or host cell in vivo. In some
embodiments, the cell is a cancer cell. In some embodiments, the
cell is a solid tumor cell. In some embodiments, the cell is
selected from the group consisting of: a gastric cancer cell,
esophageal cancer cell, intestinal cancer cell, pancreatic cancer
cell, nephroblastoma cell, lung cancer cell, ovarian cancer cell,
colon cancer cell, rectal cancer cell, liver cancer cell, head and
neck cancer cell, chronic myeloid leukemia cell and gallbladder
cancer cell.
[0029] According to one aspect of the invention, the invention
provides a method for treating a tumor in an individual in need
thereof, the method including administering to the individual an
effective amount of an antigen binding unit, chimeric antigen
receptor, composition or host cell of the invention. In some
embodiments, the tumor is a solid tumor. In some embodiments, the
tumor is gastric cancer, esophageal cancer, intestinal cancer,
pancreatic cancer, nephroblastoma, lung cancer, ovarian cancer,
colon cancer, rectal cancer, liver cancer, head and neck cancer,
chronic myelogenous leukemia or gallbladder cancer. In some
embodiments, the method further includes administering to the
individual an additional therapeutic agent. In some embodiments,
the additional therapeutic agent is at least one selected from the
group consisting of epirubicin, oxaliplatin and 5-fluorouracil.
INCORPORATION BY REFERENCE
[0030] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference as if
each of the publications, patents or patent applications is
incorporated by reference.
BRIEF DESCRIPTION OF DRAWINGS
[0031] Drawings further illustrate novel features disclosed in this
specification. Features and advantages of the present disclosure
will be better understood from the following description of the
accompanying drawings. It is to be understood, however, that the
drawings are only intended to illustrate the specific embodiments
of the application of principles disclosed herein, and are not
intended to limit the scope of the appended claims.
[0032] FIG. 1A shows the identity comparison between claudin 18A2
(SEQ ID NO:55) and claudin 18A1 (SEQ ID NO:57); FIG. 1B shows
binding of hybridoma supernatants 2B1, 3E12, 4A11 and 8E5 to HEK293
cells stably transfected with human CLD18A2 and CLD18A1 as
determined by flow cytometry.
[0033] FIG. 2 shows sequence alignment of murine anti-2B1 (heavy
chain variable region SEQ ID NO:3, light chain variable region SEQ
ID NO:1), 3E12 (heavy chain variable region SEQ ID NO:7, light
chain variable region SEQ ID NO:5), 4A11 (heavy chain variable
region SEQ ID NO:11, light chain variable region SEQ ID NO:9), 8E5
(heavy chain variable region SEQ ID NO:15, light chain variable
region SEQ ID NO:13).
[0034] FIG. 3 shows the relative binding affinity of murine
anti-2B1, 8E5 ScFv, after fused to human IgG1 Fc portion, to HEK293
cells stably transfected with human CLD18A2.
[0035] FIG. 4 shows the relative binding affinity of engineered
murine anti-2B1 antibody 2B1-N52D and 2B1-S54A, after fused to
human IgG1 Fc portion, to HEK293 cells stably transfected with
human CLD18A2.
[0036] FIG. 5 shows the relative binding affinity of humanized
hu2B1-S54A, after fused to human IgG1 Fc portion, to HEK293 cells
stably transfected with human CLD18A2.
[0037] FIG. 6 shows the relative binding affinity of humanized
hu8E5, after fused to human IgG1 Fc portion, to HEK293 cells stably
transfected with human CLD18A2.
[0038] FIG. 7 shows the relative binding affinity of engineered
humanized hu8E5-2I to HEK293 cells stably transfected with human
CLD18A2.
[0039] FIGS. 8A-8B: FIG. 8A compares CDC effects of the humanized
antibodies hu2B1-S54A, hu8E5-2I and the known chimeric antibody
ch-163E12 (see CN103509110A) on HEK293 cells transfected with
CLD18A2; and FIG. 8B compares CDC results of the humanized
antibodies hu2B1-S54A, hu8E5-2I and ch-163E12 on HEK293 cells
transfected with CLD18A1.
[0040] FIG. 9 compares ADCC effects of the humanized antibodies
hu2B1-S54A, hu8E5-2I and the chimeric antibodies ch-163E12,
ch-175D10 (see CN103509110A).
[0041] FIG. 10 compares killing activities of hu8E5-2I and
ch-175D10 in mice.
[0042] FIG. 11 compares in vitro killing activities of hu8E5-28Z,
hu8E5-BBZ and hu8E5-28BBZ T cells on different cell lines.
[0043] FIG. 12 compares in vitro killing activities of hu8E5-28Z,
hu8E5-2I-28Z and hu2B1-hs54A T cells on different cell lines.
[0044] FIGS. 13A-13B are comparison graphs of the effect of
CLDN18A2-CAR T on tumor volume over time in a subcutaneous
xenograft model of gastric cancer PDX mice (FIG. 13A) and a
comparison graph of tumor photographs (FIG. 13B).
[0045] FIG. 14 shows results of secretion assay of cytokines of
hu8E5-28Z and hu8E5-2I-28Z.
[0046] FIGS. 15A-15C are comparison graphs of the effect of
CLDN18A2-CAR T on tumor volume over time in a subcutaneous
xenograft model of gastric cancer BGC-823-A2 mice (FIG. 15A), a
comparison graph of tumor weight (FIG. 15B) and a comparison graph
of tumor photo (FIG. 15C).
[0047] FIG. 16 shows the tumor infiltration of CLDN18A2-CAR T.
[0048] FIGS. 17A-17C: FIG. 17A shows the secretion of cellular
molecules after co-expression of IFN; FIG. 17B is a comparison
graph of anti-tumor activities of CAR-T cells containing IFN and
IFN-free in subcutaneous xenografts of gastric cancer PDX model;
and FIG. 17C is a comparison graph of the number of viable cells in
the peripheral blood of a mouse at the 5, 7 and 10 days of
returning CAR-T cells.
[0049] FIG. 18A and FIG. 18B are plasmid maps for the construction
of CAR-NK cells.
[0050] FIG. 19 shows the determination of the positive rate of
hu8E5-2I-28Z CAR-NK92 and hu8E5-28BBZ CAR-NK92.
[0051] FIG. 20 is a graph showing the cytotoxicity of hu8E5-2I-28Z
CAR-NK92.
[0052] FIG. 21 is a graph showing the cytotoxicity of hu8E5-28BBZ
CAR-NK92.
MODE FOR CARRYING OUT THE INVENTION
[0053] The detailed description below discloses the embodiments
disclosed herein in detail. It should be understood that the
description is not intended to be limited to the particular
embodiments disclosed herein, which can be varied. It will be
understood by a skilled person that the present disclosure may be
variously modified or varied, and all of the modifications fall
within the scope and spirit of the disclosure. Each embodiment can
be combined arbitrarily with any other embodiment unless otherwise
stated.
[0054] Certain embodiments disclosed herein are intended to
encompass a range of values, and certain aspects of the invention
may be described by using a range. Unless otherwise stated, it
should be understood that a range of values or the description of a
scope is for the purpose of simplicity and convenience, and the
scope of the invention is not to be understood as being strictly
limited by the scope of the invention. Therefore, the description
of a scope should be construed as being specifically described as
all possible sub-ranges and all possible specific numerical points
within the range, as these sub-ranges and numerical points are
explicitly recited herein. For example, the description of the
range from 1 to 6 should be considered to specifically disclose
sub-ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6,
etc., and specific numerical points within these ranges, such as 1,
2, 3, 4, 5 and 6. Regardless of the breadth of stated values, the
above principles are equally applicable. When a range is described,
the range includes endpoints of the range.
[0055] When referring to measurable values, such as amount,
temporary duration, etc., the term "about" is meant to include a
change of .+-.20%, or in some cases .+-.10%, or in some cases
.+-.5%, or in some cases .+-.1%, or in some cases .+-.0.1% of the
specified value.
[0056] As used herein, the terms "activate" and "activation" can be
used interchangeably and they, as well as other grammatical forms
thereof, may refer to a process via which a cell transits from a
quiescent state to an active state. The process can include a
response to an antigen, migration and/or a phenotypic or genetic
change in the functionally active state. For example, the term
"activation" can refer to a process via which immune cells are
gradually activated. For example, T cells may require at least two
signals to be fully activated. The first signal can occur after the
TCR is bound by the antigen-MI-IC complex, while the second signal
can occur by the conjugation of costimulatory molecules (see
co-stimulatory molecules listed in Table 1). Anti-CD3 can in vitro
simulate the first signal and anti-CD28 can simulate the second
signal. For example, engineered T cells can be activated by the
expressed CAR. As used herein, immune cell activation may refer to
a state that has been sufficiently stimulated to induce detectable
cell proliferation, cytokine production and/or detectable effector
function.
[0057] The term "co-stimulatory molecule" as used herein refers to
a homologous binding partner on an immune cell, such as a T cell,
that specifically binds to a costimulatory ligand, thereby
mediating a costimulatory response such as, but not limited to,
proliferation. A costimulatory molecule is a molecule on cell
surface other than an antigen receptor or its ligand that promotes
an effective immune response. Costimulatory molecules include, but
are not limited to, MHC class I molecules, BTLA and Toll ligand
receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18),
ICOS (CD278) and 4-1BB (CD137). Examples of costimulatory molecules
include, but are not limited to, CDS, ICAM-1, GITR, BAFFR, HVEM
(LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19,
CD4, CD8.alpha., CD8.beta., IL2R.beta., IL2R.gamma., IL7R.alpha.,
ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,
CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX,
CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2,
TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96
(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100
(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3),
BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp,
CD19a and a ligand that specifically binds to CD83.
[0058] As used herein, "costimulatory signal" refers to a signal
that, in combination with a first signal, such as TCR/CD3, results
in T cell proliferation and/or up- or down-regulation of key
molecules.
[0059] The term "antigen binding unit" as used herein refers to an
immunoglobulin molecule and an immunologically active portion of an
immune molecule, i.e., a molecule containing an antigen binding
site that specifically binds to an antigen ("immune response"). The
term "antigen-binding unit" also includes immunoglobulin molecules
of various species, including invertebrates and vertebrates. The
simplest naturally occurring antibody (e.g., IgG) structurally
comprises four polypeptide chains, two heavy (H) chains and two
light (L) chains interconnected by disulfide bonds. Immunoglobulins
represent a large family of molecules including several types of
molecules, such as IgD, IgG, IgA, IgM and IgE. The term
"immunoglobulin molecule" includes, for example, a hybrid antibody
or altered antibody and fragments thereof. It has been shown that
the antigen binding function of an antibody can be carried out by
fragments of a naturally occurring antibody. Such fragments are
collectively named as "antigen combining unit". The term
"antigen-binding unit" also includes any polypeptide
chain-containing molecular structure having a specific shape that
conforms to an epitope and recognizes an epitope, wherein one or
more non-covalent binding interactions stabilize the complex
between the molecular structure and the epitope. Examples of such
antigen binding units include a Fab fragment, monovalent fragment
consisting of VL, VH, CL and CH1 domains, bivalent fragment
comprising two Fab fragments joined by a disulfide bridge on the
hinge region (F(ab)2 fragment); Fd fragment consisting of VH and
CH1 domains, Fv fragment consisting of VL and VH domains of a
single arm of an antibody; dAb fragment consisting of VH domain
(Ward et al., Nature, 341: 544-546, 1989); and isolated
complementarity determining regions (CDRs) or any fusion protein
comprising such antigen binding units.
[0060] The term "antibody" as used herein includes an intact
antibody and any antigen-binding fragments (i.e., "antigen-binding
portions") or single chains thereof. A naturally occurring
"antibody" is a glycoprotein comprising at least two heavy (H)
chains and two light (L) chains joined by a disulfide bond. Each
heavy chain consists of a heavy chain variable region (abbreviated
herein as VH) and a heavy chain constant region. The heavy chain
constant region consists of three domains, CH1, CH2 and CH3. Each
light chain consists of a light chain variable region (abbreviated
herein as VL) and a light chain constant region. The light chain
constant region consists of one domain CL. VH and VL regions can be
further subdivided into regions of high variability named as
complementarity determining regions (CDRs) that are spaced by more
conserved regions named as framework region (ER). Each VH and V L
consists of three CDRs and four FRs arranged in the following order
from the amino terminus to the carboxy terminus: FR1, CDR1, FR2,
CDR2, FR3, CDR3, FR4. The variable regions of the heavy chain and
light chain contain a binding domain that interacts with the
antigen. The constant region of an antibody can mediate the binding
of the immunoglobulin to host tissues or factors, including various
cells of the immune system (e.g., effector cells) and the first
component (C1q) of the classical complement system.
[0061] The term "scFv" refers to a fusion protein comprising at
least one antibody fragment comprising a variable region of a light
chain and at least one antibody fragment comprising a variable
region of a heavy chain, wherein said light chain and heavy chain
variable regions are contiguous (for example, via a synthetic
linker, such as a short flexible polypeptide linker), and can be
expressed as a single-chain polypeptide, and wherein the scFv
retains the specificity of the intact antibody from which it is
derived. Unless specified, as used herein, an scFv can have the VL
and VH variable regions in any order (e.g., relative to N-terminus
and C-terminus of the polypeptide), and the scFv can include a
VL-linker-VH or A VH-linker-VL can be included.
[0062] As used herein, the terms "complementarity determining
region" and "CDR" refer to an amino acid sequence in the variable
region of the antibody that confers antigen specificity and binding
affinity. In general, there are three CDRs (HCDR1, HCDR2, HCDR3) in
each heavy chain variable region and three CDRs (LCDR1, LCDR2,
LCDR3) in the light chain variable region.
[0063] An antigen binding unit "specifically binds" to an antigen
or is "immunoreactive" with the antigen, if the antigen binding
unit binds to the antigen with greater affinity than binding to
other reference antigens, including polypeptides or other
substances.
[0064] The term "humanized" as used herein is used for a non-human
(such as a rodent or primate) antibody, is a hybrid immunoglobulin,
immunoglobulin chain or a fragment thereof comprising a minimal
sequence derived from a non-human immunoglobulin. In most cases,
the humanized antibody is a human immunoglobulin (receptor
antibody), in which residues from the complementarity determining
regions (CDRs) of the receptor are replaced by residues from CDRs
of non-human species (donor antibody), such as mice, rats, rabbits
or primates having desired specificities, affinities and
performances. In some cases, residues in Fv framework region (FR)
of human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, a humanized antibody may comprise residues
that are present in the recipient antibody while not in the
introduced CDR or framework sequences. These modifications are made
to further improve and optimize antibody performance and minimize
immunogenicity when introduced into a human body. In some examples,
a humanized antibody will comprise substantially all, at least one,
typically two variable domains, wherein all or substantially all of
the CDR regions correspond to those of a non-human immunoglobulin,
and all or substantially all of FR regions are regions of a human
immunoglobulin sequence. A humanized antibody can also comprise at
least a portion of an immunoglobulin constant region (Fc),
typically a constant region of a human immunoglobulin. In some
embodiments, a "humanized antibody" can include a mutation, such as
a mutation introduced by random or site-directed mutagenesis in
vitro or by somatic mutation in vivo.
[0065] The term "immunoglobulin" or "Ig" as used herein may refer
to a class of proteins that can function as antibodies. Antibodies
expressed by B cells are sometimes named as chimeric antigen
receptors or antigen receptors. Five members included in this class
are IgA, IgG, IgM, IgD and IgE, with IgG being the most common
circulating antibody. It is the most potent immunoglobulin in
agglutination, complement fixation and other antibody reactions and
is important in protection against bacteria and viruses. For
example, tumor cell antigens (or "tumor antigens") or pathogen
antigens can be recognized by CAR.
[0066] As used herein, the term "isolated" refers to separation
from cellular components or other components in which
polynucleotides, peptides, polypeptides, proteins, antibodies or
fragments thereof are generally associated in a natural state. As
will be understood by a skilled person, it is not necessary to
`isolate` non-naturally occurring polynucleotides, peptides,
polypeptides, proteins, antibodies or fragments thereof so as to
distinguish them from naturally occurring counterparts.
Furthermore, a "concentrated", "isolated" or "diluted"
polynucleotide, peptide, polypeptide, protein, antibody or a
fragment thereof may be distinguished from its naturally occurring
counterpart, since the concentration or amount of a molecule per
volume is greater ("concentrated") or less ("diluted") than the
concentration of its naturally occurring counterpart. The degree of
enrichment can be measured on an absolute basis, such as the weight
per solution volume, or can be measured relative to another
potential interferent present in the source mixture. In some
embodiments, higher extent of enrichment is preferable to the
technical solutions of the present invention. Therefore, for
example, 2-fold enrichment is preferable, 10-fold enrichment is
more preferable, 100-fold enrichment is more preferable, 1000-fold
enrichment is even more preferable. "Separated" materials can also
be provided by artificial assembly methods, such as chemical
synthesis or recombinant expression.
[0067] As used herein, "antigen" refers to a substance that is
recognized and specifically bound by an antigen binding unit.
Antigens can include peptides, proteins, glycoproteins,
polysaccharides, lipids, portions thereof, and combinations
thereof. Non-limiting exemplary antigens include tumor antigens or
pathogen antigens. "Antigen" can also refer to a molecule eliciting
an immune response. This immune response may involve the production
of antibodies or the activation of specific
immunologically-competent cells, or both. A skilled person will
appreciate that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen.
[0068] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acids of any
length. The polymer may be linear, cyclic or branched, it may
comprise modified amino acids, particularly conservatively modified
amino acids, and it may be interrupted by non-amino acids. The term
also includes modified amino acid polymers such as an amino acid
polymer modified through sulfation, glycosylation, lipidation,
acetylation, phosphorylation, iodination, methylation, oxidation,
proteolytic processing, prenylation, racemization, selenoylation,
transfer-RNA-mediated amino addition (such as argination,
ubiquitination) or any other manipulation such as conjugation with
a labeled component. As used herein, the term "amino acid" refers
to natural and/or non-natural or synthetic amino acids, including
glycine and D or L optical isomers, as well as amino acid analogs
and peptidomimetics. A polypeptide or amino acid sequence "derived
from" a specified protein refers to the source of the polypeptide.
The term also encompasses polypeptides expressed by a specified
nucleic acid sequence.
[0069] The term "amino acid modification" (or "modified amino
acid") includes amino acid substitutions, insertions and/or
deletions in a polypeptide sequence. As used herein, "amino acid
substitution" or "substitution" means the replacement of an amino
acid at a particular position in a parent polypeptide sequence with
another amino acid. For example, substitution R94K means that the
arginine at position 94 is replaced by lysine. For the same
substitution at the same position in the parent polypeptide
sequence, it can also be represented by 94K, i.e., replacing the 94
position with lysine. For the purposes of this document, multiple
substitutions are typically separated by slashes. For example,
R94K/L78V refers to a double variant comprising the substitutions
R94K and L78V. As used herein, "amino acid insertion" or
"insertion" means the addition of an amino acid at a particular
position in the parent polypeptide sequence. For example,
insertion-94 indicates an insertion at position 94. As used herein,
"amino acid deletion" or "deletion" means the removal of an amino
acid at a particular position in the parent polypeptide sequence.
For example, R94-indicates deletion of arginine at position 94.
[0070] The term "conservative modification" or "conservative
sequence modification" as used herein means an amino acid
modification that does not significantly affect or alter desired
activities or properties of a peptide containing the amino acid
sequence. Such conservative modifications include amino acid
substitutions, insertions, and deletions. Modifications can be
introduced into the antibody of the invention by standard
techniques known in the art, such as site-directed mutagenesis and
PCR-mediated mutagenesis. Conservative amino acid substitutions are
substitutions in which amino acid residues are replaced with amino
acid residues having similar side chains. A family of amino acid
residues having similar side chains has been defined in the art.
These families include amino acids containing basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, serine, threonine, tyrosine, cysteine,
tryptophan), non-polar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), .beta.-branched
side chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Therefore, one or more amino acid residues in the CDR regions or
framework regions of the antibody of the invention can be replaced
with other amino acid residues with similar side chains.
[0071] The term "autologous" as used herein and other grammatical
forms thereof may refer to substances from the same source. For
example, a sample (e.g., a cell) can be removed, processed and
administered to the same individual (e.g., a patient) at a later
time. The autologous process is different from the allogeneic
process in which the donor and recipient are different
individuals.
[0072] As used herein, "xenograft" and other grammatical forms
thereof may include any procedure in which a recipient and a donor
are from different species and cells, tissues or organs are
transplanted, implanted or infused into the recipient.
Transplantation of cells, organs and/or tissues described herein
can be used as xenografts in humans. Xenografts include, but are
not limited to, vascularized xenografts, partially vascularized
xenografts, non-vascularized xenografts, xenogeneic dressings,
xenogenic bandages, and xenogenic structures.
[0073] As used herein, "allograft" and other grammatical forms
thereof (e.g., allogeneic transplantation) may include any
procedure in which a recipient and a donor are from the same
species but different individuals and cells, tissues or organs are
transplanted, implanted or infused into the recipient.
Transplantation of cells, organs and/or tissues as described herein
can be used as allografts in humans. Allografts include, but are
not limited to, vascularized allografts, partially vascularized
allografts, non-vascularized allografts, allogeneic dressings,
allogeneic bandages, and allogeneic structures.
[0074] As used herein, "autologous transplantation" and other
grammatical forms thereof (e.g., autologous transplantation) may
include any procedure in which a recipient and a donor are from the
same individual and cells, tissues or organs are transplanted,
implanted or infused into the recipient. Transplantation of cells,
organs and/or tissues described herein can be used as autografts in
humans. Autografts include, but are not limited to, vascular
autologous transplantation, partial vascular autologous
transplantation, non-vascularized autologous transplantation,
autologous dressings, autologous bandages and autologous
structures.
[0075] The term "chimeric antigen receptor" or "CAR" as used herein
refers to an engineered molecule that can be expressed by an immune
cell including, but not limited to, T cell or NK cell. CAR is
expressed in T cells and redirects T cells to induce specific
killing of target cells with a specificity determined by the
artificial receptor. The extracellular binding domain of CAR can be
derived from a murine, humanized or fully humanized monoclonal
antibody.
[0076] The term "epitope" as used herein and other grammatical
forms thereof may refer to a portion of an antigen that can be
recognized by an antibody, B cell, T cell or engineered cell. For
example, an epitope can be a tumor epitope or a pathogen epitope
recognized by a TCR. Multiple epitopes within an antigen can also
be recognized. Epitopes can also be mutated.
[0077] "Cell line" or "cell culture" means a bacterium, plant,
insect or higher eukaryotic cell grown or maintained in vitro. The
progeny of a cell may not be identical (in morphology, genotype or
phenotype) to the maternal cell.
[0078] The term "engineered" as used herein and other grammatical
forms thereof may refer to one or more changes in a nucleic acid,
such as a nucleic acid within the genome of an organism. The term
"engineered" can refer to alterations, additions, and/or deletions
of genes. Engineered cells can also refer to cells having genes
that are added, deleted, and/or altered. Engineered cells can also
refer to cells that express CAR.
[0079] The term "transfection" as used herein refers to the
introduction of an exogenous nucleic acid into a eukaryotic cell.
Transfection can be achieved by various means known in the art,
including calcium phosphate-DNA co-precipitation, DEAE-dextran
mediated transfection, polyamine mediated transfection,
electroporation, microinjection, liposome fusion, lipofection,
protoplast fusion, retroviral infection and biolistics.
[0080] The term "stable transfection" or "stably transfect" refers
to the introduction and integration of exogenous nucleic acids,
DNAs or RNAs into the genome of a transfected cell. The term
"stable transfectant" refers to a cell having exogenous nucleic
acids stably integrated into genomic DNA.
[0081] As used herein, the terms "nucleic acid molecule encoding",
"encoding DNA sequence" and "encoding DNA" refer to the order or
sequence of deoxyribonucleotides along a deoxyribonucleotide chain.
The order of these deoxyribonucleotides determines the order of the
amino acids along the polypeptide (protein) chain. Therefore, a
nucleic acid sequence encodes an amino acid sequence.
[0082] The term "individual" as used herein refers to any animal,
such as a mammal or marsupial. Individuals of the invention
include, but are not limited to, humans, non-human primates (e.g.,
rhesus monkeys or other types of macaques), mice, pigs, horses,
donkeys, cows, sheep, rats and poultry of any kind.
[0083] The term "peripheral blood lymphocytes" (PBL) and other
grammatical forms thereof as used herein may refer to lymphocytes
circulating in blood (e.g., peripheral blood). Peripheral blood
lymphocytes can refer to lymphocytes that are not restricted to
organs. Peripheral blood lymphocytes can comprise T cells, NK
cells, B cells or any combinations thereof.
[0084] The term "immune response cell" (or "immunoreactive cell",
"immune effector cell" or "immune cell") as used herein may refer
to a cell that can elicit an immune response. An immune response
cell can also refer to a cell of the lymphoid or myeloid lineage.
Examples of immune cells include, but are not limited to, T cells,
such as .alpha./.beta. T cells and .gamma./.delta. T cells, B
cells, natural killer (NK) cells, natural killer T (NKT) cells,
mast cells and bone marrow-derived phagocytic cells, respective
precursor cells and progenies thereof.
[0085] The term "T cell" and other grammatical forms thereof as
used herein may refer to T cells from any source. For example, T
cell can be a primary T cell, such as an autologous T cell or the
like. T cell can also be of human or non-human.
[0086] The term "T cell activation" or "T cell trigger" as used
herein and other grammatical forms thereof may refer to status of a
T cell that is sufficiently stimulated to induce detectable cell
proliferation, cytokine production, and/or detectable effector
function. In some embodiments, "complete T cell activation" can be
similar to triggering cytotoxicity of T cells. T cell activation
can be measured using various assays known in the art. The assay
can be an ELISA for measuring cytokine secretion, ELISPOT, a flow
cytometry assay for measuring intracellular cytokine expression
(CD107), a flow cytometry assay for measuring proliferation, and
cytotoxicity assay for determining target cell elimination (51Cr
release assay). In the assay, controls (non-engineered cells) are
typically used to compare with engineered cells (CART) to determine
the relative activation of engineered cells compared with controls.
Furthermore, the assay can be performed by comparison with
engineered cells that are incubated or contacted with target cells
that do not express the target antigen. For example, the comparison
can be a comparison with CD19-CART cells incubated with target
cells that do not express CD19.
[0087] The term "sequence" as used herein and other grammatical
forms thereof, when used in reference to a nucleotide sequence, may
include DNA or RNA, and may be single-stranded or double-stranded.
The nucleic acid sequence can be mutated. The nucleic acid sequence
can be of any length, for example, a nucleic acid having 2 to
1,000,000 or more nucleotides (or any integer value there between
or above), for example, about 100 to about 10,000 nucleotides or
about 200 to about 500 nucleotides in length.
[0088] The term "effective amount" as used herein refers to an
amount that provides therapeutic or prophylactic benefits.
[0089] The term "expression vector" as used herein, refers to a
vector comprising a recombinant polynucleotide comprising an
expression regulation sequence operably linked to a nucleotide
sequence to be expressed. The expression vector contains sufficient
cis-acting elements for expression; and other elements for
expression can be provided by host cells or in vitro expression
systems. Expression vectors include those known in the art, such as
cosmids, plasmids (e.g., naked or contained in liposomes), and
viruses (e.g., lentiviruses, retroviruses, adenoviruses, and
adeno-associated viruses).
[0090] The term "lentivirus" as used herein refers to the genus of
retroviridae family. Retroviruses are unique in retroviruses in
their ability to infect non-dividing cells; they can deliver large
amounts of genetic information into DNAs of host cells, therefore,
they are one of the most efficient methods of gene delivery
vectors. HIV, SIV and FIV are examples of lentiviruses. Vectors
derived from lentiviruses provide a means to achieve significant
levels of gene transfer in vivo.
[0091] The term "operably linked" as used herein, refers to a
functional linkage between a regulatory sequence and a heterologous
nucleic acid sequence, which results in expression of the latter.
For example, when the first nucleic acid sequence is functionally
associated to the second nucleic acid sequence, the first nucleic
acid sequence is operably linked to the second nucleic acid
sequence. For example, a promoter is operably linked to an encoding
sequence if the promoter affects the transcription or expression of
the encoding sequence. Typically, the operably linked DNA sequences
are contiguous and, where necessary, two protein encoding regions
are ligated in the same reading frame.
[0092] The term "promoter" as used herein, is defined as a DNA
sequence that is recognized by the synthetic machinery or the
introduced synthetic machinery being required to initiate specific
transcription of a polynucleotide sequence.
[0093] The term "vector," as used herein, is a composition
comprising an isolated nucleic acid and being used to deliver an
isolated nucleic acid to the interior of a cell. A number of
vectors are known in the art including, but not limited to, linear
polynucleotides, polynucleotides associated with ionic or
amphiphilic compounds, plasmids and viruses. Therefore, the term
"vector" includes autonomously replicating plasmids or viruses. The
term should also be interpreted to include non-plasmid and
non-viral compounds that facilitate the transfer of nucleic acids
into cells, such as polylysine compounds, liposomes, and the like.
Examples of viral vectors include, but are not limited to,
adenoviral vectors, adeno-associated viral vectors, retroviral
vectors, and the like.
[0094] A "host cell" includes an individual cell or cell culture
that can be or has been an acceptor of a target vector. Host cells
include the progeny of a single host cell. Due to natural,
accidental or intentional mutations, progeny may not necessarily be
identical to the original parental cell, for example, in
morphological properties or genomic DNA or total DNA. Host cells
include cells that are transfected in vivo with the vectors of the
invention. "Host cell" can refer to a prokaryotic cell, a
eukaryotic cell, or a cell line that is cultured as a single cell
entity that can be or has been used as a receptor for recombinant
vectors or other transfer polynucleotides, and includes progeny
cells that have been transfected.
[0095] The term "sequence identity" as used herein determines the
percent identity by comparing two best matched sequences over a
comparison window (e.g., at least 20 positions), wherein portions
of the polynucleotide or polypeptide sequence in the comparison
window can comprise addition or deletion (i.e., gap), for example,
20% or less of gaps (e.g., 5 to 15%, or 10 to 12%) compared with a
reference sequence (which does not contain additions or deletions)
for two best matched sequence. The percentage is usually calculated
by determining the number of positions in which nucleotides or
amino acid residues are the same in both sequences to produce the
number of correctly matched positions. The sequence identity
percentage can be obtained by dividing the number of correctly
matched positions by the total number of positions in the reference
sequence (that is, the window size) and multiplying the result by
100.
[0096] The term "disease" or "condition" or "disorder" as used
herein, refers to any alteration or disorder that impairs or
interferes with the normal function of a cell, tissue or organ. For
example, the term "disease" includes, but is not limited to, a
tumor, pathogen infection, autoimmune disease, T cell dysfunction
disease, or defect in immune tolerance (e.g., transplant
rejection).
[0097] The term "exogenous" as used herein, refers to a nucleic
acid molecule or polypeptide that is not endogenously expressed in
a cell, or the expression level of which is insufficient to achieve
the function of overexpression. Therefore, "exogenous" includes
recombinant nucleic acid molecules or polypeptides expressed in a
cell, such as exogenous, heterologous and overexpressed nucleic
acid molecules and polypeptides.
[0098] The term "regulation" as used herein refers to a positive or
negative change. Example of regulation includes 1%, 2%, 10%, 25%,
50%, 75% or 100% variation.
[0099] As used herein, the term "treatment" refers to a clinical
intervention in an attempt to alter an individual or treat a
disease caused by a cell, both prophylactically and in a clinical
pathological process. Therapeutic effects include, but are not
limited to, preventing the occurrence or recurrence of the disease,
alleviating symptoms, reducing the direct or indirect pathological
consequences of any disease, preventing metastasis, slowing the
progression of the disease, improving or ameliorating the
condition, alleviating or improving the prognosis.
[0100] The term "constitutive expression" as used herein refers to
expression under all physiological conditions.
[0101] The term "inducible expression" as used herein refers to
expression under certain conditions, such as when a T cell binds to
an antigen. One skilled in the art will know how to perform
conventional "induced expression".
[0102] In some embodiments, an antigen binding unit comprising a
light chain CDR and a heavy chain CDR is provided herein, wherein
the antigen binding unit specifically binds to claudin 18A2
peptide; and wherein the antigen binding unit does not
significantly bind to claudin 18A1 peptide.
[0103] In some embodiments, an antigen binding units comprising a
light chain CDR and a heavy chain CDR is provided herein, wherein
the antigen binding unit specifically binds to claudin 18A2
peptide; and wherein the antigen binding unit shows less
non-specific binding to claudin 18A1 peptide, as compared with a
reference antigen binding unit.
[0104] In some embodiments, an antigen binding unit described
herein comprises a light chain CDR. The light chain CDR can be a
complementarity determining region of an antigen binding unit. The
light chain CDR may comprise a contiguous sequence of amino acid
residues, or two or more contiguous sequence of amino acid residues
spaced by non-complementarity determining regions, such as
framework regions. In some embodiments, the light chain CDR
comprises two or more light chain CDRs, which may be named to as
light chain CDR-1, CDR-2, and the like. In some embodiments, the
light chain CDR comprises three light chain CDRs, which may be
named as light chain CDR-1 (LCDR1), light chain CDR-2 (LCDR2) and
light chain CDR-3 (LCDR3), respectively. In some embodiments, a set
of CDRs present on a common light chain can be collectively
referred to as a light chain CDR.
[0105] In some embodiments, an antigen binding unit described
herein comprises a heavy chain CDR. The heavy chain CDR can be a
complementarity determining region of an antigen binding unit. The
heavy chain CDR may comprise a contiguous sequence of amino acid
residues, or two or more contiguous sequence of amino acid residues
spaced by non-complementarity determining regions, such as
framework regions. In some embodiments, the heavy chain CDR
comprises two or more heavy chain CDRs, which may be named to as
heavy chain CDR-1, CDR-2, and the like. In some embodiments, the
heavy chain CDR comprises three heavy chain CDRs, which may be
named as heavy chain CDR-1 (HCDR1), heavy chain CDR-2 (HCDR2) and
heavy chain CDR-3 (HCDR3), respectively. In some embodiments, a set
of CDRs present on a common heavy chain can be collectively
referred to as a heavy chain CDR.
[0106] In some embodiments, an antigen binding unit comprising a
light chain CDR and a heavy chain CDR is provided herein, wherein
the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy
chain CDR comprises HCDR1, HCDR2 and HCDR3; the amino acid
sequences of LCDR1, LCDR2 and LCDR3 are at least 80% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52,
SEQ ID NO:53 and SEQ ID NO:54; and the amino acid sequences of
HCDR1, HCDR2 and HCDR3 are at least 80% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:31, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:83 SEQ ID NO:84 and SEQ ID NO:85.
[0107] In some embodiments, an antigen binding unit comprising a
light chain CDR and a heavy chain CDR is provided herein, wherein
the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy
chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein the amino acid
sequences of LCDR1, LCDR2 and LCDR3 are at least 60%, 65%, 70%,
75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to
an amino acid sequence selected from the following: SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ
ID NO:53 and SEQ ID NO:54; and wherein the amino acid sequences of
HCDR1, HCDR2 and HCDR3 are at least 60%, 65%, 70%, 75%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to an amino acid
sequence selected from the group consisting of: SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO: 38, SEQ ID NO:39,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49, SEQ ID
NO:50, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID
NO:85.
[0108] In some embodiments, in the antigen binding unit herein, the
light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy
chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein the LCDR1,
LCDR2 and LCDR3 respectively have an amino acid sequence selected
from the group consisting of: SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID
NO:54; and wherein said HCDR1, HCDR2 and HCDR3 respectively have an
amino acid sequence selected from the group consisting of: SEQ ID
NO:31 SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:37, SEQ ID NO:38, SEQ
ID NO:39, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:83, SEQ ID NO:84 and SEQ ID
NO:85.
[0109] In some embodiments, an antigen binding unit is provided
herein, wherein the LCDR1 comprises an amino acid sequences which
is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% identical to an amino acid sequence selected from
the group consisting of: SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46,
and SEQ ID NO:52. In some embodiments, an antigen binding unit is
provided herein, wherein the LCDR2 comprises an amino acid
sequences which is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5% or 99.9% identical to an amino acid sequence
selected from the group consisting of: SEQ ID NO:34, SEQ ID NO:41,
SEQ ID NO:47 and SEQ ID NO:53.
[0110] In some embodiments, an antigen binding unit is provided
herein, wherein the LCDR3 comprises an amino acid sequences which
is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% identical to an amino acid sequence selected from
the group consisting of: SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:48,
and SEQ ID NO:54. In some embodiments, an antigen binding unit is
provided herein, wherein the HCDR1 comprises an amino acid
sequences which is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5% or 99.9% identical to an amino acid sequence
selected from the group consisting of: SEQ ID NO:31, SEQ ID NO:37,
SEQ ID NO:43 and SEQ ID NO:49. In some embodiments, an antigen
binding unit is provided herein, wherein the HCDR2 comprises an
amino acid sequences which is at least 60%, 65%, 70%, 75%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to an amino
acid sequence selected from the group consisting of: SEQ ID NO:32,
SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:83, SEQ ID
NO:84 and SEQ ID NO:85. In some embodiments, an antigen binding
unit is provided herein, wherein the HCDR3 comprises an amino acid
sequences which is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.5% or 99.9% identical to an amino acid sequence
selected from the group consisting of: SEQ ID NO:33, SEQ ID NO:39,
SEQ ID NO:45 and SEQ ID NO:51.
[0111] In some embodiments, an antigen binding unit of the
invention binds to claudin 18A2 or a claudin 18A2 peptide. The term
"claudin 18A2" or "claudin 18A2 peptide" (CLD18.2, CLD18A2,
CLDN18A2, CLDN18.2, Claudin18.2 or Claudin18A2) herein may also
refer to a homologue, orthologue, interspecies homolog, codon
optimized form, truncated form, fragmented form, mutated form or
any other known derivative form of a known claudin 18A2 sequence,
such as a post-translational modification variant. In some
embodiments, the claudin 18A2 or claudin 18A2 peptide is a peptide
with GenBank accession number NP_001002026 (mRNA: NM_001002026). In
some embodiments, the claudin 18A2 or claudin 18A2 peptide is a
peptide comprising the amino acid sequence of SEQ ID NO:55.
[0112] In some embodiments, the antigen binding unit of the
invention does not significantly bind to claudin 18A1 peptide. The
term "claudin 18A1" or "claudin 18A1 peptide" (CLD18A1, CLD18.1,
CLDN18A1, CLDN18.1, Claudin18.1 or Claudin18A1) herein may also
refer to a homologue, orthologue, interspecies homolog, codon
optimized form, truncated form, fragmented form, mutated form or
any other known derivative form of a known claudin 18A1 sequence,
such as a post-translational modification variant. In some
embodiments, the claudin 18A1 or claudin 18A1 peptide is a peptide
with GenBank accession number NP_057453 (mRNA: NM_016369). In some
embodiments, the claudin 18A1 or claudin 18A1 peptide is a peptide
comprising an amino acid sequence of SEQ ID NO:57.
[0113] Binding specificity can be determined by complementarity
determining regions or CDRs, such as light chain CDRs or heavy
chain CDRs. In many cases, binding specificity is determined by
light chain CDRs and heavy chain CDRs. Compared with other
reference antigens or reference peptides, a given heavy chain CDR,
light chain CDR or a combination thereof provide a given binding
pocket with greater affinity and/or specificity to claudin
18A2.
[0114] Binding of the antigen binding unit to claudin 18A2 peptide
can be characterized or expressed by any method known in the art.
For example, binding can be characterized by binding affinity,
which can be the strength of the interaction between the antigen
binding unit and the antigen. Binding affinity can be determined by
any method known in the art, such as in vitro binding assays. For
example, when tested in an in vitro binding assay using cells
expressing claudin 18A2, the binding affinity of the antigen
binding unit disclosed herein can be determined. The binding
affinity of the tested antigen binding unit can be expressed as Kd,
which is the equilibrium dissociation constant between the antibody
and its respective antigen. In some cases, the antigen binding unit
disclosed herein specifically binds to claudin 18A2, with Kd
ranging from about 10 .mu.M to about 1 mM. For example, the antigen
binding unit can specifically bind to claudin 18A2 with a Kd of
less than about 10 .mu.M, 1 .mu.M, 0.1 .mu.M, 10 nM, 1 nM, 0.1 nM,
10 pM, 1 pM, 0.1 pM, 10 fM, 1 fM or less than 0.1 fM.
[0115] In some embodiments, the antigen binding unit does not
exhibit significant binding to a reference peptide. In some
examples, the binding level of the antigen binding unit to the
reference peptide is not higher than 20% of the binding level of
the antigen binding unit to claudin 18A2. For example, the binding
level may be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% of the binding
level of the antigen binding unit to claudin 18A2. In some
embodiments, the antigen binding unit herein binds to claudin 18A2
at a level that is 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold,
7 fold, 8 fold, 9 fold, 10 fold or more than 10 hold of the binding
level to the reference peptide. In some embodiments, the reference
peptide is claudin 18A1 peptide. In some embodiments, the reference
peptide is a peptide comprising an amino acid sequence of SEQ ID
NO:57. In some embodiments, the reference peptide is a peptide of
SEQ ID NO:57.
[0116] In some embodiments, compared with a reference antigen
binding unit, the antigen binding unit herein exhibits less
non-specific binding to a reference peptide. In some embodiments,
the non-specific binding level of the antigen binding unit herein
to the reference peptide is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% lower
than that of the reference antigen binding unit to the reference
peptide. In some embodiments, the non-specific binding level of the
antigen binding unit herein to the reference peptide is 1 fold, 2
fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10
fold or more than 10 hold lower than that of the reference antigen
binding unit to the reference peptide. In some embodiments, the
reference antigen binding unit comprises a light chain of SEQ ID
NO:86 or SEQ ID NO:88 and/or a heavy chain amino acid sequence of
SEQ ID NO:87 or SEQ ID NO:89. In some embodiments, the reference
antigen binding unit comprises an amino acid sequence of SEQ ID
NO:86. In some embodiments, the reference antigen binding unit
comprises an amino acid sequence of SEQ ID NO:87. In some
embodiments, the reference antigen binding unit comprises an amino
acid sequence of SEQ ID NO:88. In some embodiments, the reference
antigen binding unit comprises an amino acid sequence of SEQ ID
NO:89. In some embodiments, the reference peptide is claudin 18A1
peptide. In some embodiments, the reference peptide is a peptide
comprising an amino acid sequence of SEQ ID NO:57. In some
embodiments, the reference peptide is a peptide of SEQ ID
NO:57.
[0117] In some embodiments, the antigen binding unit is cytotoxic
to a cell comprising claudin 18A2 peptide comprising an amino acid
sequence of SEQ ID NO:55. The cytotoxicity level is at least 5%,
10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, when the ratio of the
antigen binding unit to the target cell is 20:1, 10:1, 5:1, 3:1,
2.5:1, 1:1 or 1:3.
[0118] In some embodiments, the antigen binding unit does not have
significant cytotoxicity to a cell comprising claudin 18A1 peptide
but not comprising claudin 18A2 peptide, wherein the claudin 18A1
peptide comprises an amino acid sequence of SEQ ID NO:57, and the
claudin 18A2 peptide comprises an amino acid sequence of SEQ ID
NO:55. In some embodiments, the cytotoxicity level is not higher
than 10%, 5%, 4%, 3%, 2% or 1%.
[0119] In some embodiments, an antibody specifically binding to
claudin 18A2 is provided herein, characterized in that the antibody
comprises a heavy chain CDR comprising an amino acid sequence
selected from the group consisting of SEQ ID NOS:31, 32, 33, 37,
38, 39, 43 of 44, 45, 49, 50, 51, 83, 84, 85 or a variant thereof
and/or a light chain CDR comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:34, 35, 36, 40, 41, 42, 46,
47, 48, 52, 53, 54 or a variant thereof.
[0120] In some embodiments, an antibody is provided herein which is
selected from the group consisting of (a) an antibody comprising a
heavy chain variable region, wherein the heavy chain variable
region has CDR1 comprising an amino acid sequence of SEQ ID NO:31,
SEQ ID NO:37, SEQ ID NO:43 or SEQ ID NO:49, CDR2 comprising an
amino acid sequence of SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44,
SEQ ID NO:50, SEQ ID NO:83, SEQ ID NO:84 or SEQ ID NO:85, and CDR3
comprising an amino acid sequence of SEQ ID NO:33, SEQ ID NO:39,
SEQ ID NO:45 or SEQ ID NO:51; (b) an antibody comprising a light
chain variable region, wherein the light chain variable region has
CDR1 comprising an amino acid sequence of SEQ ID NO:34, SEQ ID
NO:40, SEQ ID NO:46 or SEQ ID NO:52, CDR2 comprising an amino acid
sequence of SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47 or SEQ ID
NO:53, and CDR3 comprising an amino acid sequence of SEQ ID NO:36,
SEQ ID NO:42, SEQ ID NO:48 or SEQ ID NO:54; (c) an antibody
comprising (a) a heavy chain variable region of said antibody and
(b) a light chain variable region of said antibody; and (d) an
antibody, recognizing the same antigenic determinant site as that
of the antibody of any one of (a) to (c). In some embodiments, an
antibody is provided herein, wherein the CDR1, CDR2 and CDR3
regions of the heavy chain variable region of the antibody are SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33; or SEQ ID NO:37, SEQ ID
NO:38, SEQ ID NO:39; or SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45;
or SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51; or SEQ ID NO:31, SEQ
ID NO:83, SEQ ID NO:33; or SEQ ID NO:31, SEQ ID NO:84, SEQ ID
NO:33; or SEQ ID NO:49, SEQ ID NO:85, SEQ ID NO:51, respectively;
and/or the CDR1, CDR2 and CDR3 regions of the light chain variable
region of the antibody are SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36; or SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42; or SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48; or SEQ ID NO:52, SEQ ID NO:53,
SEQ ID NO:54, respectively.
[0121] In some embodiments, the amino acid sequences of the CDR1,
CDR2 and CDR3 of the heavy chain of the antibody of the invention
are selected from the group consisting of the amino acid sequences
set forth in the following table or variants thereof:
TABLE-US-00001 TABLE 1 HCDR1 HCDR2 HCDR3 A SEQ ID NO: 31 SEQ ID NO:
32 SEQ ID NO: 33 B SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 C SEQ
ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 D SEQ ID NO: 49 SEQ ID NO: 50
SEQ ID NO: 51 E SEQ ID NO: 31 SEQ ID NO: 83 SEQ ID NO: 33 F SEQ ID
NO: 31 SEQ ID NO: 84 SEQ ID NO: 33 G SEQ ID NO: 49 SEQ ID NO: 85
SEQ ID NO: 51
[0122] In some embodiments, an antibody is provided herein, wherein
the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are
selected from the group consisting of the amino acid sequences in
the following table or variants thereof:
TABLE-US-00002 TABLE 2 LCDR1 LCDR2 LCDR3 A SEQ ID NO: 34 SEQ ID NO:
35 SEQ ID NO: 36 B SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 C SEQ
ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 D SEQ ID NO: 52 SEQ ID NO: 53
SEQ ID NO: 54
[0123] In some embodiments, an antibody of the invention comprises
a heavy chain variable region comprising an amino acid sequence
selected from SEQ ID NOS:3, 7, 11, 15, 17, 19, 23, 27, 29 or
variants thereof, and/or a light chain variable region comprising
an amino acid sequenced sequence selected from SEQ ID NOS:1, 5, 9,
13, 21, 25 or a variant thereof. In some embodiments, the heavy
chain variable region is SEQ ID NO:3 or a variant thereof and the
light chain variable region is SEQ ID NO:1 or a variant thereof. In
some embodiments, the heavy chain variable region is SEQ ID NO:7 or
a variant thereof and the light chain variable region is SEQ ID
NO:5 or a variant thereof. In some embodiments, the heavy chain
variable region is SEQ ID NO:11 or a variant thereof and the light
chain variable region is SEQ ID NO:9 or variant thereof. In some
embodiments, the heavy chain variable region is SEQ ID NO:15 or a
variant thereof and the light chain variable region is SEQ ID NO:13
or variant thereof. In some preferred embodiments, the amino acid
sequences of the heavy chain CDR1, CDR2 and CDR3 are selected from
the table below.
TABLE-US-00003 TABLE 3 HCDR1 HCDR2 HCDR3 E SEQ ID NO: 31 SEQ ID NO:
83 SEQ ID NO: 33 F SEQ ID NO: 31 SEQ ID NO: 84 SEQ ID NO: 33 G SEQ
ID NO: 49 SEQ ID NO: 85 SEQ ID NO: 51
[0124] In some embodiments, an antibody of the invention comprises
a heavy chain variable region comprising an amino acid sequence
selected from SEQ ID NOS:17, 19, or variants thereof. In some
embodiments, the heavy chain variable region is SEQ ID NO:17 or a
variant thereof and the light chain variable region is SEQ ID NO:1
or a variant thereof. In some embodiments, the heavy chain variable
region is SEQ ID NO:19 or a variant thereof and the light chain
variable region is SEQ ID NO:1 or variant thereof.
[0125] In some embodiments, an antibody of the invention or a
functional fragment thereof comprises a heavy chain variable region
comprising an amino acid sequence selected from SEQ ID NOS:23, 27,
29 or a variant thereof, and/or a light chain variable region
comprising an amino acid sequence selected from SEQ ID NOS: 21, 25
or a variant thereof. In some embodiments, the heavy chain variable
region is SEQ ID NO:23 or a variant thereof and the light chain
variable region is SEQ ID NO:21 or a variant thereof. In some
embodiments, the heavy chain variable region is SEQ ID NO:27 or a
variant thereof and the light chain variable region is SEQ ID NO:25
or a variant thereof. In some embodiments, the heavy chain variable
region is SEQ ID NO:29 or a variant thereof and the light chain
variable region is SEQ ID NO:25 or a variant thereof.
[0126] In some embodiments, an antigen binding unit or antibody of
the invention is further linked or fused to another functional
molecule. Accordingly, the invention also encompasses formed
multifunctional immunoconjugates.
[0127] "Linked" or "fused" are used interchangeably herein. These
terms means that two or more chemical elements or components are
joined together by any means including chemical conjugation or
recombinant methods. "In-frame fusion" means that two or more
reading frames are joined in a manner maintaining the correct
reading frame of the original open reading frame (ORF) to form a
contiguous and longer ORF. Therefore, the resulting recombinant
fusion protein is a single protein containing two or more fragments
corresponding to the polypeptide encoded by the original ORF (these
fragments are usually not so ligated in a natural state). The
reading frames are contiguous throughout the fusion fragment,
however the fragments may be physically or spatially separated by,
for example, an in-frame joining sequence (e.g., "flexion").
[0128] The functional molecule is, for example used for the
diagnosis or treatment of a tumor.
[0129] The term "tumor" as used herein refers to a disease
characterized by pathological hyperplasia of cells or tissues, and
subsequent migration or invasion of other tissues or organs. Tumor
growth is usually uncontrolled and progressive and does not induce
or inhibit normal cell proliferation. Tumors can affect a variety
of cells, tissues or organs, including but not limited to, bladder,
bone, brain, breast, cartilage, glial cells, esophagus, fallopian
tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph
nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle,
skin, spinal cord, spleen, stomach, testis, thymus, thyroid,
trachea, urethra, ureter, urethra, uterus, vaginal organs, or
tissue or corresponding cells. Tumors include cancers such as
sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma
cells). The tumor of the present invention may include, but is not
limited to, leukemia (such as acute leukemia, acute lymphocytic
leukemia, acute myeloid leukemia, acute myeloid leukemia, acute
promyelocytic leukemia, acute granulocyte-monocytic leukemia, acute
monocytic leukemia, acute leukemia, chronic leukemia, chronic
myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera),
lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary
macroglobulinemia Disease, heavy chain disease, solid tumors such
as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma,
liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial
sarcoma, lymphangiosarcoma, angiosarcoma, lymphatic endothelial
sarcoma, synovial vioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer,
breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
cancer, sebaceous gland cancer, papillary cancer, papillary
adenocarcinoma, carcinoma, bronchial carcinoma, medullary
carcinoma, renal cell carcinoma, liver cancer, bile tube cancer,
choriocarcinoma, fine Tumor, embryonic carcinoma, nephroblastoma,
cervical cancer, uterine cancer, testicular cancer, lung cancer,
small cell lung cancer, bladder cancer, epithelial cancer, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, Ependymoma, pineal
tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma,
schwannomas, meningiomas, melanoma, neuroblastoma, retinoblastoma),
esophageal cancer, gallbladder carcinoma, kidney cancer, multiple
myeloma. Preferably, the "tumor" includes, but is not limited to,
pancreatic cancer, liver cancer, lung cancer, gastric cancer,
esophageal cancer, head and neck squamous cell carcinoma, prostate
cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer,
Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical
cancer and glioma.
[0130] The functional molecule includes, for example, a tumor
antigen, such as a tumor specific antigen (TSA) or a tumor
associated antigen (TAA). TSA is unique to tumor cells and does not
occur on other cells in the body. The TAA-associated antigen is not
unique to tumor cells, but is expressed on normal cells under
conditions in which the immune tolerance state to the antigen
cannot be induced. Expression of an antigen on a tumor can occur
under conditions that allow the immune system to respond to the
antigen. When the immune system is immature and unable to respond,
the TAA may be an antigen that is expressed on normal cells during
fetal development, or they may be antigens that are normally
present at very low levels on normal cells but are expressed at a
higher level on tumor cells.
[0131] Non-limiting examples of TSA or TAA antigens include the
following: differentiation antigens such as MART-1/MelanA (MART-I),
gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific
multicenter antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2,
p15; overexpressed embryonic antigens, such as CEA; overexpressed
oncogenes and mutant tumor suppressor genes, such as p53, Ras,
HER-2/neu; unique tumor antigens caused by chromosomal
translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK and
MYL-RAR; and viral antigens, such as Epstein Barr virus antigen
EBVA and human papilloma Virus (HPV) antigens E6 and E7, etc. Other
large, protein-based antigens include TSP-180, MAGE-4, MAGE-5,
MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA,
TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, .beta.-catenin,
CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein,
beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA
242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM,
HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1,
SDCCAG16, TA-90\Mac-2 binding protein\Cyclophilin C-related
protein, TAAL6, TAG72, TLP and TPS.
[0132] In some embodiments, the "tumor antigen" includes, but is
not limited to, prostate specific membrane antigen (PSMA),
carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1,
HIV-1 Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT,
mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3,
EpCAM, MUC1, MUC16, Folate receptor, CLDN6, CD30, CD138, ASGPR1,
CDH16, GD2, 5T4, 8H9, .alpha.v.beta.6 integrin, B cell mature
antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, K Kappa light
chain, CD33, CD38, CD44, CD44v6, CD44v7/8, CD70, CD123, CD171,
CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3/4, FAP, FAR, FBP, embryonic
AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11Ra, KDR, Lambda,
MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN,
TAG72, TEM1, TEM8, VEGRR2, HMW-MAA, VEGF receptor, and/or
fibronectin, tenascin or carcinoembryonic variants of tumor
necrotic regions.
[0133] In some embodiments, the functional molecule is an
interferon. In some embodiments, the interferon is a type I
interferon.
[0134] The term "type I interferon" as used herein includes
IFN.alpha., IFN.beta., IFN-.epsilon., IFN-.kappa., IFN-.omega. and
the like. All type I interferons bind to specific cell surface
receptors (so-called IFN-.alpha./.beta. receptors) consisting of
two strands of IFNAR1 and IFNAR2. In some embodiments, the term
"type I interferon" as used herein is IFN.alpha. or IFN.beta.. In
some embodiments, the term "type I interferon" as used herein is
IFN.beta.. In some embodiments, Type I interferon as used herein
includes a human, mouse or synthetic Type I interferon. In some
embodiments, the term "interferon .alpha." as used herein may be a
polypeptide having the sequence shown in NCBI aaa52724.1 or
aaa52716.1 or aaa52725.1, or a polypeptide, the sequence of which
has at least 85% identity to these sequences. In some embodiments,
the term "interferon .beta." (INF-.beta.) as used herein may be a
protein having at least 85% identity to NCBI aac41702.1 or
np_002167.1 or aah96152.1p41273 or NP 001552, or a fragment having
the function of a tumor necrosis factor (TNF) ligand. In some
embodiments, the interferon .beta. is human interferon .beta.. In
some embodiments, the interferon .beta. has an amino acid sequence
of SEQ ID NO:92.
[0135] In some embodiments, Type I interferon may be naturally
occurring, for example, isolated or purified from a mammal; or may
be artificially prepared, for example, recombinant components or
type I interferon can be produced according to conventional genetic
engineering recombination techniques. Preferably, recombinant
elements or type I interferons may be used in the present
invention.
[0136] Amino acid sequences formed based on the type I interferon
polypeptide sequence by substitution, deletion or addition of one
or more amino acid residues are also included in the present
invention. Appropriate replacement of amino acids is a technique
well known in the art that can be readily implemented and ensures
that the biological activities of a resulting molecule will not be
altered. Based on these techniques, a skilled person will
appreciate that, in general, altering a single amino acid in a
non-essential region of a polypeptide does not substantially alter
biological activities.
[0137] Polypeptides modified according to the type I interferon
polypeptide sequence can also be used in the present invention. For
example, a polypeptide modified to promote its half-life,
effectiveness, metabolism and/or potency can be used. That is, any
variation that does not affect the biological activities of a
polypeptide can be used in the present invention.
[0138] Biologically active fragments of type I interferon
polypeptide can be used in the present invention. As used herein,
the meaning of a biologically active fragment refers to a
polypeptide which, as part of a full length polypeptide, still
retains all or part of the function of the full length polypeptide.
Typically, the biologically active fragment retains at least 50% of
the activities of the full length polypeptide. Under more preferred
conditions, the active fragment is capable of retaining 60%, 70%,
80%, 90%, 95%, 99%, or 100% of the activities of the full length
polypeptide.
[0139] In another aspect, the invention provides a chimeric antigen
receptor comprising an extracellular antigen binding unit as
described herein, a transmembrane domain, and an intracellular
domain. The term "Chimeric Antigen Receptor (CAR)" as used herein
refers to a tumor antigen binding domain fused to an intracellular
signal transduction domain that activates T cells. Typically, the
extracellular binding domain of CAR is derived from a mouse or
humanized or human monoclonal antibody.
[0140] A chimeric antigen receptor typically comprises an
extracellular antigen binding region or antigen binding unit. In
some embodiments, the extracellular antigen binding unit is an
antigen binding unit as described herein above.
[0141] In some embodiments, the extracellular antigen binding
region can be of full human. In other instances, the extracellular
antigen binding region can be humanized. In other instances, the
extracellular antigen binding region can be murine or the chimera
in the extracellular antigen binding region consists of amino acid
sequences derived from at least two different animals. In some
embodiments, the extracellular antigen binding region can be of
non-human.
[0142] A variety of antigen binding regions can be designed.
Non-limiting examples include single-chain variable fragments
(scFv) derived from antibodies, fragments of antigen-binding
regions (Fabs) selected from libraries, single-domain fragments, or
natural ligands that bind to their cognate receptors. In some
embodiments, the extracellular antigen binding region can comprise
scFv, Fab or natural ligand, as well as any derivatives thereof. An
extracellular antigen binding region can refer to a molecule other
than an intact antibody, which can comprise a portion of an intact
antibody and can bind to an antigen to which the intact antibody
binds. Examples of antibody fragments can include, but are not
limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional
antibodies, linear antibodies; single-chain antibody molecules
(e.g., scFv); and multispecific antibodies formed from antibody
fragments.
[0143] An extracellular antigen binding region, such as a scFv, Fab
or natural ligand, can be a part of a CAR that determines antigen
specificity. The extracellular antigen binding region can bind to
any complementary target. The extracellular antigen binding region
can be derived from an antibody with known variable region
sequence. The extracellular antigen binding region can be obtained
from antibody sequences from available mouse hybridomas.
Alternatively, extracellular antigen binding regions can be
obtained from tumor cells or primary cells, such as tumor
infiltrating lymphocytes (TIL) through whole external cutting
sequencing.
[0144] In some embodiments, the binding specificity of the
extracellular antigen binding region can be determined by a
complementarity determining region or CDR, such as a light chain
CDR or a heavy chain CDR. In many instances, binding specificity
can be determined by light chain CDRs and heavy chain CDRs.
Compared with other reference antigens, a combination of a given
heavy chain CDR and light chain CDR can provide a given binding
pocket with greater affinity and/or specificity to an antigen.
[0145] In certain aspects of any embodiment disclosed herein, the
extracellular antigen binding region, such as scFv, can comprise a
light chain CDR specific for an antigen. The light chain CDR can be
a complementarity determining region of an antigen binding unit,
such as scFv light chain of a CAR. The light chain CDRs may
comprise contiguous sequence of amino acid residues, or two or more
contiguous sequences of amino acid residues separated by
non-complementarity determining regions (e.g., framework regions).
In some embodiments, a light chain CDR can comprise two or more
light chain CDRs, which can be named as light chain CDR-1, CDR-2,
and the like. In some embodiments, the light chain CDRs can
comprise three light chain CDRs, which can be named as light chain
CDR-1, light chain CDR-2 and light chain CDR-3, respectively. In
some examples, a set of CDRs present on a common light chain can be
collectively named as light chain CDR.
[0146] In certain aspects of any embodiment disclosed herein, the
extracellular antigen binding region, such as scFv, can comprise a
heavy chain CDR specific for an antigen. The heavy chain CDR can be
a complementarity determining region of an antigen binding unit,
such as scFv heavy chain. The heavy chain CDRs may comprise
contiguous sequence of amino acid residues, or two or more
contiguous sequences of amino acid residues separated by
non-complementarity determining regions (e.g., framework regions).
In some embodiments, a heavy chain CDR can comprise two or more
heavy chain CDRs, which can be named as heavy chain CDR-1, CDR-2,
and the like. In some embodiments, the heavy chain CDRs can
comprise three heavy chain CDRs, which can be named as heavy chain
CDR-1, heavy chain CDR-2 and heavy chain CDR-3, respectively. In
some examples, a set of CDRs present on a common heavy chain can be
collectively named as heavy chain CDR.
[0147] The extracellular antigen binding region can be modified in
various ways by genetic engineering. In some embodiments, the
extracellular antigen binding region can be mutated such that the
extracellular antigen binding region can be selected to have a
higher affinity for its target. In some embodiments, the affinity
of the extracellular antigen binding region for its target can be
optimized for targets expressed at a low level on normal tissues.
This optimization can be carried out to minimize potential
toxicities. In other instances, clones of an extracellular antigen
binding region with a higher affinity for the membrane-bound form
of a target may be preferred over the counterpart in a soluble
form. Such modifications can be performed, since different levels
of soluble forms of a target can also be detected and their
targeting can cause undesirable toxicity.
[0148] In some embodiments, the extracellular antigen binding
region comprises a hinge or spacer region. The terms "hinge" and
"spacer region" can be used interchangeably. The hinge can be
considered as a part of a CAR for rendering flexibility to the
extracellular antigen binding region. In some embodiments, the
hinge can be used to detect CAR on the surface of a cell,
especially when antibodies detecting the extracellular antigen
binding region are ineffective or available. For example, it may be
necessary to optimize the length of the hinge derived from an
immunoglobulin, depending on the location of the epitope on the
target that the extracellular antigen binding region targets.
[0149] In some embodiments, the hinge may not belong to an
immunoglobulin, but to another molecule, such as the native hinge
of a CD8.alpha. molecule. CD8.alpha. hinge may contain cysteine and
proline residues known to play a role in the interaction of CD8
co-receptor and MHC molecule. The cysteine and proline residues can
affect the performance of the CAR.
[0150] The CAR hinge can be adjustable in size. This morphology of
the immunological synapse between an immune response cell and a
target cell also defines the distance that cannot be functionally
bridged by a CAR due to the distal membrane epitope on a target
molecule at the cell surface, that is, the synaptic distance cannot
reach an approximation that a signal can be conducted even using a
CAR with short hinge. Similarly, for the membrane proximal target
antigen epitope of a CAR, signal outputs can only be observed in
the context of a CAR with long hinge. The hinge can be adjusted
depending on the used extracellular antigen binding region. The
hinge can be of any length.
[0151] The transmembrane domain can anchor a CAR to the plasma
membrane of a cell. The natural transmembrane portion of CD28 can
be used for a CAR. In other instances, the natural transmembrane
portion of CD8.alpha. can also be used in a CAR. "CD8" may be a
protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity
to NCBI reference number: NP 001759 or a fragment thereof having
stimulatory activities. A "CD8 nucleic acid molecule" may be a
polynucleotide encoding a CD8 polypeptide, and in some instances,
the transmembrane region may be a natural transmembrane portion of
CD28. "CD28" may be a protein having at least 85, 90, 95, 96, 97,
98, 99 or 100% identity to NCBI reference number: NP 006130 or a
fragment thereof having stimulatory activities. A "CD28 nucleic
acid molecule" can be a polynucleotide encoding a CD28 polypeptide.
In some embodiments, the transmembrane portion can comprise a
CD8.alpha. region.
[0152] The intracellular signaling region of a CAR may be
responsible for activating at least one effector functions of an
immune response cell into which a CAR has been placed. CAR can
induce effector functions of T cells, for example, the effector
function is cytolytic activity or helper activity, including
secretion of cytokines. Therefore, the term intracellular signaling
region refers to a protein portion that transduces effector
function signals and directs the cell to perform a specific
function. The entire intracellular signaling region can generally
be used, however, in many cases, it is not necessary to use the
entire chain of the signal domain. In some embodiments, a truncated
portion of an intracellular signaling region is used. In some
embodiments, the term intracellular signaling region is therefore
intended to include any truncated portion of an intracellular
signaling region sufficient to transduce effector function
signals.
[0153] Preferred examples of signal domains for use in CAR may
include cytoplasmic sequences of T cell receptors (TCRs) and
co-receptors that act synergistically to initiate signal
transduction after target-receptor binding, as well as any
derivatives thereof or variant sequences and any synthetic
sequences of these sequences that have the same functionality.
[0154] In some embodiments, the intracellular signaling region can
contain a known signal motif for an immunoreceptor tyrosine
activation motif (ITAM). Examples of ITAMs containing cytoplasmic
signaling sequences include those derived from TCR.zeta.,
FcR.gamma., FcR.beta., CD3.gamma., CD3.delta., CD3.epsilon., CD5,
CD22, CD79a, CD79b and CD66d. However, in a preferred embodiment,
the intracellular signal domain is derived from a CD3.zeta.
chain.
[0155] An example of a T cell signaling domain containing one or
more ITAM motifs is CD3.zeta. domain, also known as T cell receptor
T3.zeta. chain or CD247. This domain is a part of T cell
receptor-CD3 complex and plays an important role in binding antigen
recognition of several intracellular signal transduction pathways
to the main effector activation of T cells. As used herein,
CD3.zeta. refers primarily to human CD3.zeta. and isoforms thereof,
as known from Swissprot entry P20963, including proteins having
substantially identical sequences. As a part of a chimeric antigen
receptor, it is reiterated that whole T cell receptor T3.zeta.
chain is not required, and that any derivative of the signal domain
comprising T cell receptor T3.zeta. chain is suitable, including
any functional equivalent thereof.
[0156] The intracellular signaling domain can be selected from any
domains in Table 1. In some embodiments, the domain can be modified
such that identity to the reference domain can range from about 50%
to about 100%. Any domain in Table 1 can be modified such that the
modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97,
98, 99 or up to about 100% identity.
[0157] The intracellular signaling region of CAR may further
comprise one or more costimulatory domains. The intracellular
signaling region may comprise a single costimulatory domain, such
as an .zeta. chain (first generation of CAR) or it and CD28 or
4-1BB (second generation of CAR). In other examples, the
intracellular signaling region can comprise two costimulatory
domains, such as CD28/OX40 or CD28/4-1BB (third generation).
[0158] Together with intracellular signaling domains such as CD8,
these costimulatory domains can generate downstream activation of
the kinase pathway, thereby supporting gene transcription and
functional cellular responses. The co-stimulatory domain of CAR can
activate CD28 (phosphatidylinositol-4,5-diphosphate 3-kinase) or
4-1BB/OX40 (TNF-receptor-associated factor adaptor protein)
pathways as well as MAPK and Akt activation-associated proximal
signaling protein.
[0159] In some instances, signals generated by a CAR may be
combined with an auxiliary or costimulatory signal. For
costimulatory signaling domains, chimeric antigen receptor-like
complexes can be designed to contain several possible costimulatory
signal domains. As is well known in the art, in naive T cells, only
ligation of T cell receptors is not sufficient to induce complete
activation of T cells into cytotoxic T cells. A second
co-stimulatory signal is required for complete productive T cell
activation. Several receptors have been reported to provide
co-stimulation for T cell activation including, but not limited to,
CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL,
MyD88 and 4-1BB. The signal transduction pathways used by these
costimulatory molecules can act synergistically with the primary T
cell receptor activation signal. The signals provided by these
costimulatory signaling regions can act synergistically with
primary effect activation signals derived from one or more ITAM
motifs (e.g., the CD3zeta signal transduction domain) and can
fulfill the requirements for T cell activation.
[0160] In some embodiments, the addition of a costimulatory domain
to a chimeric antigen receptor-like complex can enhance the
efficacy and durability of engineered cells. In other embodiments,
the T cell signaling domain and the costimulatory domain are fused
to each other to form a signaling region.
TABLE-US-00004 TABLE 4 Costimulatory domain Gene marker
Abbreviation Name CD27 CD27; T14; S152; Tp55; CD27 molecule
TNFRSF7; S152. LPFS2 CD28 Tp44; CD28; CD28 antigen CD28 molecule
TNFRSF9 ILA; 4-1BB; CD137; Tumor necrosis factor CDw137 receptor
superfamily member 9 TNFRSF4 OX40; ACT35; CD134; Tumor necrosis
factor IMD16; TXGP1L receptor superfamily member 4 TNFRSF8 CD30;
Ki-1; D1S166E Tumor necrosis factor receptor superfamily member 8
CD40LG IGM; IMD3; TRAP; gp39; CD40 ligand CD154; CD40L; HIGM1;
T-BAM; TNFSF5; hCD40L ICOS AILIM; CD278; CVID1 Inducible T cell
costimulator ITGB2 LAD; CD18; MF17; MFI7; Integrin .beta.2
(Complement LCAMB; LFA-1; MAC-1 component 3 receptor 3 and 4
subunits) CD2 T11; SRBC; LFA-2 CD2 molecule CD7 GP40; TP41; Tp40;
LEU-9 CD7 molecule KLRC2 NKG2C; CD159c; NKG2-C Killer cell
lectin-like receptor subfamily C, member 2 TNFRSF18 AITR; GITR;
CD357; Tumor necrosis factor GITR-D receptor superfamily member 18
TNFRSF14 TR2; ATAR; HVEA; HVEM; Tumor necrosis factor CD270; LIGHTR
receptor superfamily member 14 HAVCR1 TIM; KIM1; TIM1; CD365;
Hepatitis A virus cell HAVCR; KIM-1; TIM-1; receptor 1 TIMD1;
TIMD-1; HAVCR-1 LGALS9 HUAT; LGALS9A, Lectin, galactoside binding,
Galectin-9 soluble, 9 CD83 BL11; HB15 CD83 molecule
[0161] The chimeric antigen receptor binds to the target antigen.
When T cell activation is measured in vitro or ex vivo, the target
antigen can be obtained or isolated from various sources. The
target antigen as used herein is an antigen or an immunological
epitope on an antigen that is critical in mammals for immune
recognition and ultimately elimination or control of pathogenic
factors or disease states. The immune recognition can be a cell
and/or a body fluid. In the case of intracellular pathogens and
cancer, the immune recognition can be, for example, a T lymphocyte
reaction.
[0162] In some embodiments, the target antigen comprises an antigen
associated with a pre-cancerous or proliferative state. Target
antigens may also be associated with or caused by cancer. For
example, in some embodiments, a chimeric antigen receptor of the
invention recognizes and binds to a tumor antigen comprising TSA
and TAA as described herein above.
[0163] In some embodiments, when a chimeric antigen receptor herein
is present on the plasma membrane of a cell, binds to its target
and is activated, the cell expressing the chimeric antigen receptor
can produce cytotoxicity to a cell carrying the target. For
example, in some embodiments, the chimeric antigen receptor is
present on a cytotoxic cell, such as an NK cell or a cytotoxic T
cell, and, when activated by a target, the toxicity of the
cytotoxic cell to the target cell can be increased. In some
embodiments, the chimeric antigen receptors herein can increase the
effect of immunoreactive cells on cells expressing claudin 18A2,
such as tumor cells. In some embodiments, compared with a cell that
does not express a chimeric antigen receptor herein, the cytotoxic
effect of a cell expressing a chimeric antigen receptor described
herein on cells expressing claudin 18A2 is increased by at least
10%, at least 15. %, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 1 times, at
least 1.5 times, at least 2 times, at least 2.5 times, at least 3
times, at least 3.5 times, at least 4 times, at least 4.5 times, at
least 5 times, at least 6 times, at least 7 times, at least 8
times, at least 9 times or at least 10 times.
[0164] In some embodiments, when a chimeric antigen receptor herein
is present on the plasma membrane of a cell, binds to its target
and is activated, a chimeric antigen receptor herein does not
induce significant cytotoxicity on cells comprising claudin 18A1
peptide but not claudin 18A2 peptide. In some embodiments, the
cytotoxicity level is no greater than 10%, 5%, 4%, 3%, 2% or
1%.
[0165] A transgene encoding a receptor or a CAR binding a target
antigen can be incorporated into a cell. For example, a transgene
can be incorporated into an immune response cell, such as a T cell.
When inserted into a cell, the transgene can be a complementary DNA
(cDNA) fragment that is a copy of messenger RNA (mRNA); or the gene
itself (with or without introns) located in the original region of
its genomic DNA.
[0166] A nucleic acid encoding a transgene sequence, such as DNA,
can be randomly inserted into the chromosome of a cell. Random
integration can be produced by any method that introduces a nucleic
acid, such as DNA, into a cell. For example, the method can
include, but is not limited to, electroporation, ultrasound, use of
a gene gun, lipofection, calcium phosphate transfection, use of
dendrimers, microinjection, and use of virus vector including
adenovirus, AAV, and retroviral vectors, and/or type II
ribozyme.
[0167] The DNA encoding the transgene can also be designed to
include a reporter gene such that the presence of the transgene or
its expression product can be detected by activation of the
reporter gene. Any reporter gene can be used, such as those
described above. The cells containing the transgene can be selected
by selecting cells in the cell culture in which the reporter gene
has been activated.
[0168] Expression of CAR can be verified by expression assays. such
as qPCR or by measuring the level of RNA. The expression level can
also indicate the number of copies. For example, if the expression
level is very high, this may indicate that more than one copy of a
CAR are integrated into the genome. Alternatively, high expression
may indicate that the transgene is integrated in a high transcribed
region, such as near a highly expressed promoter. Expression can
also be verified by measuring protein levels, for example by
Western blotting.
[0169] In some embodiments, an immune response cell of the
invention may comprise one or more transgenes. The one or more
transgenes can express a CAR protein that recognizes and binds to
at least one epitope on an antigen or binds to a mutant epitope on
the antigen. CAR can be a functional CAR. In some embodiments, the
immune response cells of the invention may comprise one or more
CARs, or they may comprise a single CAR and a secondary engineered
receptor.
[0170] In some embodiments, the transgene can encode a suicide
gene. As evidenced by many effective treatments for cancer
patients, CAR immune response cells can cause tumor regression
while with toxicity. In some embodiments, when the target antigen
is shared in normal tissues and tumor cells, the CAR immune
response cells may not be able to distinguish between tumors and
normal tissues ("on-target/off-target toxicity"). In other cases, a
systemic disturbance of the immune system, called cytokine release
syndrome (CRS), can occur. The CRS may comprise a systemic
inflammatory response syndrome or a cytokine storm, which may be a
consequence of rapid expansion of the CAR immune response cells in
vivo. CRS is a condition characterized by fever and hypotension,
which can lead to multiple organ failure. In most cases, the
toxicity is associated with in vivo expansion of infused CAR immune
response cells, which can cause an overall disturbance of the
immune system, as well as release high levels of pro-inflammatory
cytokines such as TNF.alpha. and IL-6. Suicide genes can induce the
elimination of CAR immunoreactive cells. The suicide gene may be
any gene that induces apoptosis in CAR immunoreactive cells. A
suicide gene can be encoded in the viral vector together with the
antigen-binding receptor. The encoding of the suicide gene allows
for the alleviation or complete abortion of the toxicity caused by
in vivo expansion of the infused CAR immune response cells under
specific conditions.
[0171] In some embodiments, CAR immunoreactive cells for an antigen
that are present in normal tissues can be produced such that they
transiently express CAR, e.g., after electroporation of the mRNA
encoding the receptor. In addition, in the case of severe target
toxicity, CAR immunoreactive cells can be substantially eliminated
through an effort to further strengthen CAR immunoreactive cells by
including a safety switch.
[0172] In some embodiments, the CAR-encoding vector can be combined
with, for example, an inducible caspase-9 gene (activated by a
dimeric chemical inducer) or a truncated form of EGF receptor R
(activated by the monoclonal antibody Cetuximab) or RQR8 safety
switch.
[0173] One or more transgenes used herein may be from different
species. For example, one or more transgenes can comprise a human
gene, a mouse gene, a rat gene, a porcine gene, a bovine gene, a
dog gene, a cat gene, a monkey gene, a chimpanzee gene, or any
combination thereof. For example, a transgene can be from a human
having a human genetic sequence. One or more transgenes may
comprise a human gene. In some cases, one or more transgenes are
not adenoviral genes.
[0174] As described above, a transgene can be inserted into the
genome of an immunoreactive cell in a random or site-specific
manner. For example, a transgene can be inserted into a random site
in the genome of an immune cell. These transgenes can be
functional, for example, fully functional when inserted into any
part of the genome. For example, a transgene can encode its own
promoter or can be inserted into a position controlled by internal
promoter. Alternatively, the transgene can be inserted into a gene,
such as an intron of a gene or an exon, promoter or non-coding
region of a gene. A transgene can be inserted to disrupt a gene,
such as an endogenous immune checkpoint.
[0175] In some embodiments, more than one copy of a transgene can
be inserted into multiple random sites within the genome. For
example, multiple copies can be inserted into random sites in the
genome. This may result in an increase in overall expression
compared with random insertion of the transgene for one time.
Alternatively, a copy of the transgene can be inserted into a gene
and another copy of the transgene can be inserted into a different
gene. The transgene can be targeted such that it can be inserted
into a specific site in the genome of an immunoreactive cell.
[0176] In some embodiments, a polynucleic acid comprising a
sequence encoding an antigen-binding receptor can take the form of
a plasmid vector. The plasmid vector may comprise a promoter. In
some cases, the promoter can be constitutive. In some embodiments,
the promoter is inducible. The promoter may be or may be derived
from CMV, U6, MND or EF1a. In some embodiments, the promoter can be
adjacent to the CAR sequence. In some embodiments, the plasmid
vector further comprises a splice acceptor. In some embodiments,
the splice acceptor can be adjacent to a CAR sequence. The promoter
sequence can be PKG or MND promoter. MND promoter may be a
synthetic promoter comprising U3 region of MoMuLV LTR modified with
myeloproliferative sarcoma virus enhancer.
[0177] In some embodiments, a polynucleic acid encoding a receptor
of interest can be designed to be delivered to a cell by non-viral
techniques. In some cases, the polynucleic acid can be a
GMP-compatible reagent.
[0178] Expression of a polynucleic acid encoding a receptor that
binds to an antigen or a CAR can be controlled by one or more
promoters. Promoters can be ubiquitous, constitutive (unrestricted
promoters, allowing for continuous transcription of related genes),
tissue-specific promoters or inducible promoters. Expression of a
transgene inserted adjacent to or proximate to a promoter can be
modulated. For example, a transgene can be inserted near or beside
a ubiquitous promoter. Some ubiquitous promoters may be CAGGS
promoter, hCMV promoter, PGK promoter, SV40 promoter or ROSA26
promoter.
[0179] Promoters can be endogenous or exogenous. For example, one
or more transgenes can be inserted adjacent to or proximate to
endogenous or exogenous ROSA26 promoter. Furthermore, the promoter
may be specific for immunoreactive cells. For example, one or more
transgenes can be inserted adjacent to or proximate to porcine
ROSA26 promoter.
[0180] Tissue-specific promoters or cell-specific promoters can be
used to control the location of expression. For example, one or
more transgenes can be inserted into proximity to a tissue-specific
promoter. The tissue-specific promoter may be FABP promoter, Lck
promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin
promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC
promoter, WAP promoter or Col2A promoter.
[0181] Inducible promoters can also be used. These inducible
promoters can be turned on and off by adding or removing an
inducer, if necessary. The inducible promoter is contemplated to
be, but not limited to, Lac, tac, trc, trp, araBAD, phoA, recA,
proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx and/or
Trex.
[0182] The term "inducible promoter" as used herein is a controlled
promoter which does not express or underexpresses a gene operably
linked thereto before the desired conditions are reached, and
expresses or expresses at high level a gene operably linked thereto
when the desired conditions are achieved under. For example, in
some embodiments, an inducible promoter of the present application
does not express or underexpress a gene operably linked thereto
under normal or hyperoxic conditions in a cell, and in response to
a reduced oxygen content in the cell, a gene operably linked
thereto is expressed or overexpressed under hypoxic conditions. In
some embodiments, an inducible promoter used herein includes
Hypoxia-Inducible Transcription factor-1.alpha. (HIF-1.alpha.). In
some embodiments, the term "inducible promoter" as used herein
refers to an "immune cell-inducible promoter" that does not express
or underexpresses a gene operably linked thereto before an immune
response cell contacts an antigen or when the immune response cell
is not activated, while only when the immune response cell contacts
the antigen or the immune response cell is activated, the promoter
drives the gene operably linked to express at a high level or
express under conditions such as hypoxia. In some embodiments, the
"immune cell-inducible promoter" comprises a NFAT (activated T cell
nuclear factor) type promoter.
[0183] As used herein, "NFAT-type promoter" refers to a class of
promoters that regulate the expression of a gene operably linked
thereto based on NFAT binding activity.
[0184] NFAT is a general term for a family of transcription factors
that play an important role in immune responses. One or more
members of the NFAT family are expressed in most cells of the
immune system. NFAT is also involved in the development of the
heart, skeletal muscle and nervous system.
[0185] The NFAT transcription factor family consists of five
members, NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. NFAT1 to NFAT4 are
regulated by calcium signals. Calcium signaling is critical for
NFAT activation since calmodulin (CaM) activates serine/threonine
phosphatase calcineurin (CN). Activated CN rapidly dephosphorylates
the serine-rich region (SRR) and SP repeats at the amino terminus
of NFAT protein, resulting in a conformational change that exposes
nuclear localization signals leading to NFAT entry into the
nucleus.
[0186] Based on the role of NFAT in the transcriptional expression
of cytokines during T cell activation, it can be used to modulate
the immune cell-inducible promoters described herein, thereby
expressing or expressing at high levels a gene operably linked
thereto when the immune response cells contact the antigen and are
activated.
[0187] A nucleic acid of the invention may comprise any suitable
nucleotide sequence encoding NFAT type promoter (or a functional
part or a functional variant thereof). As used herein, "NFAT-type
promoter" refers to one or more NFAT response elements linked to
the minimal promoter of any gene expressed by a T cell. Preferably,
the minimal promoter of the gene expressed by T cells is the
smallest human IL-2 promoter. The NFAT response element can
include, for example, NFAT1, NFAT2, NFAT3, and/or NFAT4 response
elements. In some embodiments, "NFAT-type promoter" as described
herein includes more than one NFAT binding motif. For example, the
"NFAT-type promoter" can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
NFAT binding motifs. In some embodiments, the "NFAT-type promoter"
includes up to 12 NFAT binding motifs. In some embodiments, the
"NFAT-type promoter" can be a promoter consisting of a plurality of
NFAT-binding motifs in series with a promoter, such as IL2 minimal
promoter. In some embodiments, the NFAT-type promoter described
herein comprises six NFAT binding motifs, designated (NFAT).sub.6.
For convenience purposes, the (NFAT).sub.6 is also referred to as
NFAT6. In some embodiments, the NFAT6 also represents 6 repeated
NFAT binding motifs (SEQ ID NO:94) in the NFAT-type promoter.
[0188] Furthermore, the transgenic sequences may also include
transcriptional or translational regulatory sequences, such as
promoters, enhancers, insulators, internal ribosome entry sites,
sequences encoding 2A peptides and/or polyadenylation signals,
although not essential for expression.
[0189] In some embodiments, the transgene encodes a receptor or CAR
that binds to the antigen, wherein the transgene is inserted into a
safe harbor such that the antigen-binding receptor is expressed. In
some embodiments, the transgene is inserted into PD1 and/or CTLA-4
locus. In other cases, the transgene is delivered as a lentivirus
to the cells for random insertion, while a PD1- or CTLA-4 specific
nuclease can be provided as mRNA. In some embodiments, the
transgene is delivered by a viral vector system such as retrovirus,
AAV or adenovirus and mRNA encoding a safe harbor specific nuclease
(e.g., AAVS1, CCR5, albumin or HPRT). Cells can also be treated
with mRNA encoding PD1 and/or CTLA-4 specific nucleases. In some
embodiments, the polynucleotide encoding the CAR is provided by a
viral delivery system together with an mRNA encoding an
HPRT-specific nuclease and a PD1- or CTLA-4 specific nuclease. CARs
that can be used with the methods and compositions disclosed herein
can include all types of these chimeric proteins.
[0190] In some embodiments, a transgene can be introduced into an
immunoreactive cell using a retroviral vector (.gamma.-retroviral
or lentiviral vector). For example, a transgene encoding a CAR or
any receptor that binds an antigen, or a variant or fragment
thereof, can be cloned into a retroviral vector and can be driven
from an endogenous promoter, a retroviral long terminal repeat or
target cell type-specific promoter. Non-viral vectors can also be
used. Non-viral vector delivery systems can include a DNA plasmid,
a naked nucleic acid, and a nucleic acid complexed with a delivery
vehicle such as a liposome or poloxamer.
[0191] Many virus-based systems have been developed for
transferring genes into mammalian cells. For example, retroviruses
provide a convenient platform for gene delivery systems. The
selected gene can be inserted into a vector and packaged in
retroviral particles using techniques known in the art. Vectors
derived from retroviruses such as lentiviruses are suitable tools
for achieving long-term gene transfer, since they allow long-term
stable integration of the transgene and its propagation in daughter
cells. Lentiviral vectors have additional advantages over vectors
derived from retroviruses such as murine leukemia virus, since they
can transduce non-proliferating cells. They also have additional
advantages of low immunogenicity. An advantage of adenoviral
vectors is that they do not fuse into the genome of a target cell,
thereby bypassing negative integration-related events.
[0192] Cells can be transfected with a transgene encoding the
antigen-binding receptor. The concentration of a transgene can
range from about 100 picograms to about 50 micrograms. In some
embodiments, the amount of nucleic acid (e.g., ssDNA, dsDNA or RNA)
introduced into a cell can be altered to optimize transfection
efficiency and/or cell viability. For example, 1 microgram of dsDNA
can be added to each cell sample for electroporation. In some
embodiments, the amount of nucleic acid (e.g., double-stranded DNA)
required for optimal transfection efficiency and/or cell viability
varies depending on the cell type. In some embodiments, the amount
of nucleic acid (e.g., dsDNA) used for each sample can directly
correspond to transfection efficiency and/or cell viability, for
example, a range of transfection concentrations. The transgene
encoded by the vector can be integrated into the genome of a cell.
In some embodiments, the transgene encoded by the vector is forward
integrated. In other cases, the transgene encoded by the vector is
reverse integrated.
[0193] The vector is delivered into an individual patient typically
by systemic administration (e.g., intravenous, intraperitoneal,
intramuscular, subcutaneous, or intracranial infusion) or topical
application, as described below. Alternatively, the vector can be
delivered ex vivo to a cell, such as a cell removed from an
individual patient (e.g., lymphocytes, T cells, bone marrow
aspirate, tissue biopsy), and then the cells into which the vector
is incorporated is typically selected and implanted in a patient.
Cells can be expanded before or after selection.
[0194] Suitable immunoreactive cells for expression of a receptor
that binds to an antigen may be cells that are autologous or
non-autologous to the individual in need thereof.
[0195] A suitable source of immune response cells can be obtained
from the individual. In some cases, T cells can be obtained. T
cells can be obtained from a number of sources, including PBMC,
bone marrow, lymph node tissue, cord blood, thymus tissue, and
tissues from infected sites, ascites, pleural effusion, spleen
tissue and tumor tissue. In some cases, T cells can be obtained
from blood collected from the individual using any number of
techniques known to a skilled person, such as Ficoll separation. In
some embodiments, cells from circulating blood of an individual are
obtained by apheresis. Apheresis products typically contain
lymphocytes, including T cells, monocytes, granulocytes, B cells,
other nucleated white blood cells, red blood cells, and platelets.
In some embodiments, cells collected by apheresis can be washed to
remove plasma fractions and placed in a suitable buffer or medium
for subsequent processing steps.
[0196] Alternatively, cells can be derived from a healthy donor,
from a patient diagnosed with cancer or a patient diagnosed with an
infection. In some embodiments, the cells can be a part of a mixed
cell population with different phenotypic characteristics. Cell
lines can also be obtained from transformed T cells according to
the methods previously described. Cells can also be obtained from a
cell therapy library. Modified cells that are resistant to
immunosuppressive therapy can be obtained by any of the methods
described herein. It is also possible to select an appropriate cell
population prior to modification. The engineered cell population
can also be selected after modification. Engineered cells can be
used for autologous transplantation. Alternatively, the cells can
be used for allogeneic transplantation. In some embodiments, the
cells are administered to a sample for identifying the same patient
of a cancer-associated target sequence. In other instances, the
cells are administered to a patient other than a patient whose
sample is used to identify a cancer-associated target sequence.
[0197] In some embodiments, suitable primary cells include
peripheral blood mononuclear cells (PBMC), peripheral blood
lymphocytes (PBL), and other blood cell subpopulations such as, but
not limited to, T cells, natural killer cells, monocytes, Natural
killer T cells, monocyte precursor cells, hematopoietic stem cells
or non-pluripotent stem cells. In some embodiments, the cell can be
any immune cell, including any T cell such as a tumor infiltrating
cell (TIL), such as a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, or
any other type of T cell. T cells can also include memory T cells,
memory stem T cells, or effector T cells. It is also possible to
select T cells from a large population, for example to select T
cells from whole blood. T cells can also be expanded from a large
population. T cells may also be inclined to specific populations
and phenotypes. For example, T cell can be inclined to a phenotype
comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+),
CD28(+) and/or IL-7R.alpha.(+). Suitable cells may have one or more
of the following markers: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+),
CD27(+), CD28(+) and/or IL-7R.alpha.(+). Suitable cells also
include stem cells such as, for example, embryonic stem cells,
induced pluripotent stem cells, hematopoietic stem cells, neuronal
stem cells, and mesenchymal stem cells. Suitable cells can comprise
any number of primary cells, such as human cells, non-human cells,
and/or mouse cells. Suitable cells can be progenitor cells.
Suitable cells can be derived from a subject (e.g., a patient) to
be treated.
[0198] The therapeutically effective amount of cells required in a
patient can vary depending on the viability of the cells and the
efficiency with which the cells are genetically modified (e.g., the
efficiency with which the transgene is integrated into one or more
cells, or the expression level of the protein encoded by the
transgene). In some embodiments, the product (e.g., doubling) of
the cell viability after genetic modification and the efficiency of
transgene integration may correspond to a therapeutic amount of
cells which can be used for administration to a subject. In some
embodiments, an increase in cell viability after genetic
modification may correspond to a reduction in the essential amount
of cells effective for being administered to a patient. In some
embodiments, an increase in the efficiency of integration of a
transgene into one or more cells can correspond to a reduction in
the number of cells necessary administered in a patient for
effective treatment. In some embodiments, determination of the
amount of cells necessary for effective treatment can include
determination of functions associated with changes in cells over
time. In some embodiments, determination of the amount of cells
necessary for effective treatment can include determination of
functions corresponding to changes in efficiency of integrating a
transgene into one or more cells according to a time-dependent
variable (e.g., cell culture time, electroporation time, Cell
stimulation time). In some embodiments, therapeutically effective
cells can be a population of cells comprising about 30% to about
100% of the expression of an antigen-binding receptor on the
surface of the cell. In some embodiments, the therapeutically
effective cells, as measured by flow cytometry, can express about
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more
than about 99.9% of the antigen-binding receptor on the cell
surface.
[0199] According to one aspect of the invention, the invention also
encompasses a nucleic acid encoding the antigen-binding receptor.
The invention also relates to variants of the above polynucleotides
which encode a polypeptide or a fragment, analog and derivative of
the polypeptide having the same amino acid sequence as the
invention.
[0200] The present invention also provides a vector comprising the
above nucleic acid encoding the antigen-binding receptor protein
expressed on the surface of an immune response cell.
[0201] The invention also includes viruses comprising the vectors
described above. The virus of the present invention includes an
infectious virus after packaging, and also includes a virus to be
packaged to contain components necessary for being packaged into an
infectious virus. Other viruses known in the art that can be used
to transduce exogenous genes into immune response cells and their
corresponding plasmid vectors can also be used in the present
invention.
[0202] In another aspect, a host cell is provided herein,
comprising an antigen binding unit or chimeric antigen receptor as
described herein, and optionally type I interferon. In another
aspect, a host cell is provided herein, comprising a nucleic acid
encoding an antigen binding unit or chimeric antigen receptor
described herein, and optionally type I interferon.
[0203] In some embodiments, the host cell is an immune response
cell. In some embodiments, the immune response cell is a T cell, a
natural killer cell, a cytotoxic T lymphocyte, a natural killer T
cell, a DNT cell and/or a regulatory T cell. In some embodiments,
the host cell is an NK92 cell.
[0204] In some embodiments, an expression construct can be included
in an immune response cell of the invention, and elements are
sequentially linked in the following manner: antibody, CD28
costimulatory signal domain, CD3.zeta., as well as NFAT6 and type I
interferon expression unit inversely linked with the aforementioned
elements. Preferably, the antibody and CD28 costimulatory signal
domain are joined by a CD8.alpha. transmembrane region and a
CD8.alpha. hinge region.
[0205] In some embodiments, NFAT (nuclear factor of activated T
cells) plays an important role in the transcriptional expression of
cytokines during T cell activation. Based on this consideration,
the inventors placed the IFN-beta encoding sequence under the
regulation of NFAT6 promoter, so that IFN-beta can be expressed at
a high level only when CAR-T cells contact the antigen to induce T
cell activation.
[0206] NFAT6 promoter is a promoter obtained by combining six NFAT
binding positions and a minimal promoter of IL2 (Hooijberg E,
Bakker A Q, Ruizendaal J J, Spits H. NFAT-controlled expression of
GFP permits visualization and Isolation of antigen-stimulated
primary human Tcells. Blood. 2000 Jul. 15; 96(2): 459-66), which
can be used to regulate the expression of cytokines such as IL12 in
T lymphocytes such as TCR-T (Zhang L, Kerkar S P, Yu Z, Zheng Z,
Yang S, Restifo N P, Rosenberg S A, Morgan R A. Improving adoptive
T cell therapy by targeting and controlling IL-12 expression to the
tumor environment. Mol Ther. 2011 April; 19(4): 751-9).
[0207] The immune response cell of the present invention is
transduced with a construct capable of expressing an
antigen-binding receptor and an exogenous type I interferon, or an
expression vector or a virus comprising the plasmid. Conventional
nucleic acid transduction methods in the art, including non-viral
and viral transduction methods, can be used in the present
invention.
[0208] The immune response cell of the present invention may
further carry an encoding sequence of an exogenous cytokine,
including, but not limited to, IL-12, IL-15 or IL-21 and the like.
These cytokines have further immunomodulatory or anti-tumor
activity, enhance the function of effector T cells and activated NK
cells, or directly exert anti-tumor effects. Thus, a skilled person
will appreciate that using these cytokines will help the immune
response cells to function better.
[0209] The immune response cell of the present invention may also
express another antigen-binding receptor other than the
antigen-binding receptor described above.
[0210] The immune response cells of the invention may also express
a chemokine receptor; and the chemokine receptors include, but are
not limited to, CCR2. A skilled person will appreciate that CCR2
chemokine receptor can compete with CCR2 binding in vivo, which is
advantageous for blocking tumor metastasis.
[0211] The immune response cells of the present invention can also
express siRNA which can reduce PD-1 expression or a protein which
can block PD-L1. A skilled person will appreciate that
competitively blocking the interaction of PD-L1 with its receptor
PD-1 facilitates the recovery of anti-tumor T cell responses,
thereby inhibiting tumor growth.
[0212] The immune response cells of the present invention may also
express a safety switch; and preferably, the safety switch
includes: iCaspase-9, Truncated EGFR or RQR8.
[0213] In some embodiments, the immune response cells of the
invention do not express a costimulatory ligand, such as
4-1BBL.
[0214] Accordingly, in another aspect, a method for producing an
antigen binding unit or chimeric antigen receptor described herein,
or a composition comprising the same is provided herein, comprising
culturing a host cell described herein under suitable conditions.
In some embodiments, the method includes isolating and obtaining an
expression product of the host cell.
[0215] In another aspect, a composition is provided herein
comprising an antigen binding unit, chimeric antigen receptor or
nucleic acid described herein. In some embodiments, the composition
is a pharmaceutical composition comprising the antigen binding
unit, chimeric antigen receptor or nucleic acid. In some
embodiments, the pharmaceutical composition further comprises a
pharmaceutically acceptable carrier.
[0216] In another aspect, a pharmaceutical composition is provided
herein, comprising a host cell described herein and a
pharmaceutically acceptable carrier.
[0217] The term "pharmaceutically acceptable" means that when a
molecular and composition are suitably administered to an animal or
a human, they do not produce adverse, allergic or other untoward
reactions.
[0218] In some embodiments, the composition comprises an additional
therapeutic agent. In some embodiments, the additional therapeutic
agent is a chemotherapeutic agent, such as those described in
US20140271820 and/or a pharmaceutically acceptable salt or analog
thereof. In some embodiments, the therapeutic agent includes, but
is not limited to, a mitotic inhibitor (vinca alkaloid), including
vincristine, vinblastine, vindesine, and Novibin.TM. (vinorelbine,
5'-dehydrohydrogen sulfide); topoisomerase I inhibitors, such as
camptothecin compounds, including Camptosar.TM. (irinotecan HCL),
Hycamtin.TM. (topotecan HCL), and other compounds derived from
camptothecin and the like; a podophyllotoxin derivative such as
etoposide, teniposide and midozozo; an alkylating agent cisplatin,
cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide,
carmustine, busulfan, chlorambucil, briquetazine, uracil mustard,
cloprofen and dacarbazine; antimetabolites, including cytarabine,
5-fluorouracil, methotrexate, guanidine, azathioprine and
procarbazine; antibiotics, including but not limited to
doxorubicin, bleomycin, dactinomycin, daunorubicin, mycinmycin,
mitomycin, sarcoma C and daunorubicin; as well as other
chemotherapeutic drugs, including but not limited to anti-tumor
antibodies, dacarbazine, cytidine, amushakang, melphalan,
ifosfamide and mitoxantrone. In some embodiments, the additional
therapeutic agent is selected from one or more of epirubicin,
oxaliplatin and 5-fluorouracil. In some embodiments, the additional
therapeutic agent includes, but is not limited to, an
anti-angiogenic agent, including anti-VEGF antibodies (including
humanized and chimeric antibodies, anti-VEGF aptamers and antisense
oligonucleotides), and other angiogenesis inhibitor such as
angiostatin, endostatin, interferon, interleukin 1 (including
.alpha. and .beta.), interleukin 12, retinoic acid and tissue
inhibitors of metalloproteinases-1 and -2, and the like.
[0219] Specific examples of some substances which can be used as
pharmaceutically acceptable carriers or components thereof are
sugars such as lactose, glucose and sucrose; starches such as corn
starch and potato starch; cellulose and derivatives thereof such as
carboxymethyl cellulose sodium, ethyl cellulose and methyl
cellulose; tragacanth gum powder; malt; gelatin; talc; solid
lubricants, such as stearic acid and magnesium stearate; calcium
sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame
oil, olive oil, corn oil and cocoa butter; polyols such as
propylene glycol, glycerin, sorbitol, mannitol and polyethylene
glycol; alginic acid; emulsifiers such as Tween.RTM.; wetting
agents such as sodium lauryl sulfate Colorant; flavoring agent;
compressed tablets, stabilizers; antioxidants; preservatives;
pyrogen-free water; isotonic saline solution; and phosphate
buffer.
[0220] The pharmaceutical composition described herein may comprise
one or more pharmaceutically acceptable salts. "Pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activities of the parent compound and does not produce
any adverse toxicological effects (see, for example, Berge, S. M.,
et al., 1977, J. Pharm. Sci. 66: 1-19). Examples of such salts
include acid addition salts and base addition salts.
[0221] Acid addition salts include salts derived from non-toxic
inorganic acids, such as hydrochloric acid, nitric acid, phosphoric
acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous
acid, and the like, and derived from non-toxic organic acids such
as aliphatic monocarboxylic acids and dicarboxylic acid, a
phenyl-substituted alkanoic acid, a hydroxyalkanoic acid, an
aromatic acid, an aliphatic or an aromatic sulfonic acid. Base
addition salts include salts derived from alkaline earth metals
(such as sodium, potassium, magnesium, calcium, etc.), as well as
salts derived from non-toxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucosamine, glucosamine,
chloroprocaine, choline, diethanolamine, ethylenediamine, procaine
and the like.
[0222] The pharmaceutical composition described herein may also
comprise an antioxidant. Examples of antioxidants include, but are
not limited to, water-soluble antioxidants such as ascorbic acid,
cysteine hydrochloride, sodium hydrogen sulfate, sodium
metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such
as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
etc.; and metal chelating agents such as citric acid,
ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid,
phosphoric acid, etc.
[0223] The composition of the present invention can be formulated
into various dosage forms as needed, and can be administered by a
physician in accordance with factors such as patient type, age,
body weight, general disease condition and mode of administration,
and the like in a beneficial dose to a patient. The mode of
administration can be, for example, parenteral administration
(e.g., injection) or other treatment.
[0224] "Parenteral" administration of an immunogenic composition
includes, for example, subcutaneous (s.c.), intravenous (i.v.),
intramuscular (i.m.) or intrasternal injection or infusion
techniques.
[0225] Formulations comprising an immunoreactive cell population
administered to an individual comprise a plurality of
immunoreactive cells effective to treat and/or prevent a particular
indication or disease. Therefore, a therapeutically effective
population of immunoreactive cells can be administered to an
individual. Typically, a formulation comprising from about
1.times.10.sup.4 to about 1.times.10.sup.10 immunoreactive cells is
administered. In most cases, the formulation will comprise from
about 1.times.10.sup.5 to about 1.times.10.sup.9 immunoreactive
cells, from about 5.times.10.sup.5 to about 5.times.10.sup.8
immunoreactive cells, or from about 1.times.10.sup.6 to about
1.times.10.sup.7 immunoreactive cells. However, depending on the
location, source, identity, extent and severity of a cancer, the
age and physical condition of an individual to be treated, and the
like, the number of CAR immunoreactive cells administered to the
individual will vary within a wide range. The doctor will finalize
an appropriate dose to be used.
[0226] In some embodiments, a chimeric antigen receptor is used to
stimulate an immune cell mediated immune response. For example, a T
cell mediated immune response is an immune response involving T
cell activation. Activated antigen-specific cytotoxic T cells are
capable of inducing apoptosis in target cells that exhibit an
exogenous epitope on the surface, such as cancer cells that display
tumor antigens. In other embodiments, a chimeric antigen receptor
is used to provide anti-tumor immunity in a mammal. Subjects will
develop anti-tumor immunity due to T cell-mediated immune
responses.
[0227] In certain instances, a method for treating a subject having
cancer can involve administering one or more immune response cells
of the invention to a subject in need of treatment. The immune
response cell can bind to a target molecule of a tumor and induce
death of cancer cells. As also mentioned above, the invention also
provides a method for treating pathogen infections in an
individual, comprising administering to an individual a
therapeutically effective amount of immune response cells of the
invention.
[0228] The administration frequency of the immunoreactive cells of
the present invention will depend on factors including the treated
disease, the elements of the particular immunoreactive cells, and
the mode of administration. For example, it can be administered 4
times, 3 times, 2 times a day, once a day, every other day, every
three days, every four days, every five days, every six days, once
a week, once every eight days, once every nine days, once every ten
days, once a week, or twice a month. As described herein, the
immune response cells of the present application can be
administered not only in a therapeutically effective amount which
is lower than that of a similar immune response cell without
expressing exogenous type I interferon, but also can be
administered at a lower frequency to achieve at least similar, and
preferably more pronounced therapeutic effects, since the immune
response cells of the present application have improved
viability.
[0229] In some embodiments, the compositions may be isotonic, i.e.,
they may have the same osmotic pressure as blood and tears. The
desired isotonicity of the compositions of the present invention
can be achieved using sodium chloride or other pharmaceutically
acceptable agents such as glucose, boric acid, sodium tartrate,
propylene glycol or other inorganic or organic solutes. If desired,
the viscosity of the composition can be maintained at a selected
level using a pharmaceutically acceptable thickening agent.
Suitable thickeners include, for example, methylcellulose, xanthan
gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and
the like. The preferred concentration of thickener will depend on
the reagent selected. It will be apparent that the choice of
suitable carrier and other additives will depend on the exact route
of administration and properties of the particular dosage form,
such as a liquid dosage form.
[0230] The invention also provides kits comprising an antigen
binding unit, chimeric antigen receptor, nucleic acid or immune
response cell as described herein. In some embodiments, a kit can
include a therapeutic or prophylactic composition comprising an
effective amount of an antigen binding unit, chimeric antigen
receptor, nucleic acid, or immune response cell described herein in
one or more unit dosage forms. In some embodiments, the kit
comprises a sterile container that can contain a therapeutic or
prophylactic composition; and such a container can be a cartridge,
ampule, bottle, vial, tube, bag, blister pack, or other suitable
container form known in the art. Such containers may be made of
plastic, glass, laminated paper, metal foil or other materials
suitable for holding the drug. In some embodiments, the kit
comprises an antigen binding unit, chimeric antigen receptor,
nucleic acid or immune response cell as described herein, and an
instruction for administering the antigen binding unit, chimeric
antigen receptor, nucleic acid or immune response cell described
herein to an individual. The instruction generally include methods
for treating or preventing cancer or tumors using the antigen
binding units, chimeric antigen receptors, nucleic acids or immune
response cells described herein. In some embodiments, the kit
comprises host cells as described herein and can comprise from
about 1.times.10.sup.4 cells to about 1.times.10.sup.6 cells. In
some embodiments, the kit can comprise at least about
1.times.10.sup.5 cells, at least about 1.times.10.sup.6 cells, at
least about 1.times.10.sup.7 cells, at least about 4.times.10.sup.7
cells, at least about 5.times.10.sup.7 cells, at least about
6.times.10.sup.7 cells, at least about 6.times.10.sup.7 cells,
8.times.10.sup.7 cells, at least about 9.times.10.sup.7 cells, at
least about 1.times.10.sup.8 cells, at least about 2.times.10.sup.8
cells, at least about 3.times.10.sup.8 cells, at least about
4.times.10.sup.8 cells, at least about 5.times.10.sup.8 cells, at
least about 6.times.10.sup.8 cells, at least about 6.times.10.sup.8
cells, at least about 8.times.10.sup.8 cells, at least about
9.times.10.sup.8 cells, at least about 1.times.10.sup.9 cells, at
least about 2.times.10.sup.9 cells, at least about 3.times.10.sup.9
cells, at least about 4.times.10.sup.9 cells, at least about
5.times.10.sup.9 cells, at least about 6.times.10.sup.9 cells, at
least about 8.times.10.sup.9 cells, at least about 9.times.10.sup.9
cells, at least about 1.times.10.sup.10 cells, at least about
2.times.10.sup.10 cells, at least about 3.times.10.sup.10 cells, at
least about 4.times.10.sup.10 cells, at least about
5.times.10.sup.10 cells, at least about 6.times.10.sup.10 cells, at
least at least about 9.times.10.sup.10 cells, at least about
9.times.10.sup.10 cells, at least about 1.times.10.sup.11 cells, at
least about 2.times.10.sup.11 cells, at least about
3.times.10.sup.11 cells, at least about 4.times.10.sup.11 cells, at
least about 5.times.10.sup.11 cells, at least about
8.times.10.sup.11 cells, at least about 9.times.10.sup.11 cells or
at least about 1.times.10.sup.12 cells. For example, approximately
5.times.10.sup.10 cells can be included in the kit. In another
example, the kit can include 3.times.10.sup.6 cells; and the cells
can be expanded to about 5.times.10.sup.10 cells and administered
to a subject.
[0231] In some embodiments, the kit can include allogeneic cells.
In some embodiments, the kit can include cells that can contain
genomic modifications. In some embodiments, the kit can comprise
"ready-made" cells. In some embodiments, the kit can include cells
that can be expanded for clinical use. In some cases, the kit may
contain content for research purposes.
[0232] In some embodiments, the instruction includes at least one
of: a description of a therapeutic agent; a dosage regimen and
administration for treating or preventing a tumor or a symptom
thereof; preventive measures, warnings, contraindications,
excessive information, adverse reactions, animal pharmacology,
clinical research, and/or citations. The instruction can be printed
directly on the container (if any), or as a label on the container,
or as a separate paper, booklet, card or folder in the container.
In some embodiments, the instruction provides a method for
administering an immune response cell of the invention for treating
or preventing a tumor. In some cases, the instruction provides a
method for administering an immunoreactive cell of the invention
before, after or simultaneously with the administration of a
chemotherapeutic agent.
[0233] In another aspect, a method for inducing death of a cell
comprising claudin 18A2 peptide is provided herein, the method
comprising: contacting the cell with an antigen binding unit
described herein, a chimeric antigen receptor described herein, a
compositions described herein, or a host cell described herein. In
some embodiments, the contacting is in vitro contacting. In some
embodiments, the contacting is in vivo contacting.
[0234] In some embodiments, the cell is a tumor cell. In some
embodiments, the cell is a cell of solid tumor. In some
embodiments, the cell is a cell of a cancer or a tumor as described
herein. Particular examples of such cells may include, but are not
limited to, leukemia (e.g., acute leukemia, acute lymphocytic
leukemia, acute myeloid leukemia, acute myeloid leukemia, acute
promyelocytic leukemia, acute granulocyte-monocytic leukemia, acute
monocytic leukemia, acute leukemia, chronic leukemia, chronic
myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera)
cells, lymphoma (Hodgkin's disease, non-Hodgkin's disease) cells,
primary macroglobulinemia disease cells, heavy chain disease cells,
solid tumors such as sarcoma and cancer cells (such as
fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma,
osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma,
angiosarcoma, lymphatic endothelial sarcoma, synovial vioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon cancer, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland cancer, sebaceous gland cancer,
papillary carcinoma, papillary adenocarcinoma, cancer, bronchial
carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer,
bile Tube cancer, choriocarcinoma, seminoma, embryonic carcinoma,
nephroblastoma, cervical cancer, uterine cancer, testicular cancer,
lung cancer, small cell lung cancer, bladder cancer, epithelial
cancer, glioma, astrocytoma, medulla Blastoma, craniopharyngioma,
ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma,
oligodendroglioma, schwannomas, meningioma, melanoma,
neuroblastoma, retinoblastoma), esophageal cancer cells,
gallbladder cancer cells, renal cancer cells, multiple myeloma
cells, and the like. In some embodiments, the cell is a gastric
cancer cell, an esophageal cancer cell, an intestinal cancer cell,
a pancreatic cancer cell, a nephroblastoma cell, a lung cancer
cell, an ovarian cancer cell, a colon cancer cell, a rectal cancer
cell, a liver cancer cell, a head and neck cancer cells, a chronic
myeloid leukemia cell and a gallbladder cancer cell.
[0235] In another aspect, a method for treating a tumor in an
individual in need thereof is provided herein, the method
comprising administering to the individual an effective amount of
an antigen binding unit, chimeric antigen receptor, composition,
vector or host cell described herein.
[0236] In some embodiments, the tumor includes, but is not limited
to, a tumor of bladder, bone, brain, breast, cartilage, glial
cells, esophagus, fallopian tubes, gallbladder, heart, intestine,
kidney, liver, lung, lymph nodes, nervous tissue, ovary, pancreas,
prostate, skeletal muscle, skin, spinal cord, spleen, stomach,
testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus,
vaginal organs. In some embodiments, the tumor includes, but is not
limited to, leukemia (e.g., acute leukemia, acute lymphocytic
leukemia, acute myeloid leukemia, acute myeloid leukemia, acute
promyelocytic leukemia, acute granulocyte-monocytic leukemia, acute
monocytic leukemia, acute leukemia, chronic leukemia, chronic
myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera),
lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary
macroglobulinemia disease, heavy chain disease, solid tumors such
as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma,
liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial
sarcoma, lymphangiosarcoma, angiosarcoma, lymphatic endothelial
sarcoma, synovial vioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer,
breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
cancer, sebaceous gland cancer, papillary cancer, papillary
adenocarcinoma, cancer, bronchial carcinoma, medullary carcinoma,
renal cell carcinoma, liver cancer, bile tube cancer,
choriocarcinoma, seminoma, embryonic carcinoma, nephroblastoma,
cervical cancer, uterine cancer, testicular cancer, lung cancer,
small cell lung cancer, bladder cancer, epithelial cancer, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal
tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma,
schwannomas, meningiomas, melanoma, neuroblastoma, retinoblastoma),
esophageal cancer, gallbladder cancer, kidney cancer, multiple
myeloma. In some embodiments, the tumor is gastric cancer,
esophageal cancer, intestinal cancer, pancreatic cancer,
nephroblastoma, lung cancer, ovarian cancer, colon cancer, rectal
cancer, liver cancer, head and neck cancer, chronic myelogenous
leukemia, or gallbladder cancer.
[0237] In some embodiments, immunoreactive cells can be
administered to a subject, wherein the immunoreactive cells that
can be administered can be from about 1 to about 35 days of age.
For example, the administered cells may be 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34 or up to about 40 days of age.
The age of CAR immunoreactive cells can be calculated from the time
of stimulation. The age of the immunoreactive cells can be
calculated from the time of blood collection. The age of
immunoreactive cells can be calculated from the time of
transduction. In some embodiments, the immunoreactive cells that
can be administered to a subject are from about 10 to about 14 or
about 20 days of age. In some embodiments, the "age" of an
immunoreactive cell can be determined by the telomere length. For
example, a "young" immune response cell can have a longer telomere
length than that of "depleted" or "old" immunoreactive cells.
Without being bound by a particular theory, it is believed that
immunoreactive cells lose an estimated telomere length of about 0.8
kb per week in culture, and a young immunoreactive cell culture can
have a longer telomere than an immunoreactive cell of about 44 days
about 1.4 kb. Without being bound by a particular theory, it is
believed that a longer telomere length can be associated with a
positive objective clinical response in a patient and persistence
of cells in vivo.
[0238] Cells (e.g., engineered cells or engineered primary T cells)
can be functional before, after and/or during transplantation. For
example, the transplanted cells may function at least about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or
100 days after transplantation. The transplanted cells can function
at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after
transplantation. The transplanted cells can function at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 years after
transplantation. In some embodiments, the transplanted cells can
function during the life of the recipient.
[0239] In addition, the transplanted cells can function at 100% of
normally expected function. The transplanted cells can also exert
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or up to about
100% of their normally expected function.
[0240] Transplanted cells can also exert more than 100% of their
normally expected function. For example, the transplanted cells can
exert about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,
300, 400, 500, 600, 700, 800, 900, 1000 or up to about 5000% of
their normally expected function.
[0241] Transplant can be any type of transplant. Local position may
include, but is not limited to, subhepatic sac space, subsplenic
sac space, subcapsular space, omentum, gastric or intestinal
submucosa, small intestinal vascular segment, venous sac, testis,
brain, spleen, or cornea. For example, the transplant can be a
subcapsular transplant. The transplant can also be an intramuscular
transplant. The transplant can be a portal vein transplant.
[0242] The transplant rejection can be improved after treatment
with the immune response cells of the present invention as compared
with the situation when one or more wild type cells are
transplanted to a recipient. For example, transplant rejection can
be a hyperacute rejection. Transplant rejection can also be an
acute rejection. Other types of rejection may include chronic
rejection. Transplant rejection can also be cell-mediated rejection
or T cell-mediated rejection. Transplant rejection can also be a
natural killer cell mediated rejection.
[0243] Improvement in transplantation may refer to alleviation of
hyperacute rejection, which may include reduction, alleviation or
lowering of adverse effects or symptoms. Transplantation can refer
to adoptive transplantation of cellular products.
[0244] Another sign of successful transplantation may be the number
of days for which the recipient does not need immunosuppressive
therapy. For example, after providing the immune response cells of
the invention, the recipient may not require at least about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more days of immunosuppressive therapy.
This can indicate successful transplantation. This can also
indicate that the transplanted cells, tissues and/or organs are not
rejected.
[0245] In some cases, the recipient does not require
immunosuppressive therapy for at least 1 day. The recipient may not
require immunosuppressive therapy for at least 7 days. The
recipient does not require immunosuppressive therapy for at least
14 days. The recipient does not require immunosuppressive therapy
for at least 21 days. The recipient does not require
immunosuppressive therapy for at least 28 days. The recipient does
not require immunosuppressive therapy for at least 60 days. In
addition, the recipient may not require immunosuppressive therapy
for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
[0246] Another sign of successful transplants may be the reduced
number of days for which a recipient needs an immunosuppressive
therapy. For example, after the treatment provided herein, the
recipient may require a reduced immunosuppressive therapy for at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate
successful transplantation. This may also indicate that there are
no or only minimal rejection of the transplanted cells, tissues
and/or organs.
[0247] For example, a recipient may require a reduced
immunosuppressive therapy for at least 1 day. The recipient may
also require a reduced immunosuppressive therapy for at least 7
days. The recipient may require a reduced immunosuppressive therapy
for at least 14 days. The recipient requires a reduced
immunosuppressive therapy for at least 21 days. The recipient
requires a reduced immunosuppressive therapy for at least 28 days.
The recipient requires a reduced immunosuppressive therapy for at
least 60 days. In addition, the recipient may require a reduced
immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more years.
[0248] A reduced immunosuppressive therapy can refer to less
requirement on immunosuppressive therapy as compared with the
situation when one or more wild type cells are transplanted to a
recipient.
[0249] Immunosuppressive therapy can include any treatment that
inhibits the immune system. Immunosuppressive therapy can
facilitate alleviation, reduction or elimination of transplant
rejection in patients. For example, immunosuppressants can be used
before, during, and/or after transplantation, including MMF
(Mycophenolate mofetil, Cellcept), ATG (anti-thymocyte globulin),
anti-CD154 (CD4OL), anti-CD40 (2C10), immunosuppressive drugs,
anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies
(sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), anti-IL-6
antibodies (ASKP1240, CCFZ533X2201)), amphetamine (Campath),
anti-CD20 (rituximab), bevacizumab (LEA29Y), sirolimus (Rapimune),
everolimus, tacrolimus (Prograf), Zemegab, Zemilect, Remicade,
cyclosporin, deoxygenin, soluble complement receptor 1, cobra
venom, anti-C5 antibody Eculizumab/Soliris), methylprednisolone,
FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin,
anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody and
anti-CD122 antibody. In addition, one or more immunosuppressive
agents/drugs may be used together or sequentially. One or more
immunosuppressive agents/drugs can be used to induce therapy or to
maintain therapy. Same or different drugs can be used in the
induction and maintenance phases. In some cases, daclizumab
(Zenapax) can be used for induction therapy, and Tacrolimus
(Prograf) and Sirolimus (Rapimune) can be used to maintain
treatment. Non-pharmacological regimens can also be used to achieve
immunosuppression, including but not limited to whole body
irradiation, thymic irradiation, and total and/or partial
splenectomy. These techniques can also be used in combination with
one or more immunosuppressive drugs.
[0250] In some embodiments, an antigen binding unit, chimeric
antigen receptor, composition, vector or host cell described herein
can be administered in combination with another therapeutic agent.
In some embodiments, the additional therapeutic agent is a
chemotherapeutic agent, such as those described in US20140271820.
Chemotherapeutic agents that can be used in combination with the
immune response cells of the invention include, but are not limited
to, mitotic inhibitors (vinca alkaloids), including vincristine,
vinblastine, vindesine, and Novibin.TM. (vinorelbine),
5'-dehydrohydrogen sulfide); topoisomerase I inhibitors, such as
camptothecin compounds, including Camptosar.TM. (Irinotecan HCL),
Hycamtin.TM. (topotecan HCL), and other compounds derived from
camptothecin and analogs thereof; podophyllotoxin derivatives such
as etoposide, teniposide and midozozo; alkylating agents cisplatin,
cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide,
nitrogen mustard, busulfan, chlorambucil, briquetazine, uracil
mustard, cloprofen and dacarbazine; antimetabolites, including
cytarabine, 5-fluorouracil, methotrexate, anthraquinone,
azathioprine and procarbazine; antibiotics including, but not
limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin,
mycinmycin, mitomycin, sarcoma C and daunorubicin; and other
chemotherapeutic drugs, including but not limited to anti-tumor
antibodies, dacarbazine, cytidine, amushakang, melphalan,
ifosfamide and mitoxantrone. In some embodiments, the additional
therapeutic agent is selected from one or more of epirubicin,
oxaliplatin and 5-fluorouracil.
[0251] In some embodiments, chemotherapeutic agents that can be
used in combination with the immune response cells of the invention
include, but are not limited to, an anti-angiogenic agent,
including anti-VEGF antibodies (including humanized and chimeric
antibodies, anti-VEGF aptamers and antisense oligonucleotides), and
other angiogenesis inhibitor such as angiostatin, endostatin,
interferon, interleukin 1 (including .alpha. and .beta.),
interleukin 12, retinoic acid and tissue inhibitors of
metalloproteinases-1 and -2, and the like.
EXAMPLES
[0252] The invention is further illustrated below in conjunction
with specific embodiments. It is to be understood that the examples
are intended to demonstrate the invention while not intended to
limit the scope of the invention. The experimental methods in the
following examples, specific conditions of which are not specified
are usually prepared according to conventional conditions such as
conditions described in J. Sambrook et al., Molecular Cloning
Experimental Guide, Third Edition, Science Press, 2002, or
according to the conditions suggested by the manufacturer.
[0253] In the following examples of the invention, when the
antigen-binding receptor or CAR is constructed, CD28 costimulatory
signal domain is abbreviated as 28; CD3.zeta. is abbreviated as Z;
4-1BB or CD137 is abbreviated as BB. For example, a chimeric
antigen receptor constructed by a scFv with a code of 8E5-2I and
CD3.zeta. as well as CD28 co-stimulatory signal domains as an
intracellular signal domain can be referred to as 8E5-2I-28Z. CARs
for different antigens are constructed as such.
Example 1. Production and Characterization of Mouse Antibody
Against CLD18A2
[0254] Antibody fragments were obtained using standard biological
protocols. Briefly, 8-week old Balb/c mice were immunized with a
eukaryotic expression vector containing human CLD18A2 full-length
sequence (NCBI Reference Sequence: NM_001002026.2). The spleen of
the immunized mouse was removed, and a monoclonal antibody was
obtained using a conventional biological scheme in the art.
[0255] Individual cells were screened for anti-CLD18A2 monoclonal
antibody by flow cytometry, and HEK293 cells (HEK-CLD18A2) stably
expressing human CLD18A2 were used for primary screening by flow
cytometry using a Guava easyCyte.TM. HT System instrument. The
binding of the antibody to human CLD18A1 and CLD18A2 transformants
was then compared by flow cytometry. The instrument used was the
Guava easyCyte.TM. HT System.
[0256] After multiple rounds of preparation and screening of
hybridomas, the inventors found several antibodies with relatively
ideal binding properties. FIG. 1B shows an example of the binding
of hybridoma supernatants 2B1, 3E12, 4A11, 8E5 to HEK293 cells
stably transfected with human CLD18A2 or CLD18A1 as determined by
flow cytometry. As shown in FIG. 1B, after two rounds of
subcloning, most of subclones of antibodies 2B1, 3E12, 4A11 and 8E5
specifically bound to human CLD18A2 but not human CLD18A1, and the
average fluorescence intensities differed by more than 5 times.
[0257] The monoclonal antibody-secreting hybridoma cell strain was
cultured, and total RNA was extracted from the cell pellet
according to instructions of TRIzol.RTM. Plus RNA Purification kit
(Invitrogen, 12183-555). The cDNA was reverse-transcribed using
total RNA as template according to the instructions of High
capacity RNA to cDNA kit (Invitrogen, 4387406). The cDNA was used
as a template, and 5'-Full RACE kit (TAKARA, D315) and primers of
the constant region of the antibody were used for amplification.
PCR products were separated on 1.5% agarose gel, and the DNA
fragments were purified and recovered. The sequencing results were
as follows:
TABLE-US-00005 TABLE 5 Sequencing results Amino acid sequence
Nucleotide sequence 2B1 VL SEQ ID NO: 1 SEQ ID NO: 2 2B1 VH SEQ ID
NO: 3 SEQ ID NO: 4 3E12 VL SEQ ID NO: 5 SEQ ID NO: 6 3E12 VH SEQ ID
NO: 7 SEQ ID NO: 8 4A11 VH SEQ ID NO: 9 SEQ ID NO: 10 4A11VL SEQ ID
NO: 11 SEQ ID NO: 12 8E5 VL SEQ ID NO: 13 SEQ ID NO: 14 8E5 VH SEQ
ID NO: 15 SEQ ID NO: 16
[0258] The antibody sequences were aligned and the results are
shown in FIG. 2.
Example 2. Construction of Anti-Claudin 18A2 scFv_Fc Fusion
Antibody and its Transient Expression in Eukaryotic Cells
[0259] For VH and VL fragments of 2B1, 8E5, a flexible amino acid
GGGGSGGGGSGGGGS (SEQ ID NO:93) was introduced as a linker to
constitute scFv; an appropriate restriction site and protective
bases were introduced upstream to VH, and an appropriate
restriction site and protective bases were introduced downstream to
VL; and digested and ligated into an eukaryotic expression vector
(see vector pH or vector pK used in CN101602808). 293Fectin.TM.
Transfection reagent (Invitrogen, 12347-019) was used in transient
transfection, the supernatant was collected and subjected to
affinity purification, and the obtained antibody was quantitatively
and qualitatively analyzed by SDS PAGE.
[0260] Binding of anti-Claudin 18A2 scFv_Fc fusion antibody to
HEK293 cells stably transfected with CLD18A2 was determined by flow
cytometry. Experimental data was analyzed using GraphPad Prism and
Guava easyCyte.TM. HT System instrument as described in Example 1
to obtain EC50 value. FIG. 3 shows the relative binding affinity of
scFvs of monoclonal antibody 2B1, 8E5, after fused to human IgG1 Fc
portion, to HEK293 cells stably transfected with human CLD18A2. It
can be seen that the EC50 value of 2B1 is 3.56 nM, and the EC50
value of 8E5 is 49.19 nM.
Example 3. Preparation of Variants of Anti-Claudin 18A2
Antibodies
[0261] Antibody 2B1 was subject to site-directed mutagenesis by
bridge PCR. Mutations were introduced at position 52 or 54
(N-glycosylation site) on the heavy chain of antibody 2B1 for
preparing two 2B1 mutants 2B1-N52D (VH amino acid sequence: SEQ ID
NO:17; nucleotide sequence: SEQ ID NO:18) and 2B1-S54A (VH amino
acid sequence: SEQ ID NO:19; nucleotide sequence: SEQ ID
NO:20).
[0262] The amino acid sequences and nucleotide sequences of the
light chains of 2B1-N52D and 2B1-S54A are identical to the
corresponding sequences of 2B1.
[0263] Expression vectors of ScFv Fc-form of the two mutants were
constructed as described in Example 2, and according to the
procedure of Example 2, and the experimental data was analyzed
using GraphPad Prism and Guava easyCyte.TM. HT System instrument to
obtain EC50.
[0264] FIG. 4 shows the relative binding affinity of 2B1-N52D and
2B1-S54A, after fused to human IgG1 Fc portion, to HEK293 cells
stably transfected with human CLD18A2. It can be seen that the EC50
value of 2B1-N52D is 6.11 nM, and the EC50 value of 2B1-S54A is
3.85 nM.
Example 4. Preparation of Humanized Antibody of 2B1-S54A
[0265] Sequences of 6 CDRs of the antibody light and heavy chain
were determined according to Kabat, Chothia and IMGT Naming
schemes. By using sequence similarity alignment, the antibody
sequence with the highest similarity to 2B1-S54A was selected as
the antibody template. In this example, IGHV1-46*01 in IMGT
database was selected as an antibody template for hu2B1-S54A heavy
chain. IGKV4-1*01 was used as an antibody template for hu2B1-S54A
light chain. The light and heavy chain CDR regions of 2B1-S54A were
replaced with CDR regions of the antibody template.
[0266] Determination of reverse mutation sites: (1) Aligning a
designed humanized antibody with the starting antibody, and
checking which amino acids in the antibody framework region are
different. (2) Checking whether these different amino acids are
amino acids that support the loop structure of the antibody or
amino acids that affect the binding of variable regions of the
light and heavy chains, and if yes, these regions are relatively
conserved regions. (3) Checking whether there are some potential
post-translational modification sites in humanized antibodies, such
as deamidation sites (Asn-Gly), isomerization sites (Asp-Gly),
surface-exposed methionine, N glycosylation site (Asn-X-Ser/Thr, X
is not proline). (4) There are six potential reverse mutation sites
in the heavy chain of humanized antibody (hu2B1-S54A), namely M481,
V68A, M70L, R72A, T74K, T91S, respectively. There is a potential
reverse mutation site in the light chain of humanized antibody
(hu2B1-S54A), L84V.
[0267] Expression and purification of humanized antibodies: (1) A
nucleotide sequence was designed and synthesized based on the amino
acid sequence of humanized antibody (hu2B1-S54A). A light chain
nucleotide sequence (SEQ ID NO:62) was synthesized; and a heavy
chain nucleotide sequence (SEQ ID NO:60) was synthesized. (2) The
synthetic antibody nucleotide sequence including the signal
peptide, variable region of the antibody and constant region is
inserted into a mammalian cell expression vector to construct
antibody expression vectors containing the heavy chain and the
light chain, respectively, sequenced and identified.
[0268] The heavy chain amino acid sequence of hu2B1-S54A is set
forth in SEQ ID NO:59; and the nucleotide sequence is set forth in
SEQ ID NO:60. The amino acid sequence of hu2B1-S54A light chain is
set forth in SEQ ID NO:61; and the nucleotide sequence is set forth
in SEQ ID NO:62. The amino acid sequence of hu2B1-S54A heavy chain
variable region is set forth in SEQ ID NO:23, the nucleotide
sequence is set forth in SEQ ID NO:24; and the amino acid sequence
of light chain variable region is set forth in SEQ ID NO:21, and
the nucleoside sequence is shown in SEQ ID NO:22.
[0269] 293F cells were transiently transfected by 293Fectin and the
HCDR of hu2B1-S54A and the same sequence of LCDR were
expressed.
[0270] The binding activity assay was performed as described in
Example 2, and experimental data was analyzed using HEK293 cells
(HEK-CLD18A2) stably expressing human CLD18A2, GraphPad Prism and
Guava easyCyte.TM. HT System instrument to obtain EC50, and the
results are shown in FIG. 5. The relative binding affinity EC50
value of the scFv of hu2B1-S54A, after fused to human IgG1 Fc
portion, to HEK293 cell stably transfected with human CLD18A2 was
18.59 nM, indicating that hu2B1-S54A also exhibited a good binding
to HEK-CLD18A2.
Example 5. Preparation and Optimization of Humanized Antibody of
Monoclonal Antibody 8E5
[0271] Following the procedure of Example 4, 8E5 was humanized
while the N-glycosylation site in monoclonal antibody 8E5 was
removed by point-mutation of S62A to obtain humanized antibody
hu8E5 (or hu8E5-S62A). The specific method is described as
follows:
[0272] (1) IGHV4-30*03 was selected as the antibody template of 8E5
heavy chain, and IGKV4-1*01 was selected as the antibody template
of 8E5 light chain. The light chain or heavy chain CDR regions of
8E5 antibody are replaced by the CDR regions of the antibody
template.
[0273] (2) Reverse mutation sites are determined, there are six
potential reverse mutation sites in the heavy chain of humanized
antibody (hu8E5), namely G27Y, G45K, L46M, I49M, V68I, V72R, A97T,
respectively. There is a potential reverse mutation site in the
light chain of humanized antibody (hu8E5), L84V.
[0274] (3) Nucleotide sequences were designed, light chain
nucleotide sequence was synthesized and a heavy chain nucleotide
sequence was synthesized, based on the amino acid sequence of
humanized antibody hu8E5.
[0275] The heavy chain amino acid sequence of hu8E5-S62A is set
forth in SEQ ID NO:67; and the nucleotide sequence is set forth in
SEQ ID NO:68. The amino acid sequence of hu8E5-S62A light chain is
set forth in SEQ ID NO:65; and the nucleotide sequence is set forth
in SEQ ID NO:66. The amino acid sequence of heavy chain variable
region of hu8E5-S62A is set forth in SEQ ID NO:27, the nucleotide
sequence is set forth in SEQ ID NO:28; the amino acid sequence of
light chain variable region of hu8E5-S62A is set forth in SEQ ID
NO:25, the nucleotide sequence is set forth in SEQ ID NO:26; HCDR2
in hu8E5-S62A is different from that in 8E5, the sequence of which
is set forth in SEQ ID NO:85; and the other HCDRs and LCDR are the
same as 8E5.
[0276] (4) The synthetic antibody nucleotide sequence including the
signal peptide, variable region of the antibody and constant region
are inserted into a mammalian cell expression vector to construct
an antibody expression vector containing a heavy chain and a light
chain, respectively, sequenced and identified. 293F cells were
transiently transfected by 293Fectin and expressed.
[0277] (5) The binding activity was tested, and experimental data
was analyzed using HEK293 cells (HEK-CLD18A2) stably expressing
human CLD18A2, GraphPad Prism and Guava easyCyte.TM. HT System
instrument, and the results are shown in FIG. 6. It is shown that
the relative binding affinity of the scFv of hu8E5, after fused to
human IgG1 Fc portion, to HEK293 cell stably transfected with human
CLD18A2 was 107 nM.
[0278] The 3D model of hu8E5 was established by Discovery studio
software, and the potential aggregation sites were analyzed. It was
found that the 12.sup.th and 93.sup.rd valine of the heavy chain
tend to cause aggregation of antibodies, which, in turn affects the
stability of the antibody. By analysis on point mutation, it was
found that when the two sites were mutated to I, the antibody was
more stable. The results of the molecular sieve showed that after
these two sites were mutated to I (hu8E5-2I), the proportion of the
monomeric form in scFv_Fc fusion antibody was increased from the
initial 74% (hu8E5) to 87% (hu8E5-2I).
[0279] The amino acid sequence of hu8E5-2I heavy chain of is set
forth in SEQ ID NO:63; and the nucleotide sequence is set forth in
SEQ ID NO:64. The amino acid sequence of hu8E5-2I light chain is
set forth in SEQ ID NO:65; and the nucleotide sequence is set forth
in SEQ ID NO:66. HCDR2 in hu8E5-2I is different from that in 8E5,
but is identical to that in hu8E5, and its sequence is shown in SEQ
ID NO:85; other HCDRs and LCDR are the same as 8E5.
[0280] The mutant hu8E5-2I of the humanized antibody hu8E5 was
constructed as described in Example 3. Experimental data was
analyzed using HEK293 cells (HEK-CLD18A2) stably expressing human
CLD18A2, GraphPad Prism and Guava easyCyte.TM. HT System
instrument, and the results are shown in FIG. 7. The relative
binding affinity, EC50 value of the scFv of hu8E5-2I, after fused
to human IgG1 Fc portion, to HEK293 cell stably transfected with
human CLD18A2 was 9.22 nM. Compared with the parent antibody 8E5,
the affinity of mutant hu8E5-2I had a 5-fold increase.
Example 6. In Vitro Functional Assay and In Vivo Functional Assay
of Humanized Antibody hu2B1-S54A and Humanized Antibody
hu8E5-2I
[0281] The humanized antibody hu2B1-S54A (light chain sequence: SEQ
ID NO:62, heavy chain sequence: SEQ ID NO:60); humanized antibody
hu8E5-2I (Light chain sequence: SEQ ID NO:66, heavy chain sequence:
SEQ ID NO:64) were cloned into a eukaryotic expression vector by
standard methods known to a skilled person. 293F cells in
logarithmic growth phase were transiently transfected with
293Fectin.TM. Transfection reagent (Invitrogen, 12347-019), and the
culture supernatant was collected and subjected to affinity
purification. The obtained antibodies were quantitatively and
qualitatively analyzed by SDS PAGE. The above eukaryotic expression
vector uses the vector pH or vector pK used in CN101602808B.
[0282] 1. Complement Dependent Cytotoxicity (CDC)
[0283] Blood was collected from healthy volunteers and serum was
prepared by centrifugation. A CCK-8 cell proliferation-toxicity
assay kit (Dojindo, #CK04) was used. HEK293 cells stably
transfected with CLD18A2 or CLD18A1 were used as target cells. The
cells were washed twice, resuspended in complete medium at a
density of 1.times.10.sup.5 cells/ml, seeded in a 96-well culture
plate at 100 .mu.l per well and cultured overnight at 37.degree. C.
The next day, antibody was added to each well at a final
concentration of 20 .mu.g/ml, and incubate for 30 min at 37.degree.
C. in an incubator. Then, serum at a final concentration of 10% was
added and incubated at 37.degree. C. for 1.5 hours. 10 ul of CCK-8
solution was added to each well, incubated at 37.degree. C. for 3.5
h (adjusted as appropriate), and the absorbance at 450 nm was
measured with a microplate reader. The experiment was divided into
six groups and duplicate wells were set up, as shown in the
following table.
TABLE-US-00006 TABLE 6 Grouping antibody complement cell cell
Experimental experiment Lysis control control control Blank Blank
Materials well well well well well well well CLD18A2 100 ul 100 ul
100 ul 100 ul 100 ul 0 0 cell 1*10.sup.6/ml DMEM -50 ul 0 -50 ul
-25 ul 0 50 ul 100 ul Ch-163E12 25 ul Lysate 25 ul 0 0 25 ul 0
antibody 10 ul 60 ug/ml Complement 25 ul 25 ul 25 ul 0 25 ul 0
(serum) 40% Inactivated complement CCK-8 10 ul 10 ul 10 ul 10 ul 10
ul 10 ul 10 ul solution
[0284] lysis percentage is calculated as follows:
Lysis percentage=(cell control well-experiment well)/cell control
well-(cell control well-antibody control well)/cell control
well.
[0285] FIG. 8A compares CDC effects of the humanized antibodies
hu2B1-S54A, hu8E5-2I and the chimeric antibody ch-163E12 on HEK293
cells transfected with CLD18A2. The experiment results showed that
when the concentration of hu2B1-S54A and hu8E5-2I were 20 .mu.g/ml,
CDC effects against HEK293-CLD18A2 were 79.88% and 82.65%,
respectively, and CDC effects of ch-163E12 under the same reaction
conditions was lower than 55%. FIG. 8B compares CDC results of the
humanized antibodies hu2B1-S54A, hu8E5-2I and chimeric antibody
ch-163E12 on HEK293 cells transfected with CLD18A1. The results
showed that ch-163E12 also had a certain killing on cells
expressing 18A1, and hu2B1-S54A and hu8E5-2I did not kill cells
expressing 18A1.
[0286] 2. Antibody-Dependent Cytotoxicity (ADCC) Activity
[0287] The ADCC activity of the humanized antibody, claudin 18A2
antibody was measured by a lactate dehydrogenase (LDH) release
assay using a cytoTox 96 non-radioactive cytotoxicity assay kit
(Promega, Madison, USA). Human peripheral blood mononuclear cells
(PBMC) were purified from citrated whole blood by standard
Ficoll-paque separation and resuspended in complete medium
(RPMI-1640 culture, Gibco) supplemented with 10% fetal bovine serum
(FBS, Gibco) at a density of 8.times.10.sup.6 cells/ml. HEK293
cells stably transfected with CLD18A2 were used as target cells.
The cells were washed twice and resuspended in a complete culture
at a density of 2.times.10.sup.5 cells/ml. PBMCs were incubated
with an antibody at a final concentration of 20 ug/ml for 30
minutes at 37.degree. C., and then 50 .mu.l of antibody and
effector cells were added into 50 .mu.l of target cells at a
effector-to-target ratio of 50:1, 20:1, 10:1 (total
1.times.10.sup.4 target cells). After incubated for 4 hours at
37.degree. C., the cells were centrifuged, and 50 .mu.l of
cell-free supernatant sample was collected, transferred to a
flat-bottomed 96-well plate, and assayed. The percentage of lysis
was calculated as follows: (sample release-target spontaneous
release-spontaneous release of effector cells)/(maximum
release-target spontaneous release)*100; wherein the target
spontaneous release is fluorescence in wells containing only target
cells, spontaneous release of effector cells is fluorescence in
wells containing only effector cells, and maximum release is
fluorescence in wells containing target cells that have been
treated with lysis buffer.
[0288] FIG. 9 compares ADCC effects of the humanized antibodies
hu2B1-S54A, hu8E5-2I and the known chimeric antibodies ch-163E12,
ch-175D10 (see CN103509110A). The experimental results showed that
the humanized antibodies hu2B1-S54A and hu8E5-2I, at an antibody
concentration of 20 .mu.g/ml and a effector-to-target ratio of
50:1, 20:1 and 10:1, exhibited significantly higher ADCC effects
than ch-175D10 and ch-163E12, and the ADCC effects against
HEK293-CLD18A2 at a effector-to-target ratio of 50:1 were 62.84%
and 72.88%, respectively. While the ADCC effects of ch-163E12 and
ch-175D10 against HEK293-CLD18A2 under the same reaction conditions
were only 33.39% and 43.74%. The antibody of the present invention
exhibits significantly better killing effects than ch-163E12 and
ch-175D10.
[0289] 3. In Vivo Experiment in Mice
[0290] Establishment of a PDX model of gastric cancer: Tumors of
about 3 mm.times.3 mm.times.3 mm in size were inoculated
subcutaneously into the right ankle of BALB/c nude mice. The day of
tumor cell inoculation was recorded as DO days, the tumor volume
was measured at D27 from the inoculation of tumor, and the mice
were randomly divided into 5 groups. The specific groups are as
follows: (1) PBS (phosphate buffer) control group; (2) hu8E5-2I
antibody treatment group (40 mg/kg); (3) EOF treatment group (E is
epirubicin: 1.25 mg/Kg; O is oxaliplatin: 3.25 mg/kg; F is
5-fluorouracil: 56.25 mg/kg)+PBS; (4) hu8E5-2I antibody (40
mg/kg)+EOF treatment group (1.25 mg/kg Epirubicin+3.25 mg/kg
oxaliplatin+56.25 mg/kg 5-fluorouracil); (5) ch175D10 antibody (40
mg/kg)+EOF treatment group (1.25 mg/kg epirubicin+3.25 mg/kg
oxaliplatin+56.25 mg/kg 5-fluorouracil). Dosage: EOF was
administered once a week for 2 weeks; hu8E5-2I and ch175D10
antibodies were administered 3 times per week for 2 weeks.
[0291] The results are shown in FIG. 10. The tumor was inoculated
for D56 days, the EOF was injected twice, the antibody was injected
6 times, and the mice were sacrificed by cervical dislocation.
Compared with the PBS control group, the tumor inhibition rates
were: 21.13% in the hu8E5-2I monoclonal antibody group, 47.89% in
the EOF+PBS treatment group, 81.69% in the EOF+mAb hu8E5-2I
treatment group, and 71.83% in the EOF+mAb 175D10 treatment group,
respectively. From the tumor weighing, the EOF+mAb hu8E5-2I
treatment group was statistically different over the EOF+PBS group,
P=0.033; while the EOF+mAb 175D10 treatment group was not
statistically different over the EOF+PBS group, P=0.097.
Example 7. Construction of Humanized Antibody Chimeric Antigen
Receptor Plasmid (CAR Plasmid)
[0292] 1. Construction of Humanized Antibody hu8E5 Chimeric Antigen
Receptor Plasmid
[0293] Using PRRLSIN-cPPT.EF-1.alpha. as a vector, lentiviral
plasmids encoding the second and third generation of chimeric
antigen receptors of humanized antibody hu8E5 were constructed,
including PRRLSIN-cPPT.EF-1.alpha.-hu8E5-28Z,
PRRLSIN-cPPT.EF-1.alpha.-hu8E5-BBZ and
PRRLSIN-cPPT.EF-1.alpha.-hu8E5-28BBZ.
[0294] Hu8E5-28Z mainly includes (from 5' to 3' end): encoding
sequence of CD8.alpha. signal peptide (SEQ ID NO:70), hu8E5 scFV
(VH: SEQ ID NO:27, VL: SEQ ID NO:25, Linker: SEQ ID NO:93), CD8
hinge (SEQ ID NO:72), CD28 transmembrane region (SEQ ID NO:74) and
intracellular signaling domain (SEQ ID NO:76) as well as
intracellular segment CD3 .zeta. of CD3 (SEQ ID NO:78).
[0295] hu8E5-BBZ mainly includes (from 5' to 3' end): encoding
sequence of CD8.alpha. signal peptide (SEQ ID NO:70), hu8E5 scFV
(VH: SEQ ID NO:27, VL: SEQ ID NO:25, Linker: SEQ ID NO:93), CD8
hinge (SEQ ID NO:72), CD8 transmembrane region (SEQ ID NO:80),
CD137 intracellular signaling domain (SEQ ID NO:82) and CD3.zeta.
(SEQ ID NO:78).
[0296] hu8E5-28BBZ mainly includes (from 5' to 3' end): encoding
sequence of CD8.alpha. signal peptide (SEQ ID NO:70), hu8E5-scFV,
CD8 hinge (SEQ ID NO:72), CD28 transmembrane region (SEQ ID NO:74)
and intracellular segment (SEQ ID NO:76), CD137 intracellular
signaling domain (SEQ ID NO:82) and CD3.zeta. (SEQ ID NO:78).
[0297] 2. Construction of Humanized Antibody hu8E5-2I Chimeric
Antigen Receptor Plasmid
[0298] Using PRRLSIN-cPPT.EF-1.alpha. as a vector, lentiviral
plasmid PRRLSIN-cPPT.EF-1.alpha.-hu8E5-2I-28Z encoding the second
generation of chimeric antigen receptors of humanized antibody
hu8E5 was constructed.
[0299] hu8E5-2I-28Z mainly includes (from 5' to 3' end): encoding
sequence of CD8.alpha. signal peptide (SEQ ID NO:70), hu8E5-2I scFV
(VH: SEQ ID NO:29, VL: SEQ ID NO:25, Linker: SEQ ID NO:93), CD8
hinge (SEQ ID NO:72), CD28 transmembrane region (SEQ ID NO:74) and
intracellular signaling domain (SEQ ID NO:76) and intracellular
signaling domain CD3.zeta. of CD3 (SEQ ID NO:78).
[0300] 3. Construction of Humanized Antibody hu2B1-S54A Chimeric
Antigen Receptor Plasmid
[0301] Using PRRLSIN-cPPT.EF-1.alpha. as a vector, lentiviral
plasmid PRRLSIN-cPPT.EF-1.alpha.-hu2B1-S54A-28Z encoding the second
generation of chimeric antigen receptors of humanized antibody
hu2B1-S54A was constructed.
[0302] hu2B1-S54A-28Z mainly includes (from 5' end to 3' end):
encoding sequence of CD8.alpha. signal peptide (SEQ ID NO:70),
hu2B1-S54A scFV (VH: SEQ ID NO:23; VL: SEQ ID NO:21; Linker: SEQ ID
NO:93), CD8 hinge (SEQ ID NO:72), CD28 transmembrane region (SEQ ID
NO:74) and intracellular segment (SEQ ID NO:76), and intracellular
CD3Z of CD3 (SEQ ID NO:78).
Example 8. Lentiviral Packaging and Titer Determination
[0303] The 293T cells cultured to the 6.sup.th to 10.sup.th passage
were inoculated at a virus density of 5.times.10.sup.6 in a 10 cm
culture dish, and cultured overnight at 37.degree. C., 5% CO.sub.2
for transfection, and the medium was DMEM containing 10% fetal
bovine serum (Gibico).
[0304] The target gene plasmid PRRLSIN-cPPT.EF-1.alpha.-EGFP (Mock)
and different CAR plasmids prepared in Example 9 (5.4 .mu.g) and
packaging plasmid pRsv-REV (6.2 .mu.g), RRE-PMDLg (6.2 .mu.g) and
Vsvg (2.4 .mu.g) were dissolved in 800 .mu.L of blank DMEM medium;
60 .mu.g of PEI (1 .mu.g/p1) was dissolved in 800 .mu.l of
serum-free DMEM medium and mixed for 5 min at room temperature.
[0305] The plasmid mixture was added to PEI mixture, and mixed for
20 min at room temperature to form a transfection complex; 1.6 ml
of the transfection complex was added dropwise to a 10 cm culture
dish containing 11 ml of DMEM medium; after 4-5 hours, 10% FBS DMEM
medium was used to change the medium for the transfected 293T
cells, and incubated at 37.degree. C. for 72 h. The virus
supernatant was collected and concentrated, and the titer was
determined. The number of cells with a positive rate of 5.about.20%
was preferred, and the titer (U/mL) was calculated as =cell
number.times.positive rate/virus volume.
[0306] Upon concentration, the virus titers were:
[0307] hu8E5-28Z: 2.3.times.10.sup.7 U/ml;
[0308] hu8E5-BBZ: 6.65.times.10.sup.7 U/ml;
[0309] hu8E5-28BBZ: 6.67.times.10.sup.7 U/ml;
[0310] hu8E5-2I-28Z: 1.54.times.10.sup.8 U/ml;
[0311] hu2B1-S54A-28Z: 1.14.times.10.sup.8 U/ml.
Example 9. Cytotoxicity Assay of Lentiviral-Transduced T
Lymphocytes and CAR-T Cells
[0312] 1. Lentivirus-Infected T Lymphocytes
[0313] (1) Lymphocyte culture medium was added at a density of
about 1.times.10.sup.6/mL for culture, and magnetic beads
(Invitrogen) coated with anti-CD3 and CD28 antibodies and
recombinant human IL-2 with a final concentration of 300 U/mL were
added according to a magnetic bead:cell ratio of 1:1 for
stimulation and culture for 48h;
[0314] (2) Retronectin coated 24-well plates: 380 .mu.l of 5
.mu.g/ml retronectin solution (PBS) was added to each well, and
after incubation at 4.degree. C. overnight, the retronectin
solution (PBS) in a 24-well plate was discarded, and washed twice
with 1 ml of PBS;
[0315] (3) Cells were seeded in a 24-well plate coated with
retronectin. The number of cells per well was 3.times.10.sup.5, and
the volume of the culture solution was 600 .mu.l. The concentrated
lentivirus was added to PBMCs cells at MOI=10, centrifuged at
32.degree. C. for 40 min and transferred to a cell culture
incubator;
[0316] (4) Expansion culture: The infected cells were passaged
every other day at a density of 5.times.10.sup.5/mL, and
recombinant human IL-2 was supplemented in the lymphocyte culture
solution at a final concentration of 300 U/mL.
[0317] 2. T Lymphocyte Chimeric Antigen Receptor Expression
[0318] (1) On the 7.sup.th day of culture of lentivirus-infected T
lymphocytes, 1.times.10.sup.6 T cells were taken, aliquoted into a
2 ml centrifuge tube, centrifuged at 4.degree. C., 5000 rpm for 5
min, the supernatant was discarded, and PBS was washed twice.
[0319] (2) Control cells were added 50 .mu.l of PE-SA (1:200
dilution) antibody and incubated for 45 min on ice, washed twice
with PBS (2% NBS), and resuspended as a control; cells in the test
group+50 .mu.l: 50 diluted biotin-Goat anti human IgG, F(ab')2
antibody, incubated on ice for 45 min; washed twice with PBS (2%
NBS).
[0320] (3) 50 .mu.l of PE-SA (1:200 dilution) antibody was added
and incubated for 45 min on ice; 2 ml of PBS (2% NBS) was added for
resuspending the cells, and the supernatant was discarded upon
centrifugation.
[0321] (4) Proportion of CAR-positive T cells was detected by Flow
cytometry. The infection positive rates of three CAR T cells,
hu8E5-28Z, hu8E5-BBZ and hu8E5-28BBZ and Mock control cells were
52.1%, 47.8%, 44.6% and 71.7%, respectively.
[0322] 3. Cytotoxicity Assay of CLD18A2-Targeting CAR T Cells
[0323] (1) Target cells: 75 .mu.L of 2.times.10.sup.5/mL 293T-A1
cells, 293T-A2 cells, gastric adenocarcinoma cell line AGS, AGS-A2,
gastric cancer cell lines BGC-823, and BGC-823-A2 cells were
inoculated respectively. Gastric adenocarcinoma cell line AGS,
gastric cancer cell line BGC-823 were purchased from ATCC cell
bank, 293T-A1 cells, 293T-A2 cells, AGS-A2, BGC-823-A2 cells were
constructed with reference to CN101602808B, among which 293T-A2
cells, AGS-A2, and BGC-823-A2 cells were CLD18A2-positive cells,
and the rest were CLD18A2-negative cells.
[0324] (2) Effector cells: T-Mock and CAR T cells expressing
different chimeric antigen receptors were added at an
effector-to-target ratio of 3:1, 1:1 or 1:3;
[0325] (3) 4 duplicate wells were set for each group, and the
average of 4 replicate wells was taken. The detection time was
18h.
[0326] Each experimental group and each control group are as
follows:
[0327] Each experimental group: each target cell+CAR T expressing
different chimeric antigen receptors;
[0328] Control group 1: maximum release of LDH from target
cells;
[0329] Control group 2: spontaneous release of LDH from target
cells;
[0330] Control group 3: spontaneous release of LDH from effector
cells;
[0331] (4) Detection method: CytoTox 96 non-radioactive
cytotoxicity test kit (Promega) was used. CytoTox 96.RTM. assay
quantitatively measures lactate dehydrogenase (LDH), in particular,
referring to instructions of CytoTox 96 Non-Radioactive
Cytotoxicity Assay Kit.
[0332] (5) The calculation formula for cytotoxicity is:
Cytotoxicity %=(Experimental group-Control group 2-Control group
3)/(Control group 1-Control group 2)*100%
[0333] The cell killing results at different effector-to-target
ratios are shown in FIG. 11 and FIG. 12. The results showed that
the CAR-T products showed good killing effects on 293T-A2, AGS-A2
and BGC-823-A2 cells with positive CLD18A2 expression. Among them,
hu8E5-28Z and hu8E5-2I-28Z cells can kill more than 60% of AGS-A2
cells at an effector-to-target ratio of 3:1, and the killing
effects on BGC-823-A2 cells can reach higher than 90%. At the same
time, the results also showed that the killing effects of each CAR
T cell (except Hu2B1-S54A-28Z) on cells with negative expression of
CLD18A2 was not obvious.
Example 10. In Vivo Activity of CLD18A2 CAR-T Cell
[0334] Anti-tumor treatment experiments of untransfected T cells
(UTD) and hu8E5-2I-28Z T cells on subcutaneously transplanted
tumors in gastric cancer PDX were observed.
[0335] 1) Establishment of PDX model of gastric cancer: a gastric
cancer PDX tumor of about 2.times.2.times.2 mm was inoculated in
the right axillary area of female NOD/SCID mice of 6-8 weeks old,
and the day of tumor cell inoculation was recorded as DO day.
[0336] 2) Experimental group: The tumors were inoculated for 15
days, and NOD-SCID mice were randomly divided into 3 groups, 7 in
each group, untransfected T cell group and hu8E5-2I-28Z T cell
group.
[0337] 3) Adoptive transfer of T cells: 100 mg/kg of
cyclophosphamide was intraperitoneally injected when a tumor volume
was 30 mm.sup.3, and 1.0.times.10.sup.7 CAR-T cells were infused
through the tail vein 24 hours after injection, while untransfected
T cell groups were used as control. The growth of subcutaneous
xenografts of gastric cancer PDX was observed and measured. The
experiment results are shown in FIGS. 13A and 13B. On the D32 day
after CAR T injection, the mice were sacrificed by cervical
dislocation. Compared with the UTD group, the anti-tumor effect of
the hu8E5-2I-28Z treatment group was significant, and the
inhibition rate was 79.2%. From the tumor weighing, the
hu8E5-2I-28Z treatment group was statistically different over the
UTD group, P=0.01.
Example 11. Cytokine Release Assay Induced by CLD18A2 CAR-T Cells
In Vitro
[0338] To verify whether the constructed hu8E5-28Z and hu8E5-2I-28Z
T cells can be efficiently activated under stimulation of target
cell, we examined secretion of cytokines from hu8E5-28Z and
hu8E5-2I-28Z T cells after co-incubation with target cells.
[0339] Cytokines released by transfected T cells (Mock), hu8E5-28Z
and hu8E5-2I-28Z T cells were detected respectively. The above two
T cells of good growth within 1-2 weeks after lentivirus infection
were collected, inoculated in a 24-well plate at
5.times.10.sup.4/200 .mu.L (positive cell number), and
5.times.10.sup.4/200 .mu.L/24 well of target cells were inoculated
at an effector-totarget ratio of 1:1. The target cells include
293T-A1, 293T-A2, AGS, AGS-A2, BGC-823 and BGC-823-A2 cells. The
supernatant was collected after 24 hours of co-cultivation. The
sandwich ELISA method was used to detect IL2, IFN-.gamma. and
TNF-.alpha. in the supernatant released during the co-culture of
CAR T lymphocytes with target cells.
[0340] The experiment results are shown in FIG. 14. The results
showed that when hu8E5-28Z, hu8E5-2I-28Z were incubated with
CLD18A2 positive 293T-A2, AGS-A2 and BGC-823-A2 cells, the
secretion of IL-2, IFN-.gamma. and TNF-.alpha. cytokines was
activated, while in the Mock control group, the secretion of these
cytokines can not be activated and there were significant
differences; when hu8E5-2I-28Z was incubated with 293T-A1, AGS and
BGC-823 cells with negative expression of CLD18A2, the secretion of
IL-2, IFN-.gamma. and TNF-.alpha. cytokines can not be activated,
and in the Mock control group, the secretion of the above cytokines
can not be activated either. The above experimental results
indicate that cells with positive expression of CLD18A2 can
effectively activate hu8E5-2I-28Z CAR T cells.
Example 12. In Vivo Killing Activity of CLD18A2 CAR-T Cells
[0341] Anti-tumor treatment experiments of untransfected T cells
(Mock), hu8E5-28Z and hu8E5-2I-28Z T cells on subcutaneous
xenografts of BGC-823-A2 cells were determined.
[0342] 1) Inoculation of BGC-823-A2 subcutaneous xenografts:
BGC-823-A2 cells collected in logarithmic growth phase and grown
well were adjusted to a density of 2.5.times.10.sup.7/mL using
physiological saline, and a volume of the cell suspension (200
.mu.L, 5.times.10.sup.6/animal) was injected subcutaneously in the
right side of the mouse. The day of tumor cell inoculation was
recorded as day 0.
[0343] 2) Experiment groups: On the 11th day of tumor inoculation,
the volume of BGC-823-A2 tumor was measured, and the NOD-SCID mice
were randomly divided into 3 groups, 6 mice of each group. The
groups were untransfected T cell group, hu8E5-28Z T cell, and
hu8E5-2I-28Z T cell group, respectively.
[0344] 3) Adoptive transfer of T cells: 100 mg/kg of
cyclophosphamide was intraperitoneally injected when the tumor
volume was 100-150 mm.sup.3 (Day 11), and 1.times.10.sup.7 CAR T
cells (Mock cells, hu8E5-28Z T cells or hu8E5-2I-28Z T cells) were
infused through the tail vein 24 hours after the injection, and the
untransfected T cell group (Mock group) was used as a control to
observe the growth of subcutaneous xenografts.
[0345] The results of animal experiments are shown in FIG. 15. The
results showed that on the 17.sup.th day of treatment of hu8E5-28Z
and hu8E5-2I-28Z CAR T cells, the tumor inhibition rates of
BGC-823-A2 xenografts were 81.3% and 89.2%, respectively; and there
were significant differences in the therapeutic effects on
BGC-823-A2 xenografts between the hu8E5-28Z, hu8E5-2I-28Z treatment
group and the Mock control group. After the 17.sup.th day of
treatment, the mice were sacrificed and the tumors were removed and
weighed. The average tumor weight of the transplanted tumors of
BGC-823-A2 in the hu8E5-28Z and hu8E5-2I-28Z treatment groups were
0.1 and 0.06 g, respectively, while the average weight of tumor in
the Mock control group was 0.53 g, and there were significant
differences between the CAR T cell treatment group and the Mock
control group, and the P values were 0.0013 and <0.0001,
respectively.
Example 13. Effect of CLD18A2 CAR-T Cells on Tumor Infiltration In
Vivo
[0346] According to the animal model of BGC-823-A2 cell
subcutaneous xenograft established in Example 12, 17 days after
Mock, hu8E5-28Z, and hu8E5-2I-28Z cells were returned, tumor
tissues were taken and CD3+ cells were detected by
histochemistry.
[0347] Results are shown in FIG. 16. Almost no T cell infiltration
was observed around the tumor tissue in Mock T cell group, almost
no T cell infiltration was observed around the tumor tissue in Mock
T cell group; in hu8E5-28Z and hu8E5-2I-28Z cell groups,
infiltration of CD3+ T cells can be observed at the edge of the
tumor tissue; and more T cell infiltration can be observed at the
edge of the tumor tissue in hu8E5-2I-28Z treatment group.
Example 14. Preparation of CAR-T Cells Co-Expressing IFN
[0348] According to the procedure of Examples 7-9, a plasmid of
hu8E5-28Z-IFNb CAR expressing IFNb cytokine was constructed based
on hu8E5-28Z, and hu8E5-2I-28Z-IFN CAR plasmid which can express
IFNb cytokine was constructed based on hu8E5-2I-28Z CAR, which were
packaged and infected with lentivirus, so that hu8E5-28Z-IFNb CAR T
cells co-expressing IFNb (also labeled as hu8E5-28Z&IFNB) and
hu8E5-2I-28Z-IFN CAR T cells co-expressing IFNb (also labeled as
hu8E5-2I-28Z&IFNB) were obtained. hu8E5-28Z-IFNb CAR is encoded
by the nucleotide sequence of SEQ ID NO:90; and hu8E5-2I-28Z-IFN
CAR is encoded by the nucleotide sequence of SEQ ID NO:91.
[0349] According to the procedure of Example 11, in vitro induction
of cytokine release assay was carried out. The results are shown in
FIG. 17A, and the presence of IFN resulted in an increase in
IFN-.gamma. cytokine secretion when hu8E5-28Z CAR T cells were
co-incubated with target cells.
[0350] Anti-tumor treatment experiments of untransfected T cells
(UTD), hu8E5-28Z T cells and hu8E5-2I-28Z-IFN T cells on
subcutaneous xenografts of gastric cancer PDX model were observed.
A gastric cancer PDX tumor of about 2.times.2.times.2 mm was
subcutaneously inoculated in the right axilla of 6-8 weeks old
female NOD/SCID mice, and the day of tumor cell inoculation was
recorded as DO day. On D15 day of tumor inoculation, NOD-SCID mice
were randomly divided into 3 groups, 7 in each group, untransfected
T cell group, hu8E5-28Z T cell group and hu8E5-2I-28Z-IFN T cell
group. When the tumor volume was 30 mm.sup.3, 100 mg/kg of
cyclophosphamide was intraperitoneally injected, and
1.0.times.10.sup.7 CAR-T cells (hu8E5-28Z T cells or
hu8E5-2I-28Z-IFN T cells) were infused through the tail vein 24
hours after the injection. At the same time, the untransfected T
cell group was used as a control. The growth of subcutaneous
xenografts of gastric cancer PDX were observed and measured. The
results were shown in FIG. 17B, and in one of the 7 mice in the
hu8E5-2I-28Z-IFN-treated group, the tumor completely regressed.
[0351] CAR-T cells was determined for in vivo survival using PDX
model described above. Peripheral blood was collected from the
saphenous vein of mice at D5, D7 and D10 days after CAR-T infusion,
and CAR-T cells (blank T cells (Mock), hu8E5-28Z T cells or
hu8E5-2I-28Z-IFN T cells) were detected for in vivo survival. The
results were shown in FIG. 17C, and, the survival number of T cell
in the hu8E5-2I-28Z-IFN T cell treated group was significantly
higher than that of the hu8E5-28Z-28Z T cell treatment group.
Example 15. Construction of CAR-NK Cell
[0352] As shown in the plasmid maps shown in FIGS. 18A and 18B,
using PRRLSIN-cPPT.EF-1.alpha. as a vector, lentiviral plasmids
encoding the chimeric antigen receptors of humanized antibody hu8E5
were constructed, including PRRLSIN-cPPT.EF-1.alpha.-hu8E5-28BBZ
and PRRLSIN-cPPT.EF-1.alpha.-hu8E5-2I-28Z. The hu8E5-28BBZ sequence
consists of a CD8.alpha. signal peptide (SEQ ID NO:70), hu8E5-scFV,
CD8 hinge (SEQ ID NO:72), a CD28 transmembrane region (SEQ ID
NO:74) and an intracellular segment (SEQ ID NO:76), CD137
intracellular signaling domain segment (SEQ ID NO:82) and CD3.zeta.
(SEQ ID NO:78); and hu8E5-2I-28Z sequence consists of CD8.alpha.
signal peptide (SEQ ID NO:70), hu8E5-2I scFV, CD8 hinge (SEQ ID
NO:72), CD28 transmembrane region (SEQ ID NO:74) and CD28
intracellular signaling domain (SEQ ID NO:76) and CD3.zeta.
intracellular segment of CD3 (SEQ ID NO:78).
[0353] 1. Preparation of CAR-Positive NK-92 Cell Line
[0354] 1) Retronectin coated 24-well plates: 380 .mu.l of 5
.mu.g/ml retronectin solution (PBS) was added to each well, and
incubated at 4.degree. C. overnight. Cells were seeded in the
24-well plate coated with retronectin. The number of cells per well
was 5.times.10.sup.5, and the volume of the culture solution was
500 .mu.l;
[0355] 2) The concentrated lentivirus was added to NK92 cells at
MOI=30, centrifuged at 32.degree. C. for 90 min and transferred to
a cell culture incubator;
[0356] 3) Expansion culture: The infected cells were passaged every
other day at a density of 5.times.10.sup.5/mL, and recombinant
human IL-2 was supplemented in the lymphocyte culture solution at a
final concentration of 500 U/mL.
[0357] 2. Expression of NK-92 Cell Chimeric Antigen Receptor
[0358] (1) On the 7.sup.th day of culture of lentivirus-infected
NK92 cells, 1.times.10.sup.6 cells were taken, aliquoted into a 2
ml centrifuge tube;
[0359] (2) Control cells were added 50 .mu.l of PE-SA (1:200
dilution) antibody and incubated on ice and resuspended as a
control; cells in the test group+50 .mu.l 1:50 diluted biotin-Goat
anti human IgG; F(ab')2 antibody, incubated on ice for 45 min;
washed twice with PBS (2% NBS); 50 .mu.l of PE-SA (1:200 dilution)
antibody was added and incubated on ice;
[0360] (3) 2 ml of PBS (2% NBS) was added for resuspending the
cells, and the supernatant was discarded upon centrifugation at
4.degree. C.; 500 .mu.l of PBS (2% NBS) was added and transferred
to a flow tube. The PE channel was detected by flow cytometry to
determine the proportion of CAR positive NK92 cells. The results
are shown in FIG. 19.
[0361] Cytotoxicity assay: Target cells: 10.sup.4 of AGS, AGS-A2,
BGC-823, BGC-823-A2 cells were inoculated into 96-well plates,
respectively; Effector cells: NK92 and CAR NK92 cells were added at
an effector-to-target ratio of 6:1, 3:1 or 1.5:1; 5 duplicate wells
were set for each group, and the average of 5 replicate wells was
taken. The detection time was 4h. Each experimental group and each
control group are as follows:
[0362] Each experimental group: each target cell+above effector
cells; Control group 1: maximum release of LDH from target cells;
Control group 2: spontaneous release of LDH from target cells; and
Control group 3: spontaneous release of LDH from effector
cells.
[0363] Detection method: CytoTox 96 non-radioactive cytotoxicity
test kit (Promega) was used, in particular, referring to
instructions of CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit.
The calculation formula for cytotoxicity is:
Cytotoxicity %=(Experimental group-Control group 2-Control group
3)/(Control group 1-Control group 2)*100%
[0364] The results are shown in FIGS. 20 and 21, in which
hu8E5-2I-28Z is hu8E5-2I-28Z CAR-NK92 cells and hu8E5-28BBZ is
hu8E5-28BBZ CAR-NK92 cells. The results showed that hu8E5 CAR-NK92
cells had significant in vitro killing activities against cells
overexpressing CLDN18A2 and almost no killing toxicities against
CLDN18A2-negative cells.
TABLE-US-00007 TABLE 7 Sequences used herein SEQ ID: Sequence 1
divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpqqppklliywastresgvpdrftgsg-
sgtdfthiss vqaedlavyycqndysypltfgagtklelkr 2
GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGA
AGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCA
AAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTG
TTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAG
GCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGA
AGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCG
GTGCTGGGACCAAGCTGGAGCTGAAACGG 3
qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyinpssgytnynqkfkdkatlta-
dkssstay mqlssltsedsavyycariyygnsfaywgqgttvtvss 4
CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAG
TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACACGATGCA
CTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAAT
CCTAGCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACAT
TGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGAC
ATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCGT
TTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 5
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLAS
GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPYTFGGGTKLEIKR 6
CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGA
AGGTCACCATGACCTGCAGTGCCAGCTCAAGTATAAGTTACATGCACTGGTA
CCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAA
CTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTA
TTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGC
CATCAGCGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAA ATAAAACGG 7
QVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWVKQRPGQGLEWIGWIYPG
DGSTKYNEKFKGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGGYRYDEAM DYWGQGTTVTVSS
8 CAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTTAG
TGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAGCTACGATATAAA
CTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTAT
CCTGGAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGCCACA
CTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGA
CTTCTGAGAACTCTGCAGTCTATTTCTGTGCAAGAGGGGGCTATAGGTACGA
CGAGGCTATGGACTACTGGGGTCAAGGGACCACGGTCACCGTCTCCTCA 9
divmtqspsslsysagekvtmsckssqsllnsgnqknylawyqqkpgqppklliygastresgvpdrftgsg-
sgtdftltiss vqaedlavyycqndhsypltfgagtklelkr 10
GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGA
AGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCA
AAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTG
TTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAG
GCAGTGGATCTGGAACCGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGA
AGACCTGGCAGTTTATTACTGTCAGAATGATCATAGTTATCCGCTCACGTTCG
GTGCTGGGACCAAGCTGGAGCTGAAACGG 11
qiqlvqsgpelkkpgetvkisckasgytftnygmnwvkqapgkglkwmgwintntgeptyaeefkgrfafs-
letsastay lqinnlknedtatyfcarfsygnsfaywgqgttvtvss 12
CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACA
GTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAAACTATGGAATGA
ACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAA
ACACCAACACTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGTTTG
CCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTC
AAAAATGAGGACACGGCTACATATTTCTGTGCTAGATTCTCTTATGGTAACTC
CTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 13
divmtqspssltvtpgekvtmtckssqslfnsgnqknyltwyqqrpgqppkmliywastresgvpdrftgs-
gsgtdftltis svqaedlavfycqnaysfpytfgggtkleikr 14
GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACACCAGGAGAGA
AGGTCACTATGACCTGCAAGTCCAGTCAGAGTTTGTTTAATAGTGGAAATCA
AAAGAACTACTTGACCTGGTACCAACAGAGACCTGGCCAGCCCCCTAAAATG
TTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAG
GCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGA
AGACCTGGCAGTTTTTTACTGTCAGAATGCTTATAGTTTTCCGTACACGTTCG
GAGGGGGGACCAAGCTGGAAATAAAACGG 15
dvqlqesgpdlvkpsqslsltctvtgysitsgynwhwirqfpgnkmewmgyihytgstnynpslrsrisit-
rdtsknqfflql nsvttddtatyyctriyngnsfpywgqgtsvtvss 16
GATGTGCAACTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCAC
TTTCACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAACTGG
CACTGGATCCGGCAGTTTCCAGGAAACAAAATGGAATGGATGGGCTACATAC
ACTACACTGGTAGCACTAATTACAACCCATCTCTCAGAAGTCGAATCTCTATC
ACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACCA
CTGATGACACAGCCACATATTACTGTACAAGAATCTACAATGGTAACTCTTTT
CCTTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA 17
qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyidpssgytnynqkfkdkatlt-
adkssstay mqlssltsedsavyycariyygnsfaywgqgttvtvss 18
CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAG
TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACACGATGCA
CTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTGA
CCCTAGCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACA
TTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGA
CATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCG
TTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 19
qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyinpasgytnynqkfkdkatlt-
adkssstay mqlssltsedsavyycariyygnsfaywgqgttvtvss 20
CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAG
TGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACACGATGCA
CTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAAT
CCTGCCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACAT
TGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGAC
ATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCGT
TTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 21
divmtqspdslavslgeratinckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgs-
gsgtdftltissl qaedvavyycqndysypltfgggtkveikr 22
GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAG
CGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAAC
CAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAG
CTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTCA
GCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGC
CGAGGACGTGGCCGTGTACTACTGCCAGAACGACTACAGCTACCCCCTGACC
TTCGGCGGCGGCACCAAGGTGGAGATCAAGCGG 23
qvqlvqsgaevkkpgasvkvsckasgytftsytmhwvrqapgqglewmgyinpasgytnynqkfkdrvtmt-
rdtstst aymelsslrsedtavyycariyygnsfaywgqgtlvtvss 24
CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGC
GTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACACCATGC
ACTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTACATCA
ACCCCGCCAGCGGCTACACCAACTACAACCAGAAGTTCAAGGACCGGGTGA
CCATGACCCGGGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCT
GCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGATCTACTACGGCAAC
AGCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC 25
divmtqspdslavslgeratinckssqslfnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgs-
gsgtdftltissl qaedvavyycqnaysfpytfgggtkleikr 26
GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAG
CGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCAACAGCGGCAAC
CAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAG
CTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTCA
GCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGC
CGAGGACGTGGCCGTGTACTACTGCCAGAACGCCTACAGCTTCCCCTACACC
TTCGGCGGCGGCACCAAGCTGGAGATCAAGCGG 27
qvqlqesgpglvkpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtis-
vdtsknqfslkl ssvtaadtavyycariyngnsfpywgqgttvtvss 28
CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACC
CTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCTACAACT
GGCACTGGATCCGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACAT
CCACTACACCGGCAGCACCAACTACAACCCCGCCCTGCGGAGCCGGGTGACC
ATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTG
ACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGGATCTACAACGGCAACA
GCTTCCCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC 29
QVQLQESGPGLIKPSQTLSLTCTVSGGSISSGYNWHWIRQPPGKGLEWIGYIHYT
GSTNYNPALRSRVTISVDTSKNQFSLKLSSVTAADTAIYYCARIYNGNSFPYWGQ GTTVTVSS 30
CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGATCAAGCCCAGCCAGACC
CTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCTACAACT
GGCACTGGATCCGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACAT
CCACTACACCGGCAGCACCAACTACAACCCCGCCCTGCGGAGCCGGGTGACC
ATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTG
ACCGCCGCCGACACCGCCATCTACTACTGCGCCCGGATCTACAACGGCAACA
GCTTCCCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC 31 SER TYR THR MET HIS
32 TYR ILE ASN PRO SER SER GLY TYR THR ASN TYR ASN GLN LYS PHE LYS
ASP 33 ILE TYR TYR GLY ASN SER PHE ALA TYR 34 LYS SER SER GLN SER
LEU LEU ASN SER GLY ASN GLN LYS ASN TYR LEU THR 35 TRP ALA SER THR
ARG GLU SER 36 GLN ASN ASP TYR SER TYR PRO LEU THR 37 SYDIN 38
WIYPGDGSTKYNEKFKG 39 GGYRYDEAMDY 40 SASSSISYMH 41 DTSKLAS 42
HQRSSYPYT 43 ASN TYR GLY MET ASN 44 TRP ILE ASN THR ASN THR GLY GLU
PRO THR TYR ALA GLU GLU PHE LYS GLY 45 PHE SER TYR GLY ASN SER PHE
ALA TYR 46 LYS SER SER GLN SER LEU LEU ASN SER GLY ASN GLN LYS ASN
TYR LEU ALA 47 GLY ALA SER THR ARG GLU SER 48 GLN ASN ASP HIS SER
TYR PRO LEU THR 49 SER GLY TYR ASN TRP HIS 50 TYR ILE HIS TYR THR
GLY SER THR ASN TYR ASN PRO SER LEU ARG SER 51 ILE TYR ASN GLY ASN
SER PHE PRO TYR 52 LYS SER SER GLN SER LEU PHE ASN SER GLY ASN GLN
LYS ASN TYR LEU THR 53 TRP ALA SER THR ARG GLU SER 54 GLN ASN ALA
TYR SER PHE PRO TYR THR 55
MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSC
VRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSM
EDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGM
VQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHAS
GHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV 56
atggccgtgactgcctgtcagggcttggggttcgtggtttcactgattgggattgcgggcatcattgctgc-
cacctgcatggacca
gtggagcacccaagacttgtacaacaaccccgtaacagctgttttcaactaccaggggctgtggcgctcctgt-
gtccgagagag
ctctggcttcaccgagtgccggggctacttcaccctgctggggctgccagccatgctgcaggcagtgcgagcc-
ctgatgatcgt
aggcatcgtcctgggtgccattggcctcctggtatccatctttgccctgaaatgcatccgcattggcagcatg-
gaggactctgcca
aagccaacatgacactgacctccgggatcatgttcattgtctcaggtctttgtgcaattgctggagtgtctgt-
gtttgccaacatgct
ggtgactaacttctggatgtccacagctaacatgtacaccggcatgggtgggatggtgcagactgttcagacc-
aggtacacattt
ggtgcggctctgttcgtgggctgggtcgctggaggcctcacactaattgggggtgtgatgatgtgcatcgcct-
gccggggcctg
gcaccagaagaaaccaactacaaagccgtttcttatcatgcctcaggccacagtgttgcctacaagcctggag-
gcttcaaggcc
agcactggctttgggtccaacaccaaaaacaagaagatatacgatggaggtgcccgcacagaggacgaggtac-
aatcttatcc ttccaagcacgactatgtgtaa 57
MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSC
VRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSME
DSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMV
QTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASG
HSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPS 58
atgtccaccaccacatgccaagtggtggcgttcctcctgtccatcctggggctggccggctgcatcgcggc-
caccgggatgga
catgtggagcacccaggacctgtacgacaaccccgtcacctccgtgttccagtacgaagggctctggaggagc-
tgcgtgagg
cagagttcaggcttcaccgaatgcaggccctatttcaccatcctgggacttccagccatgctgcaggcagtgc-
gagccctgatg
atcgtaggcatcgtcctgggtgccattggcctcctggtatccatctttgccctgaaatgcatccgcattggca-
gcatggaggactc
tgccaaagccaacatgacactgacctccgggatcatgttcattgtctcaggtattgtgcaattgctggagtgt-
ctgtgtttgccaac
atgctggtgactaacttctggatgtccacagctaacatgtacaccggcatgggtgggatggtgcagactgttc-
agaccaggtaca
catttggtgcggctctgttcgtgggctgggtcgctggaggcctcacactaattgggggtgtgatgatgtgcat-
cgcctgccgggg
cctggcaccagaagaaaccaactacaaagccgtttcttatcatgcctcaggccacagtgttgcctacaagcct-
ggaggcttcaa
ggccagcactggattgggtccaacaccaaaaacaagaagatatacgatggaggtgcccgcacagaggacgagg-
tacaatct tatccttccaagcacgactatgtgtaa 59
qvqlvqsgaevkkpgasvkvsckasgytftsytmhwvrqapgqglewmgyinpasgytnynqkfkdrvtmt-
rdtstst
aymelsslrsedtavyycariyygnsfaywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyf-
pepvtvswn
sgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpap-
ellggpsvflf
ppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng-
keykc
kvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennyktt-
ppvldsdg sfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 60
caggtgcagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaaggc-
cagcggct
acaccttcaccagctacaccatgcactgggtgcggcaggcccccggccagggcctggagtggatgggctacat-
caaccccg
ccagcggctacaccaactacaaccagaagttcaaggaccgggtgaccatgacccgggacaccagcaccagcac-
cgcctaca
tggagctgagcagcctgcggagcgaggacaccgccgtgtactactgcgcccggatctactacggcaacagctt-
cgcctactg
gggccagggcaccctggtgaccgtgagcagcgctagcaccaaaggcccatcggtcttccccctggcaccctcc-
tccaagag
cacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtgg-
aactcaggc
gccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtgg-
tgaccgtgccc
tccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaaga-
aagttgagc
ccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtctt-
cctcttcccccc
aaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa-
gaccctgag
gtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaca-
acagcacg
taccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtct-
ccaacaaagc
cctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg-
cccccatc
ccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgcc-
gtggagtgg
gagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcc-
tctatagcaa
gctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcac-
aaccactaca cgcagaagagcctctccctgtctccgggtaaa 61
divmtqspdslavslgeratinckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgs-
gsgtdftltissl
qaedvavyycqndysypltfgggtkveikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvd-
nalqsgns qesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec 62
gacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagcgggccaccatcaactgcaa-
gagcagcca
gagcctgctgaacagcggcaaccagaagaactacctgacctggtaccagcagaagcccggccagccccccaag-
ctgctgat
ctactgggccagcacccgggagagcggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcacc-
ctgacca
tcagcagcctgcaggccgaggacgtggccgtgtactactgccagaacgactacagctaccccctgaccttcgg-
cggcggcac
caaggtggagatcaagcggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaa-
tctggaactgcc
tctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctcc-
aatcgggtaact
cccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaa-
agcagac
tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca-
acaggggag agtgt 63
Qvqlqesgpglikpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtis-
vdtsknqfslkl
ssvtaadtaiyycariyngnsfpywgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvt-
vswnsgalts
gvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggp-
svflfppkp
kdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeyk-
ckvsn
kalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvl-
dsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 64
caggtgcagctgcaggagagcggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgt-
gagcggcgg
cagcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctggagtggatcggctac-
atccacta
caccggcagcaccaactacaaccccgccctgcggagccgggtgaccatcagcgtggacaccagcaagaaccag-
ttcagcct
gaagctgagcagcgtgaccgccgccgacaccgccatctactactgcgcccggatctacaacggcaacagcttc-
ccctactgg
ggccagggcaccaccgtgaccgtgagcagcgctagcaccaaaggcccatcggtcttccccctggcaccctcct-
ccaagagc
acctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtgga-
actcaggcg
ccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggt-
gaccgtgccct
ccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaa-
agttgagcc
caaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc-
ctcttcccccca
aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaag-
accctgagg
tcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa-
cagcacgt
accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctc-
caacaaagcc
ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc-
ccccatcc
cgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg-
tggagtggg
agagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcct-
ctatagcaag
ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca-
accactacac gcagaagagcctctccctgtctccgggtaaa 65
divmtqspdslavslgeratinckssqslfnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgs-
gsgtdftltissl
qaedvavyycqnaysfpytfgggtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkvd-
nalqsgns qesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec 66
gacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagcgggccaccatcaactgcaa-
gagcagcca
gagcctgttcaacagcggcaaccagaagaactacctgacctggtaccagcagaagcccggccagccccccaag-
ctgctgat
ctactgggccagcacccgggagagcggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcacc-
ctgacca
tcagcagcctgcaggccgaggacgtggccgtgtactactgccagaacgcctacagcttcccctacaccttcgg-
cggcggcac
caagctggagatcaagcggacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaa-
tctggaactgcc
tctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctcc-
aatcgggtaact
cccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaa-
agcagac
tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca-
acaggggag agtgt 67
qvqlqesgpglvkpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtis-
vdtsknqfslkl
ssvtaadtaiyycariyngnsfpywgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvt-
vswnsgalts
gvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapellggp-
svflfppkp
kdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsyltvlhqdwlngkeyk-
ckvsn
kalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvl-
dsdgsfflys kltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 68
caggtgcagctgcaggagagcggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgt-
gagcggcgg
cagcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctggagtggatcggctac-
atccacta
caccggcagcaccaactacaaccccgccctgcggagccgggtgaccatcagcgtggacaccagcaagaaccag-
ttcagcct
gaagctgagcagcgtgaccgccgccgacaccgccatctactactgcgcccggatctacaacggcaacagcttc-
ccctactgg
ggccagggcaccaccgtgaccgtgagcagcgctagcaccaaaggcccatcggicttccccctggcaccctcct-
ccaagagc
acctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtgga-
actcaggcg
ccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggt-
gaccgtgccct
ccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaa-
agttgagcc
caaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc-
ctcttcccccca
aaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaag-
accctgagg
tcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaa-
cagcacgt
accgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctc-
caacaaagcc
ctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgc-
ccccatcc
cgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccg-
tggagtggg
agagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcct-
ctatagcaag
ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcaca-
accactacac gcagaagagcctctccctgtctccgggtaaa 69 malpvtalllplalllhaarp
70 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg
71 Tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacd 72
accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgccc-
agaggcgtg ccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat
73 Fwvlvvvggvlacysllvtvafiifwv 74
ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattat-
tttctgggtg 75 Rskrsrllhsdymnmtprrpgptrkhyqpyapprdfaayrs 76
aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggccaacccg-
caagcattac cagccctatgccccaccacgcgacttcgcagcctatcgctcc 77
rvkfsrsadapayqqgqnqlynelnlgrreeydvldkrrgrdpemggkpqrrknpqeglynelqkdkmaea-
yseigmk gerrrgkghdglyqglstatkdtydalhmqalppr
78
agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagct-
caatctagga
cgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaa-
ggaaga
accctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaa-
aggcgagc
gccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccct-
tcacatgc aggccctgccccctcgc 79 Iyiwaplagtcgvlllslvit 80
Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc 81
Krgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel 82
aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaaga-
ggaagatggctgt agctgccgatttccagaagaagaagaaggaggatgtgaactg 83
YIDPSSGYTNYNQKFKD 84 YINPASGYTNYNQKFKD 85 yihytgstnynpalrs 86
Divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrftgs-
gsgtdftltiss
vqaedlavyycqndysypltfgagtklelkrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkv-
dnalqsgns qesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
87
Qiqlvqsgpelkkpgetvkisckasgytftnygmnwvkqapgkglkwmgwintntgeptyaeefkgrfafs-
letsasta
ylqinnlknedtatyfcarlgfgnamdywgqgtsvtvssastkgpsvfplapsskstsggtaalgclvkdyfp-
epvtvswns
galtsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpape-
llggpsvflf
ppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlng-
keykc
kvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennyktt-
ppvldsdg sfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 88
Divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrftgs-
gsgtdftltiss
vqaedlavyycqndysypftfgsgtkleikrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwkv-
dnalqsgns qesvteqdskdstyslsstltlskadyekhkvyacevthqglsspvtksfnrgec
89
Qvqlqqpgaelvrpgasvklsckasgytftsywinwvkqrpgqglewigniypsdsytnynqkfkdkatlt-
vdkssstay
mqlssptsedsavyyctrswrgnsfdywgqgttltvssastkgpsvfplapsskstsggtaalgclvkdyfpe-
pvtvswnsg
altsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppcpapel-
lggpsvflfp
pkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngk-
eykck
vsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttp-
pvldsdgsf flyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 90
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgca-
gctgcaggagag
cggccccggcctggtgaagcccagccagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagc-
ggctaca
actggcactggatccggcagccccccggcaagggcctggagtggatcggctacatccactacaccggcagcac-
caactaca
accccgccctgcggagccgggtgaccatcagcgtggacaccagcaagaaccagttcagcctgaagctgagcag-
cgtgacc
gccgccgacaccgccgtgtactactgcgcccggatctacaacggcaacagcttcccctactggggccagggca-
ccaccgtga
ccgtgagcagcggtggaggcggttcaggcggaggtggttctggcggtggcggatcggacatcgtgatgaccca-
gagccccg
acagcctggccgtgagcctgggcgagcgggccaccatcaactgcaagagcagccagagcctgttcaacagcgg-
caaccag
aagaactacctgacctggtaccagcagaagcccggccagccccccaagctgctgatctactgggccagcaccc-
gggagagc
ggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccg-
aggacgt
ggccgtgtactactgccagaacgcctacagcttcccctacaccttcggcggcggcaccaagctggagatcaag-
cggaccacg
acgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgt-
gccggcc
agcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggt-
ggagtcctgg
cttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgca-
cagtgactacatga
acatgactccccgccgccccgggccaacccgcaagcattaccagccctatgccccaccacgcgacttcgcagc-
ctatcgctc
cagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctc-
aatctagg
acgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagaga-
aggaag
aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatga-
aaggcgag
cgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccc-
ttcacatg
caggccctgccccctcgctaggtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattc-
ttaactatgttgctc
cttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcatttt-
ctcctccttgtataaatcctg
gttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgac-
gcaacccccactg
gttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgga-
actcatcgccgcc
tgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctga-
cgtcctttccat
ggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatcc-
agcggaccttcct
tcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccc-
tttgggccgcct
ccccgcctggaattcgctagcctcgagctcacacaaaaaaccaacacacagatgtaatgaaaataaagatatt-
ttattgcggccg
ctttagtttcggaggtaacctgtaagtctgttaatgaagtaaaagttccttaggatttccactctgactatgg-
tccaggcacagtgact
gtactccttggccttcaggtaatgcagaatcctcccataatatcttttcaggtgcagactgctcatgagtttt-
cccctggtgaaatctt
ctttctccagtttttcttccaggactgtcttcagatggtttatctgatgatagacattagccaggaggttctc-
aacaatagtctcattcca
gccagtgctagatgaatcttgtctgaaaatagcaaagatgttctggagcatctcatagatggtcaatgcggcg-
tcctccttctgga
actgctgcagctgcttaatctcctcagggatgtcaaagttcatcctgtccttgaggcagtattcaagcctccc-
attcaattgccaca
ggagcttctgacactgaaaattgctgcttctttgtaggaatccaagcaagttgtagctcatggaaagagctgt-
agtggagaagca
caacaggagagcaataggaggagacacttgttggtcatggtggcgaccggtagcgctaggtcatatgcaggag-
ttgaggttac
tgtgagtagtgattaaagagagtgatagggaactcttgaacaagagatgcaatttatactgttaattctggaa-
aaatattatggggg
tgtcaaaatgtcccgggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaaca-
gtttttcctccacgcc
ttctgtatgaaacagtttttcctccgtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgc-
cttctgtatgaaacagt ttttcctccacgccttctgtatgaaacagtttttcctcc 91
atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgca-
gctgcaggagag
cggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagc-
ggctaca
actggcactggatccggcagccccccggcaagggcctggagtggatcggctacatccactacaccggcagcac-
caactaca
accccgccctgcggagccgggtgaccatcagcgtggacaccagcaagaaccagttcagcctgaagctgagcag-
cgtgacc
gccgccgacaccgccatctactactgcgcccggatctacaacggcaacagcttcccctactggggccagggca-
ccaccgtga
ccgtgagcagcggtggaggcggttcaggcggaggtggttctggcggtggcggatcggacatcgtgatgaccca-
gagccccg
acagcctggccgtgagcctgggcgagcgggccaccatcaactgcaagagcagccagagcctgttcaacagcgg-
caaccag
aagaactacctgacctggtaccagcagaagcccggccagccccccaagctgctgatctactgggccagcaccc-
gggagagc
ggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccg-
aggacgt
ggccgtgtactactgccagaacgcctacagcttcccctacaccttcggcggcggcaccaagctggagatcaag-
cggaccacg
acgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgt-
gccggcc
agcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatttttgggtgctggtggtggttggt-
ggagtcctgg
cttgctatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcaggctcctgca-
cagtgactacatga
acatgactccccgccgccccgggccaacccgcaagcattaccagccctatgccccaccacgcgacttcgcagc-
ctatcgctc
cagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctc-
aatctagg
acgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagaga-
aggaag
aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatga-
aaggcgag
cgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccc-
ttcacatg
caggccctgccccctcgctaggtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattc-
ttaactatgttgctc
cattttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattt-
tctcctccttgtataaatcctg
gttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgac-
gcaacccccactg
gttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgga-
actcatcgccgcc
tgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctga-
cgtcctttccat
ggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatcc-
agcggaccttcct
tcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccc-
tttgggccgcct
ccccgcctggaattcgctagcctcgagctcacacaaaaaaccaacacacagatgtaatgaaaataaagatatt-
ttattgcggccg
ctttagtttcggaggtaacctgtaagtctgttaatgaagtaaaagttccttaggatttccactctgactatgg-
tccaggcacagtgact
gtactccttggccttcaggtaatgcagaatcctcccataatatcttttcaggtgcagactgctcatgagtttt-
cccctggtgaaatctt
ctttctccagtttttcttccaggactgtcttcagatggtttatctgatgatagacattagccaggaggttctc-
aacaatagtctcattcca
gccagtgctagatgaatcttgtctgaaaatagcaaagatgttctggagcatctcatagatggtcaatgcggcg-
tcctccttctgga
actgctgcagctgcttaatctcctcagggatgtcaaagttcatcctgtccttgaggcagtattcaagcctccc-
attcaattgccaca
ggagcttctgacactgaaaattgctgcttctttgtaggaatccaagcaagttgtagctcatggaaagagctgt-
agtggagaagca
caacaggagagcaatttggaggagacacttgttggtcatggtggcgaccggtagcgctaggtcatatgcagga-
gttgaggttac
tgtgagtagtgattaaagagagtgatagggaactcttgaacaagagatgcaatttatactgttaattctggaa-
aaatattatggggg
tgtcaaaatgtcccgggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaaca-
gtttttcctccacgcc
ttctgtatgaaacagtttttcctccgtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgc-
cttctgtatgaaacagt ttttcctccacgccttctgtatgaaacagtttttcctcc 92
MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLK
DRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLA
NVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCA
WTIVRVEILRNFYFINRLTGYLRN 93 GGGGSGGGGSGGGGS 94
Acgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgccttctgt-
atgaaacagtttttcctcc
gtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctc-
cacgccttctgtatga aacagtttttcctcc
Sequence CWU 1
1
941114PRTArtificial Sequencesynthetic polypeptide 1Asp Ile Val Met
Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly1 5 10 15Glu Lys Val
Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn
Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro
Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55
60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65
70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
Asn 85 90 95Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
Glu Leu 100 105 110Lys Arg2342DNAArtificial Sequencesynthetic
polynucleotide 2gacattgtga tgacacagtc tccatcctcc ctgactgtga
cagcaggaga gaaggtcact 60atgagctgca agtccagtca gagtctgtta aacagtggaa
atcaaaagaa ctacttgacc 120tggtaccagc agaaaccagg gcagcctcct
aaactgttga tctactgggc atccactagg 180gaatctgggg tccctgatcg
cttcacaggc agtggatctg gaacagattt cactctcacc 240atcagcagtg
tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat
300ccgctcacgt tcggtgctgg gaccaagctg gagctgaaac gg
3423118PRTArtificial Sequencesynthetic polypeptide 3Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met
His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly
Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55
60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln
Gly Thr 100 105 110Thr Val Thr Val Ser Ser 1154354DNAArtificial
Sequencesynthetic polynucleotide 4caggtccagc tgcagcagtc tggggctgaa
ctggcaagac ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact
agctacacga tgcactgggt aaaacagagg 120cctggacagg gtctggaatg
gattggatac attaatccta gcagtggtta tactaattac 180aatcagaagt
tcaaggacaa ggccacattg actgcagaca aatcctccag cacagcctac
240atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc
aagaatctac 300tatggtaact cgtttgctta ctggggccaa gggaccacgg
tcaccgtctc ctca 3545107PRTArtificial Sequencesynthetic polypeptide
5Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly1 5
10 15Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr
Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Thr Ser Pro Lys Arg Trp
Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe
Ser Gly Ser 50 55 60Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Met Glu Ala Glu65 70 75 80Asp Ala Ala Thr Tyr Tyr Cys His Gln Arg
Ser Ser Tyr Pro Tyr Thr 85 90 95Phe Gly Gly Gly Thr Lys Leu Glu Ile
Lys Arg 100 1056321DNAArtificial Sequencesynthetic polynucleotide
6caaattgttc tcacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc
60atgacctgca gtgccagctc aagtataagt tacatgcact ggtaccagca gaagccaggc
120acctccccca aaagatggat ttatgacaca tccaaactgg cttctggagt
ccctgctcgc 180ttcagtggca gtgggtctgg gacctcttat tctctcacaa
tcagcagcat ggaggctgaa 240gatgctgcca cttattactg ccatcagcgg
agtagttacc cgtacacgtt cggagggggg 300accaagctgg aaataaaacg g
3217120PRTArtificial Sequencesynthetic polypeptide 7Gln Val Gln Leu
Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1 5 10 15Leu Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Asp Ile
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly
Trp Ile Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe 50 55
60Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65
70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asn Ser Ala Val Tyr Phe
Cys 85 90 95Ala Arg Gly Gly Tyr Arg Tyr Asp Glu Ala Met Asp Tyr Trp
Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser 115
1208360DNAArtificial Sequencesynthetic polynucleotide 8caggttcagc
tgcagcagtc tggacctgag ctggtgaagc ctggggcttt agtgaagata 60tcctgcaagg
cttctggtta caccttcaca agctacgata taaactgggt gaagcagagg
120cctggacagg gacttgagtg gattggatgg atttatcctg gagatggtag
tactaagtac 180aatgagaaat tcaagggcaa ggccacactg actgcagaca
aatcctccag cacagcctac 240atgcagctca gcagcctgac ttctgagaac
tctgcagtct atttctgtgc aagagggggc 300tataggtacg acgaggctat
ggactactgg ggtcaaggga ccacggtcac cgtctcctca 3609114PRTArtificial
Sequencesynthetic polypeptide 9Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Ser Val Ser Ala Gly1 5 10 15Glu Lys Val Thr Met Ser Cys Lys
Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asp His
Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105
110Lys Arg10342DNAArtificial Sequencesynthetic polynucleotide
10gacattgtga tgacacagtc tccatcctcc ctgagtgtgt cagcaggaga gaaggtcact
60atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttggcc
120tggtaccagc agaaaccagg gcagcctcct aaactgttga tctacggggc
atccactagg 180gaatctgggg tccctgatcg cttcacaggc agtggatctg
gaaccgattt cactcttacc 240atcagcagtg tgcaggctga agacctggca
gtttattact gtcagaatga tcatagttat 300ccgctcacgt tcggtgctgg
gaccaagctg gagctgaaac gg 34211118PRTArtificial Sequencesynthetic
polypeptide 11Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys
Pro Gly Glu1 5 10 15Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asn Tyr 20 25 30Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys
Gly Leu Lys Trp Met 35 40 45Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro
Thr Tyr Ala Glu Glu Phe 50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu
Thr Ser Ala Ser Thr Ala Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys
Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Phe Ser Tyr Gly
Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val
Ser Ser 11512354DNAArtificial Sequencesynthetic polynucleotide
12cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc
60tcctgcaagg cttctgggta taccttcaca aactatggaa tgaactgggt gaagcaggct
120ccaggaaagg gtttaaagtg gatgggctgg ataaacacca acactggaga
gccaacatat 180gctgaagagt tcaagggacg gtttgccttc tctttggaaa
cctctgccag cactgcctat 240ttgcagatca acaacctcaa aaatgaggac
acggctacat atttctgtgc tagattctct 300tatggtaact cctttgctta
ctggggccaa gggaccacgg tcaccgtctc ctca 35413114PRTArtificial
Sequencesynthetic polypeptide 13Asp Ile Val Met Thr Gln Ser Pro Ser
Ser Leu Thr Val Thr Pro Gly1 5 10 15Glu Lys Val Thr Met Thr Cys Lys
Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Arg Pro Gly Gln 35 40 45Pro Pro Lys Met Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Thr
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Val Gln Ala Glu Asp Leu Ala Val Phe Tyr Cys Gln Asn 85 90 95Ala Tyr
Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg14342DNAArtificial Sequencesynthetic polynucleotide
14gacattgtga tgacacagtc tccatcctcc ctgactgtga caccaggaga gaaggtcact
60atgacctgca agtccagtca gagtttgttt aatagtggaa atcaaaagaa ctacttgacc
120tggtaccaac agagacctgg ccagccccct aaaatgttga tctactgggc
atccactagg 180gaatctgggg tccctgatcg cttcacaggc agtggatctg
gaacagattt cactctcacc 240atcagcagtg tgcaggctga agacctggca
gttttttact gtcagaatgc ttatagtttt 300ccgtacacgt tcggaggggg
gaccaagctg gaaataaaac gg 34215118PRTArtificial Sequencesynthetic
polypeptide 15Asp Val Gln Leu Gln Glu Ser Gly Pro Asp Leu Val Lys
Pro Ser Gln1 5 10 15Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser
Ile Thr Ser Gly 20 25 30Tyr Asn Trp His Trp Ile Arg Gln Phe Pro Gly
Asn Lys Met Glu Trp 35 40 45Met Gly Tyr Ile His Tyr Thr Gly Ser Thr
Asn Tyr Asn Pro Ser Leu 50 55 60Arg Ser Arg Ile Ser Ile Thr Arg Asp
Thr Ser Lys Asn Gln Phe Phe65 70 75 80Leu Gln Leu Asn Ser Val Thr
Thr Asp Asp Thr Ala Thr Tyr Tyr Cys 85 90 95Thr Arg Ile Tyr Asn Gly
Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110Ser Val Thr Val
Ser Ser 11516354DNAArtificial Sequencesynthetic polynucleotide
16gatgtgcaac ttcaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc
60acctgcactg tcactggcta ctccatcacc agtggttata actggcactg gatccggcag
120tttccaggaa acaaaatgga atggatgggc tacatacact acactggtag
cactaattac 180aacccatctc tcagaagtcg aatctctatc actcgagaca
catccaagaa ccagttcttc 240ctgcagttga attctgtgac cactgatgac
acagccacat attactgtac aagaatctac 300aatggtaact cttttcctta
ctggggccaa ggaacctcag tcaccgtctc ctca 35417118PRTArtificial
Sequencesynthetic polypeptide 17Gln Val Gln Leu Gln Gln Ser Gly Ala
Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Tyr Ile Asp Pro Ser
Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Asp Lys Ala Thr
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser 11518354DNAArtificial Sequencesynthetic
polynucleotide 18caggtccagc tgcagcagtc tggggctgaa ctggcaagac
ctggggcctc agtgaagatg 60tcctgcaagg cttctggcta cacctttact agctacacga
tgcactgggt aaaacagagg 120cctggacagg gtctggaatg gattggatac
attgacccta gcagtggtta tactaattac 180aatcagaagt tcaaggacaa
ggccacattg actgcagaca aatcctccag cacagcctac 240atgcaactga
gcagcctgac atctgaggac tctgcagtct attactgtgc aagaatctac
300tatggtaact cgtttgctta ctggggccaa gggaccacgg tcaccgtctc ctca
35419118PRTArtificial Sequencesynthetic polypeptide 19Gln Val Gln
Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala1 5 10 15Ser Val
Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
Tyr65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp
Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11520354DNAArtificial Sequencesynthetic polynucleotide 20caggtccagc
tgcagcagtc tggggctgaa ctggcaagac ctggggcctc agtgaagatg 60tcctgcaagg
cttctggcta cacctttact agctacacga tgcactgggt aaaacagagg
120cctggacagg gtctggaatg gattggatac attaatcctg ccagtggtta
tactaattac 180aatcagaagt tcaaggacaa ggccacattg actgcagaca
aatcctccag cacagcctac 240atgcaactga gcagcctgac atctgaggac
tctgcagtct attactgtgc aagaatctac 300tatggtaact cgtttgctta
ctggggccaa gggaccacgg tcaccgtctc ctca 35421114PRTArtificial
Sequencesynthetic polypeptide 21Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr
Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105
110Lys Arg22342DNAArtificial Sequencesynthetic polynucleotide
22gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgggccacc
60atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc
120tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc
cagcacccgg 180gagagcggcg tgcccgaccg gttcagcggc agcggcagcg
gcaccgactt caccctgacc 240atcagcagcc tgcaggccga ggacgtggcc
gtgtactact gccagaacga ctacagctac 300cccctgacct tcggcggcgg
caccaaggtg gagatcaagc gg 34223118PRTArtificial Sequencesynthetic
polypeptide 23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln Ala Pro Gly Gln
Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Ala Ser Gly Tyr Thr
Asn Tyr Asn Gln Lys Phe 50 55 60Lys Asp Arg Val Thr Met Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Ile Tyr Tyr Gly
Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110Leu Val Thr Val
Ser Ser 11524354DNAArtificial Sequencesynthetic polynucleotide
24caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
60agctgcaagg ccagcggcta caccttcacc agctacacca tgcactgggt gcggcaggcc
120cccggccagg gcctggagtg gatgggctac atcaaccccg ccagcggcta
caccaactac 180aaccagaagt tcaaggaccg ggtgaccatg acccgggaca
ccagcaccag caccgcctac 240atggagctga gcagcctgcg gagcgaggac
accgccgtgt actactgcgc ccggatctac 300tacggcaaca gcttcgccta
ctggggccag ggcaccctgg tgaccgtgag cagc 35425114PRTArtificial
Sequencesynthetic polypeptide 25Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95Ala Tyr
Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg26342DNAArtificial Sequencesynthetic polynucleotide
26gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgggccacc
60atcaactgca agagcagcca gagcctgttc aacagcggca accagaagaa ctacctgacc
120tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc
cagcacccgg 180gagagcggcg tgcccgaccg gttcagcggc agcggcagcg
gcaccgactt caccctgacc 240atcagcagcc tgcaggccga ggacgtggcc
gtgtactact gccagaacgc ctacagcttc 300ccctacacct tcggcggcgg
caccaagctg gagatcaagc gg 34227118PRTArtificial Sequencesynthetic
polypeptide 27Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Tyr Asn Trp
His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly
Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60Arg
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75
80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly
Thr 100 105 110Thr Val Thr Val Ser Ser 11528354DNAArtificial
Sequencesynthetic polynucleotide 28caggtgcagc tgcaggagag cggccccggc
ctggtgaagc ccagccagac cctgagcctg 60acctgcaccg tgagcggcgg cagcatcagc
agcggctaca actggcactg gatccggcag 120ccccccggca agggcctgga
gtggatcggc tacatccact acaccggcag caccaactac 180aaccccgccc
tgcggagccg ggtgaccatc agcgtggaca ccagcaagaa ccagttcagc
240ctgaagctga gcagcgtgac cgccgccgac accgccgtgt actactgcgc
ccggatctac 300aacggcaaca gcttccccta ctggggccag ggcaccaccg
tgaccgtgag cagc 35429118PRTArtificial Sequencesynthetic polypeptide
29Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln1
5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
Gly 20 25 30Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp 35 40 45Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn
Pro Ala Leu 50 55 60Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys
Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr Ala Ala Asp
Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Ile Tyr Asn Gly Asn Ser Phe
Pro Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser
11530354DNAArtificial Sequencesynthetic polynucleotide 30caggtgcagc
tgcaggagag cggccccggc ctgatcaagc ccagccagac cctgagcctg 60acctgcaccg
tgagcggcgg cagcatcagc agcggctaca actggcactg gatccggcag
120ccccccggca agggcctgga gtggatcggc tacatccact acaccggcag
caccaactac 180aaccccgccc tgcggagccg ggtgaccatc agcgtggaca
ccagcaagaa ccagttcagc 240ctgaagctga gcagcgtgac cgccgccgac
accgccatct actactgcgc ccggatctac 300aacggcaaca gcttccccta
ctggggccag ggcaccaccg tgaccgtgag cagc 354315PRTArtificial
Sequencesynthetic polypeptide 31Ser Tyr Thr Met His1
53217PRTArtificial Sequencesynthetic polypeptide 32Tyr Ile Asn Pro
Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10
15Asp339PRTArtificial Sequencesynthetic polypeptide 33Ile Tyr Tyr
Gly Asn Ser Phe Ala Tyr1 53417PRTArtificial Sequencesynthetic
polypeptide 34Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys
Asn Tyr Leu1 5 10 15Thr357PRTArtificial Sequencesynthetic
polypeptide 35Trp Ala Ser Thr Arg Glu Ser1 5369PRTArtificial
Sequencesynthetic polypeptide 36Gln Asn Asp Tyr Ser Tyr Pro Leu
Thr1 5375PRTArtificial Sequencesynthetic polypeptide 37Ser Tyr Asp
Ile Asn1 53817PRTArtificial Sequencesynthetic polypeptide 38Trp Ile
Tyr Pro Gly Asp Gly Ser Thr Lys Tyr Asn Glu Lys Phe Lys1 5 10
15Gly3911PRTArtificial Sequencesynthetic polypeptide 39Gly Gly Tyr
Arg Tyr Asp Glu Ala Met Asp Tyr1 5 104010PRTArtificial
Sequencesynthetic polypeptide 40Ser Ala Ser Ser Ser Ile Ser Tyr Met
His1 5 10417PRTArtificial Sequencesynthetic polypeptide 41Asp Thr
Ser Lys Leu Ala Ser1 5429PRTArtificial Sequencesynthetic
polypeptide 42His Gln Arg Ser Ser Tyr Pro Tyr Thr1
5435PRTArtificial Sequencesynthetic polypeptide 43Asn Tyr Gly Met
Asn1 54417PRTArtificial Sequencesynthetic polypeptide 44Trp Ile Asn
Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys1 5 10
15Gly459PRTArtificial Sequencesynthetic polypeptide 45Phe Ser Tyr
Gly Asn Ser Phe Ala Tyr1 54617PRTArtificial Sequencesynthetic
polypeptide 46Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys
Asn Tyr Leu1 5 10 15Ala477PRTArtificial Sequencesynthetic
polypeptide 47Gly Ala Ser Thr Arg Glu Ser1 5489PRTArtificial
Sequencesynthetic polypeptide 48Gln Asn Asp His Ser Tyr Pro Leu
Thr1 5496PRTArtificial Sequencesynthetic polypeptide 49Ser Gly Tyr
Asn Trp His1 55016PRTArtificial Sequencesynthetic polypeptide 50Tyr
Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ser Leu Arg Ser1 5 10
15519PRTArtificial Sequencesynthetic polypeptide 51Ile Tyr Asn Gly
Asn Ser Phe Pro Tyr1 55217PRTArtificial Sequencesynthetic
polypeptide 52Lys Ser Ser Gln Ser Leu Phe Asn Ser Gly Asn Gln Lys
Asn Tyr Leu1 5 10 15Thr537PRTArtificial Sequencesynthetic
polypeptide 53Trp Ala Ser Thr Arg Glu Ser1 5549PRTArtificial
Sequencesynthetic polypeptide 54Gln Asn Ala Tyr Ser Phe Pro Tyr
Thr1 555261PRTHomo sapiens 55Met Ala Val Thr Ala Cys Gln Gly Leu
Gly Phe Val Val Ser Leu Ile1 5 10 15Gly Ile Ala Gly Ile Ile Ala Ala
Thr Cys Met Asp Gln Trp Ser Thr 20 25 30Gln Asp Leu Tyr Asn Asn Pro
Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45Leu Trp Arg Ser Cys Val
Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60Gly Tyr Phe Thr Leu
Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg65 70 75 80Ala Leu Met
Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95Ser Ile
Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105
110Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser
115 120 125Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met
Leu Val 130 135 140Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr
Gly Met Gly Gly145 150 155 160Met Val Gln Thr Val Gln Thr Arg Tyr
Thr Phe Gly Ala Ala Leu Phe 165 170 175Val Gly Trp Val Ala Gly Gly
Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190Cys Ile Ala Cys Arg
Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205Val Ser Tyr
His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220Phe
Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile225 230
235 240Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro
Ser 245 250 255Lys His Asp Tyr Val 26056786DNAHomo sapiens
56atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg gattgcgggc
60atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa caaccccgta
120acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag
ctctggcttc 180accgagtgcc ggggctactt caccctgctg gggctgccag
ccatgctgca ggcagtgcga 240gccctgatga tcgtaggcat cgtcctgggt
gccattggcc tcctggtatc catctttgcc 300ctgaaatgca tccgcattgg
cagcatggag gactctgcca aagccaacat gacactgacc 360tccgggatca
tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc
420aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg
catgggtggg 480atggtgcaga ctgttcagac caggtacaca tttggtgcgg
ctctgttcgt gggctgggtc 540gctggaggcc tcacactaat tgggggtgtg
atgatgtgca tcgcctgccg gggcctggca 600ccagaagaaa ccaactacaa
agccgtttct tatcatgcct caggccacag tgttgcctac 660aagcctggag
gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata
720tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa
gcacgactat 780gtgtaa 78657261PRTHomo sapiens 57Met Ser Thr Thr Thr
Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu1 5 10 15Gly Leu Ala Gly
Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30Gln Asp Leu
Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45Leu Trp
Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60Pro
Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg65 70 75
80Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val
85 90 95Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp
Ser 100 105 110Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe
Ile Val Ser 115 120 125Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe
Ala Asn Met Leu Val 130 135 140Thr Asn Phe Trp Met Ser Thr Ala Asn
Met Tyr Thr Gly Met Gly Gly145 150 155 160Met Val Gln Thr Val Gln
Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175Val Gly Trp Val
Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190Cys Ile
Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200
205Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly
210 215 220Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys
Lys Ile225 230 235 240Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val
Gln Ser Tyr Pro Ser 245 250 255Lys His Asp Tyr Val 26058786DNAHomo
sapiens 58atgtccacca ccacatgcca agtggtggcg ttcctcctgt ccatcctggg
gctggccggc 60tgcatcgcgg ccaccgggat ggacatgtgg agcacccagg acctgtacga
caaccccgtc 120acctccgtgt tccagtacga agggctctgg aggagctgcg
tgaggcagag ttcaggcttc 180accgaatgca ggccctattt caccatcctg
ggacttccag ccatgctgca ggcagtgcga 240gccctgatga tcgtaggcat
cgtcctgggt gccattggcc tcctggtatc catctttgcc 300ctgaaatgca
tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc
360tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc
tgtgtttgcc 420aacatgctgg tgactaactt ctggatgtcc acagctaaca
tgtacaccgg catgggtggg 480atggtgcaga ctgttcagac caggtacaca
tttggtgcgg ctctgttcgt gggctgggtc 540gctggaggcc tcacactaat
tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600ccagaagaaa
ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac
660aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa
caagaagata 720tacgatggag gtgcccgcac agaggacgag gtacaatctt
atccttccaa gcacgactat 780gtgtaa 78659448PRTArtificial
Sequencesynthetic polypeptide 59Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Thr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Tyr Ile Asn Pro Ala
Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Asp Arg Val Thr
Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Ile Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr 100 105
110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445601344DNAArtificial Sequencesynthetic polynucleotide
60caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg
60agctgcaagg ccagcggcta caccttcacc agctacacca tgcactgggt gcggcaggcc
120cccggccagg gcctggagtg gatgggctac atcaaccccg ccagcggcta
caccaactac 180aaccagaagt tcaaggaccg ggtgaccatg acccgggaca
ccagcaccag caccgcctac 240atggagctga gcagcctgcg gagcgaggac
accgccgtgt actactgcgc ccggatctac 300tacggcaaca gcttcgccta
ctggggccag ggcaccctgg tgaccgtgag cagcgctagc 360accaaaggcc
catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca
420gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt
gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc 540tactccctca gcagcgtggt gaccgtgccc
tccagcagct tgggcaccca gacctacatc 600tgcaacgtga atcacaagcc
cagcaacacc aaggtggaca agaaagttga gcccaaatct 660tgtgacaaaa
ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca
720gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 900taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 960aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
1020aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga
tgagctgacc 1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 1140gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct
ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag
1320agcctctccc tgtctccggg taaa 134461220PRTArtificial
Sequencesynthetic polypeptide 61Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr
Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105
110Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 22062660DNAArtificial Sequencesynthetic polynucleotide
62gacatcgtga tgacccagag ccccgacagc ctggccgtga gcctgggcga gcgggccacc
60atcaactgca agagcagcca gagcctgctg aacagcggca accagaagaa ctacctgacc
120tggtaccagc agaagcccgg ccagcccccc aagctgctga tctactgggc
cagcacccgg 180gagagcggcg tgcccgaccg gttcagcggc agcggcagcg
gcaccgactt caccctgacc 240atcagcagcc tgcaggccga ggacgtggcc
gtgtactact gccagaacga ctacagctac 300cccctgacct tcggcggcgg
caccaaggtg gagatcaagc ggacggtggc tgcaccatct 360gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc
420ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga
taacgccctc 480caatcgggta actcccagga gagtgtcaca gagcaggaca
gcaaggacag cacctacagc 540ctcagcagca ccctgacgct gagcaaagca
gactacgaga aacacaaagt ctacgcctgc 600gaagtcaccc atcagggcct
gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 66063448PRTArtificial
Sequencesynthetic polypeptide 63Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Ile Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30Tyr Asn Trp His Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile His Tyr
Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60Arg Ser Arg Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu
Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg
Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105
110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440
445641344DNAArtificial Sequencesynthetic polynucleotide
64caggtgcagc tgcaggagag cggccccggc ctgatcaagc ccagccagac cctgagcctg
60acctgcaccg tgagcggcgg cagcatcagc agcggctaca actggcactg gatccggcag
120ccccccggca agggcctgga gtggatcggc tacatccact acaccggcag
caccaactac 180aaccccgccc tgcggagccg ggtgaccatc agcgtggaca
ccagcaagaa ccagttcagc 240ctgaagctga gcagcgtgac cgccgccgac
accgccatct actactgcgc ccggatctac 300aacggcaaca gcttccccta
ctggggccag ggcaccaccg tgaccgtgag cagcgctagc 360accaaaggcc
catcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca
420gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt
gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg
tcctacagtc ctcaggactc 540tactccctca gcagcgtggt gaccgtgccc
tccagcagct tgggcaccca gacctacatc 600tgcaacgtga atcacaagcc
cagcaacacc aaggtggaca agaaagttga gcccaaatct 660tgtgacaaaa
ctcacacatg cccaccgtgc ccagcacctg aactcctggg gggaccgtca
720gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac
ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg
tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca
aagccgcggg aggagcagta caacagcacg 900taccgtgtgg tcagcgtcct
caccgtcctg caccaggact ggctgaatgg caaggagtac 960aagtgcaagg
tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc
1020aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga
tgagctgacc 1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct
atcccagcga catcgccgtg 1140gagtgggaga gcaatgggca gccggagaac
aactacaaga ccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct
ctatagcaag ctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct
tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag
1320agcctctccc tgtctccggg taaa 134465220PRTArtificial
Sequencesynthetic polypeptide 65Asp Ile Val Met Thr Gln Ser Pro Asp
Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg Ala Thr Ile Asn Cys Lys
Ser Ser Gln Ser Leu Phe Asn Ser 20 25 30Gly Asn Gln Lys Asn Tyr Leu
Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45Pro Pro Lys Leu Leu Ile
Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95Ala Tyr
Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105
110Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
115 120 125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205Ser Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 22066660DNAArtificial
Sequencesynthetic polynucleotide 66gacatcgtga tgacccagag ccccgacagc
ctggccgtga gcctgggcga gcgggccacc 60atcaactgca agagcagcca gagcctgttc
aacagcggca accagaagaa ctacctgacc 120tggtaccagc agaagcccgg
ccagcccccc aagctgctga tctactgggc cagcacccgg 180gagagcggcg
tgcccgaccg gttcagcggc agcggcagcg gcaccgactt caccctgacc
240atcagcagcc tgcaggccga ggacgtggcc gtgtactact gccagaacgc
ctacagcttc 300ccctacacct tcggcggcgg caccaagctg gagatcaagc
ggacggtggc tgcaccatct 360gtcttcatct tcccgccatc tgatgagcag
ttgaaatctg gaactgcctc tgttgtgtgc 420ctgctgaata acttctatcc
cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480caatcgggta
actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc
540ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt
ctacgcctgc 600gaagtcaccc atcagggcct gagctcgccc gtcacaaaga
gcttcaacag gggagagtgt 66067448PRTArtificial Sequencesynthetic
polypeptide 67Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser
Ile Ser Ser Gly 20 25 30Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly
Lys Gly Leu Glu Trp 35 40 45Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr
Asn Tyr Asn Pro Ala Leu 50 55 60Arg Ser Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe Ser65 70 75 80Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95Ala Arg Ile Tyr Asn Gly
Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135
140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230 235 240Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250
255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375
380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445681344DNAArtificial
Sequencesynthetic polynucleotide 68caggtgcagc tgcaggagag cggccccggc
ctgatcaagc ccagccagac cctgagcctg 60acctgcaccg tgagcggcgg cagcatcagc
agcggctaca actggcactg gatccggcag 120ccccccggca agggcctgga
gtggatcggc tacatccact acaccggcag caccaactac 180aaccccgccc
tgcggagccg ggtgaccatc agcgtggaca ccagcaagaa ccagttcagc
240ctgaagctga gcagcgtgac cgccgccgac accgccatct actactgcgc
ccggatctac 300aacggcaaca gcttccccta ctggggccag ggcaccaccg
tgaccgtgag cagcgctagc 360accaaaggcc catcggtctt ccccctggca
ccctcctcca agagcacctc tgggggcaca 420gcggccctgg gctgcctggt
caaggactac ttccccgaac cggtgacggt gtcgtggaac 480tcaggcgccc
tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc
540tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcaccca
gacctacatc 600tgcaacgtga atcacaagcc cagcaacacc aaggtggaca
agaaagttga gcccaaatct 660tgtgacaaaa ctcacacatg cccaccgtgc
ccagcacctg aactcctggg gggaccgtca 720gtcttcctct tccccccaaa
acccaaggac accctcatga tctcccggac ccctgaggtc 780acatgcgtgg
tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg
840gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta
caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctg caccaggact
ggctgaatgg caaggagtac 960aagtgcaagg tctccaacaa agccctccca
gcccccatcg agaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc
acaggtgtac accctgcccc catcccggga tgagctgacc 1080aagaaccagg
tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg
1140gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc
cgtgctggac 1200tccgacggct ccttcttcct ctatagcaag ctcaccgtgg
acaagagcag gtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat
gaggctctgc acaaccacta cacgcagaag 1320agcctctccc tgtctccggg taaa
13446921PRTArtificial Sequencesynthetic polypeptide 69Met Ala Leu
Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala
Ala Arg Pro 207063DNAArtificial Sequencesynthetic polynucleotide
70atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg
60ccg 637145PRTArtificial Sequencesynthetic polypeptide 71Thr Thr
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala1 5 10 15Ser
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25
30Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40
4572135DNAArtificial Sequencesynthetic polynucleotide 72accacgacgc
cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60tccctgcgcc
cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg
120gacttcgcct gtgat 1357327PRTArtificial Sequencesynthetic
polypeptide 73Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
Tyr Ser Leu1 5 10 15Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20
257481DNAArtificial Sequencesynthetic polynucleotide 74ttttgggtgc
tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60gcctttatta
ttttctgggt g 817541PRTArtificial Sequencesynthetic polypeptide
75Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr1
5 10 15Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
Pro 20 25 30Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35
4076123DNAArtificial Sequencesynthetic polynucleotide 76aggagtaaga
ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60gggccaaccc
gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120tcc
12377113PRTArtificial Sequencesynthetic polypeptide 77Arg Val Lys
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40
45Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
Glu65 70 75 80Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
Leu Ser Thr 85 90 95Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro 100 105 110Arg78339DNAArtificial Sequencesynthetic
polynucleotide 78agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc
agcagggcca gaaccagctc 60tataacgagc tcaatctagg acgaagagag gagtacgatg
ttttggacaa gagacgtggc 120cgggaccctg agatgggggg aaagccgcag
agaaggaaga accctcagga aggcctgtac 180aatgaactgc agaaagataa
gatggcggag gcctacagtg agattgggat gaaaggcgag 240cgccggaggg
gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac
300acctacgacg cccttcacat gcaggccctg ccccctcgc 3397921PRTArtificial
Sequencesynthetic polypeptide 79Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr
208063DNAArtificial Sequencesynthetic polynucleotide 80atctacatct
gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60acc
638142PRTArtificial Sequencesynthetic polypeptide 81Lys Arg Gly Arg
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu
Glu Glu Glu Gly Gly Cys Glu Leu 35 4082126DNAArtificial
Sequencesynthetic polynucleotide 82aaacggggca
gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag
aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg
1268317PRTArtificial Sequencesynthetic polypeptide 83Tyr Ile Asp
Pro Ser Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10
15Asp8417PRTArtificial Sequencesynthetic polypeptide 84Tyr Ile Asn
Pro Ala Ser Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys1 5 10
15Asp8516PRTArtificial Sequencesynthetic polypeptide 85Tyr Ile His
Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu Arg Ser1 5 10
1586220PRTArtificial Sequencesynthetic polypeptide 86Asp Ile Val
Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly1 5 10 15Glu Lys
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser 20 25 30Gly
Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40
45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr
Cys Gln Asn 85 90 95Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr
Lys Leu Glu Leu 100 105 110Lys Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln Ser Gly
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170 175Ser
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 180 185
190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
22087448PRTArtificial Sequencesynthetic polypeptide 87Gln Ile Gln
Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu1 5 10 15Thr Val
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30Gly
Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40
45Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe
50 55 60Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala
Tyr65 70 75 80Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr
Tyr Phe Cys 85 90 95Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp
Gly Gln Gly Thr 100 105 110Ser Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310
315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425
430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 44588220PRTArtificial Sequencesynthetic polypeptide 88Asp
Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly1 5 10
15Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly
Gln 35 40 45Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val 50 55 60Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr65 70 75 80Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val
Tyr Tyr Cys Gln Asn 85 90 95Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile 100 105 110Lys Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp 115 120 125Glu Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140Phe Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu145 150 155 160Gln
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 165 170
175Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
180 185 190Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser 195 200 205Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 22089448PRTArtificial Sequencesynthetic polypeptide 89Gln
Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala1 5 10
15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln
Lys Phe 50 55 60Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser
Ala Val Tyr Tyr Cys 85 90 95Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110Thr Leu Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170
175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295
300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410
415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys 435 440 445903075DNAArtificial Sequencesynthetic
polynucleotide 90atggccttac cagtgaccgc cttgctcctg ccgctggcct
tgctgctcca cgccgccagg 60ccgcaggtgc agctgcagga gagcggcccc ggcctggtga
agcccagcca gaccctgagc 120ctgacctgca ccgtgagcgg cggcagcatc
agcagcggct acaactggca ctggatccgg 180cagccccccg gcaagggcct
ggagtggatc ggctacatcc actacaccgg cagcaccaac 240tacaaccccg
ccctgcggag ccgggtgacc atcagcgtgg acaccagcaa gaaccagttc
300agcctgaagc tgagcagcgt gaccgccgcc gacaccgccg tgtactactg
cgcccggatc 360tacaacggca acagcttccc ctactggggc cagggcacca
ccgtgaccgt gagcagcggt 420ggaggcggtt caggcggagg tggttctggc
ggtggcggat cggacatcgt gatgacccag 480agccccgaca gcctggccgt
gagcctgggc gagcgggcca ccatcaactg caagagcagc 540cagagcctgt
tcaacagcgg caaccagaag aactacctga cctggtacca gcagaagccc
600ggccagcccc ccaagctgct gatctactgg gccagcaccc gggagagcgg
cgtgcccgac 660cggttcagcg gcagcggcag cggcaccgac ttcaccctga
ccatcagcag cctgcaggcc 720gaggacgtgg ccgtgtacta ctgccagaac
gcctacagct tcccctacac cttcggcggc 780ggcaccaagc tggagatcaa
gcggaccacg acgccagcgc cgcgaccacc aacaccggcg 840cccaccatcg
cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg
900ggcgcagtgc acacgagggg gctggacttc gcctgtgatt tttgggtgct
ggtggtggtt 960ggtggagtcc tggcttgcta tagcttgcta gtaacagtgg
cctttattat tttctgggtg 1020aggagtaaga ggagcaggct cctgcacagt
gactacatga acatgactcc ccgccgcccc 1080gggccaaccc gcaagcatta
ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140tccagagtga
agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag
1200ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga
caagagacgt 1260ggccgggacc ctgagatggg gggaaagccg cagagaagga
agaaccctca ggaaggcctg 1320tacaatgaac tgcagaaaga taagatggcg
gaggcctaca gtgagattgg gatgaaaggc 1380gagcgccgga ggggcaaggg
gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440gacacctacg
acgcccttca catgcaggcc ctgccccctc gctaggtcga caatcaacct
1500ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
tccttttacg 1560ctatgtggat acgctgcttt aatgcctttg tatcatgcta
ttgcttcccg tatggctttc 1620attttctcct ccttgtataa atcctggttg
ctgtctcttt atgaggagtt gtggcccgtt 1680gtcaggcaac gtggcgtggt
gtgcactgtg tttgctgacg caacccccac tggttggggc 1740attgccacca
cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
1800gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
gttgggcact 1860gacaattccg tggtgttgtc ggggaagctg acgtcctttc
catggctgct cgcctgtgtt 1920gccacctgga ttctgcgcgg gacgtccttc
tgctacgtcc cttcggccct caatccagcg 1980gaccttcctt cccgcggcct
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 2040cctcagacga
gtcggatctc cctttgggcc gcctccccgc ctggaattcg ctagcctcga
2100gctcacacaa aaaaccaaca cacagatgta atgaaaataa agatatttta
ttgcggccgc 2160tttagtttcg gaggtaacct gtaagtctgt taatgaagta
aaagttcctt aggatttcca 2220ctctgactat ggtccaggca cagtgactgt
actccttggc cttcaggtaa tgcagaatcc 2280tcccataata tcttttcagg
tgcagactgc tcatgagttt tcccctggtg aaatcttctt 2340tctccagttt
ttcttccagg actgtcttca gatggtttat ctgatgatag acattagcca
2400ggaggttctc aacaatagtc tcattccagc cagtgctaga tgaatcttgt
ctgaaaatag 2460caaagatgtt ctggagcatc tcatagatgg tcaatgcggc
gtcctccttc tggaactgct 2520gcagctgctt aatctcctca gggatgtcaa
agttcatcct gtccttgagg cagtattcaa 2580gcctcccatt caattgccac
aggagcttct gacactgaaa attgctgctt ctttgtagga 2640atccaagcaa
gttgtagctc atggaaagag ctgtagtgga gaagcacaac aggagagcaa
2700tttggaggag acacttgttg gtcatggtgg cgaccggtag cgctaggtca
tatgcaggag 2760ttgaggttac tgtgagtagt gattaaagag agtgataggg
aactcttgaa caagagatgc 2820aatttatact gttaattctg gaaaaatatt
atgggggtgt caaaatgtcc cgggacaatt 2880gacgccttct gtatgaaaca
gtttttcctc cacgccttct gtatgaaaca gtttttcctc 2940cacgccttct
gtatgaaaca gtttttcctc cgtcgaggac aattgacgcc ttctgtatga
3000aacagttttt cctccacgcc ttctgtatga aacagttttt cctccacgcc
ttctgtatga 3060aacagttttt cctcc 3075913075DNAArtificial
Sequencesynthetic polynucleotide 91atggccttac cagtgaccgc cttgctcctg
ccgctggcct tgctgctcca cgccgccagg 60ccgcaggtgc agctgcagga gagcggcccc
ggcctgatca agcccagcca gaccctgagc 120ctgacctgca ccgtgagcgg
cggcagcatc agcagcggct acaactggca ctggatccgg 180cagccccccg
gcaagggcct ggagtggatc ggctacatcc actacaccgg cagcaccaac
240tacaaccccg ccctgcggag ccgggtgacc atcagcgtgg acaccagcaa
gaaccagttc 300agcctgaagc tgagcagcgt gaccgccgcc gacaccgcca
tctactactg cgcccggatc 360tacaacggca acagcttccc ctactggggc
cagggcacca ccgtgaccgt gagcagcggt 420ggaggcggtt caggcggagg
tggttctggc ggtggcggat cggacatcgt gatgacccag 480agccccgaca
gcctggccgt gagcctgggc gagcgggcca ccatcaactg caagagcagc
540cagagcctgt tcaacagcgg caaccagaag aactacctga cctggtacca
gcagaagccc 600ggccagcccc ccaagctgct gatctactgg gccagcaccc
gggagagcgg cgtgcccgac 660cggttcagcg gcagcggcag cggcaccgac
ttcaccctga ccatcagcag cctgcaggcc 720gaggacgtgg ccgtgtacta
ctgccagaac gcctacagct tcccctacac cttcggcggc 780ggcaccaagc
tggagatcaa gcggaccacg acgccagcgc cgcgaccacc aacaccggcg
840cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc
agcggcgggg 900ggcgcagtgc acacgagggg gctggacttc gcctgtgatt
tttgggtgct ggtggtggtt 960ggtggagtcc tggcttgcta tagcttgcta
gtaacagtgg cctttattat tttctgggtg 1020aggagtaaga ggagcaggct
cctgcacagt gactacatga acatgactcc ccgccgcccc 1080gggccaaccc
gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc
1140tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg
ccagaaccag 1200ctctataacg agctcaatct aggacgaaga gaggagtacg
atgttttgga caagagacgt 1260ggccgggacc ctgagatggg gggaaagccg
cagagaagga agaaccctca ggaaggcctg 1320tacaatgaac tgcagaaaga
taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380gagcgccgga
ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag
1440gacacctacg acgcccttca catgcaggcc ctgccccctc gctaggtcga
caatcaacct 1500ctggattaca aaatttgtga aagattgact ggtattctta
actatgttgc tccttttacg 1560ctatgtggat acgctgcttt aatgcctttg
tatcatgcta ttgcttcccg tatggctttc 1620attttctcct ccttgtataa
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 1680gtcaggcaac
gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
1740attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
tattgccacg 1800gcggaactca tcgccgcctg ccttgcccgc tgctggacag
gggctcggct gttgggcact 1860gacaattccg tggtgttgtc ggggaagctg
acgtcctttc catggctgct cgcctgtgtt 1920gccacctgga ttctgcgcgg
gacgtccttc tgctacgtcc cttcggccct caatccagcg 1980gaccttcctt
cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
2040cctcagacga gtcggatctc cctttgggcc gcctccccgc ctggaattcg
ctagcctcga 2100gctcacacaa aaaaccaaca cacagatgta atgaaaataa
agatatttta ttgcggccgc 2160tttagtttcg gaggtaacct gtaagtctgt
taatgaagta aaagttcctt aggatttcca 2220ctctgactat ggtccaggca
cagtgactgt actccttggc cttcaggtaa tgcagaatcc 2280tcccataata
tcttttcagg tgcagactgc tcatgagttt tcccctggtg aaatcttctt
2340tctccagttt ttcttccagg actgtcttca gatggtttat ctgatgatag
acattagcca 2400ggaggttctc aacaatagtc tcattccagc cagtgctaga
tgaatcttgt ctgaaaatag 2460caaagatgtt ctggagcatc tcatagatgg
tcaatgcggc gtcctccttc tggaactgct 2520gcagctgctt aatctcctca
gggatgtcaa agttcatcct gtccttgagg cagtattcaa 2580gcctcccatt
caattgccac aggagcttct gacactgaaa attgctgctt ctttgtagga
2640atccaagcaa gttgtagctc atggaaagag ctgtagtgga gaagcacaac
aggagagcaa 2700tttggaggag acacttgttg gtcatggtgg cgaccggtag
cgctaggtca tatgcaggag 2760ttgaggttac tgtgagtagt gattaaagag
agtgataggg aactcttgaa caagagatgc 2820aatttatact gttaattctg
gaaaaatatt atgggggtgt caaaatgtcc cgggacaatt 2880gacgccttct
gtatgaaaca gtttttcctc cacgccttct gtatgaaaca gtttttcctc
2940cacgccttct gtatgaaaca gtttttcctc cgtcgaggac aattgacgcc
ttctgtatga 3000aacagttttt cctccacgcc ttctgtatga aacagttttt
cctccacgcc ttctgtatga 3060aacagttttt cctcc 307592187PRTHomo sapiens
92Met Thr Asn Lys Cys Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser1
5 10 15Thr Thr Ala Leu Ser Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln
Arg 20 25 30Ser Ser Asn Phe Gln Cys Gln Lys Leu Leu Trp Gln Leu Asn
Gly Arg 35 40 45Leu Glu Tyr Cys Leu Lys Asp Arg Met Asn Phe Asp Ile
Pro Glu Glu 50 55 60Ile Lys Gln Leu Gln Gln Phe Gln Lys Glu Asp Ala
Ala Leu Thr Ile65 70 75 80Tyr Glu Met Leu Gln Asn Ile Phe Ala Ile
Phe Arg Gln Asp Ser Ser 85 90 95Ser Thr Gly Trp Asn Glu Thr Ile Val
Glu Asn Leu Leu Ala Asn Val 100 105 110Tyr His Gln Ile Asn His Leu
Lys Thr Val Leu Glu Glu Lys Leu Glu 115 120 125Lys Glu Asp Phe Thr
Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys 130 135 140Arg Tyr Tyr
Gly Arg Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser145 150 155
160His Cys Ala Trp Thr Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr
165 170 175Phe Ile Asn Arg Leu Thr Gly Tyr Leu Arg Asn 180
1859315PRTArtificial Sequencesynthetic polypeptide 93Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10
1594194DNAArtificial Sequencesynthetic polynucleotide 94acgccttctg
tatgaaacag tttttcctcc acgccttctg tatgaaacag tttttcctcc 60acgccttctg
tatgaaacag tttttcctcc gtcgaggaca attgacgcct tctgtatgaa
120acagtttttc ctccacgcct tctgtatgaa acagtttttc ctccacgcct
tctgtatgaa 180acagtttttc ctcc 194
* * * * *