U.S. patent application number 17/182956 was filed with the patent office on 2022-01-20 for tumor-specific anti-egfr antibody and application thereof.
The applicant listed for this patent is CAFA THERAPEUTICS LIMITED. Invention is credited to Bo SONG, Huamao WANG.
Application Number | 20220017625 17/182956 |
Document ID | / |
Family ID | 1000005872075 |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220017625 |
Kind Code |
A1 |
WANG; Huamao ; et
al. |
January 20, 2022 |
TUMOR-SPECIFIC ANTI-EGFR ANTIBODY AND APPLICATION THEREOF
Abstract
The present invention provides a tumor-specific anti-EGFR
antibody and application thereof. The antibody can be used for
preparing targeted antitumor drugs and tumor diagnosis drugs.
Inventors: |
WANG; Huamao; (Shanghai,
CN) ; SONG; Bo; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CAFA THERAPEUTICS LIMITED |
DUBLIN |
|
IE |
|
|
Family ID: |
1000005872075 |
Appl. No.: |
17/182956 |
Filed: |
February 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15746711 |
Jan 22, 2018 |
10927176 |
|
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PCT/CN2016/090892 |
Jul 21, 2016 |
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17182956 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70517 20130101;
C07K 16/30 20130101; C07K 16/468 20130101; A61K 2039/572 20130101;
C07K 2317/565 20130101; C12N 2320/32 20130101; C07K 16/2809
20130101; A61P 35/00 20180101; C07K 2317/569 20130101; A61K 39/395
20130101; C12N 15/1138 20130101; C07K 14/70578 20130101; C07K
2319/02 20130101; C12N 2320/31 20130101; A61K 38/00 20130101; C07K
14/70521 20130101; C07K 14/54 20130101; C07K 16/46 20130101; C07K
2319/30 20130101; C07K 16/2863 20130101; C07K 2317/56 20130101;
C07K 2317/73 20130101; C12N 5/10 20130101; C07K 2319/33 20130101;
C12N 15/63 20130101; C07K 2317/92 20130101; C07K 14/5434 20130101;
C12N 15/62 20130101; C07K 19/00 20130101; C12N 2310/14 20130101;
C07K 14/7158 20130101; C07K 14/5443 20130101; C07K 2317/622
20130101; G01N 33/574 20130101; C07K 2317/31 20130101; C07K 2319/03
20130101; A61K 48/00 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/574 20060101 G01N033/574; C07K 16/30 20060101
C07K016/30; A61K 39/395 20060101 A61K039/395; C12N 15/63 20060101
C12N015/63; C12N 15/62 20060101 C12N015/62; C12N 5/10 20060101
C12N005/10; A61K 48/00 20060101 A61K048/00; C07K 19/00 20060101
C07K019/00; C07K 16/46 20060101 C07K016/46; A61P 35/00 20060101
A61P035/00; C07K 14/54 20060101 C07K014/54; C07K 14/705 20060101
C07K014/705; C07K 14/715 20060101 C07K014/715; C12N 15/113 20060101
C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 2015 |
CN |
201510431481.6 |
Claims
1-36. (canceled)
37. A multi-functional immunoconjugate, comprising: an antibody;
and a functional molecule linked thereto; and the functional
molecule is selected from a group consisting of a molecule that
targets a tumor surface marker, a tumor-suppressing molecule, a
molecule that targets a surface marker of an immune cell, a
detectable label; wherein the antibody specifically recognizing
EGFRvIII expressed or EGFR overexpressed by tumor cells, wherein
the antibody comprises a light chain variable region and a heavy
chain variable region, and CDR1 of the light chain variable region
has an amino acid sequence selected from a group consisting of SEQ
ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55; CDR2 of the light chain
variable region has an amino acid sequence selected from a group
consisting of SEQ ID NO: 42, SEQ ID NO: 53; CDR3 of the light chain
variable region has an amino acid sequence selected from a group
consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 57; CDR1 of the heavy chain variable region has
the amino acid sequence of SEQ ID NO: 44; CDR2 of the heavy chain
variable region has an amino acid sequence selected from a group
consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID
NO: 52; CDR3 of the heavy chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID
NO: 50.
38. The multi-functional immunoconjugate of claim 37, wherein the
molecule that targets the tumor surface marker is an antibody or
ligand that binds to a tumor surface marker; or the
tumor-suppressing molecule is an anti-tumor cytokine or an
anti-tumor toxin; preferably, the cytokines include but are not
limited to: IL-12, IL-15, IFN-beta, TNF-alpha.
39. The multi-functional immunoconjugate of claim 37, wherein the
detectable label includes a fluorescent label and a chromogenic
label.
40. The multi-functional immunoconjugate of claim 37, wherein the
molecule targeting the surface marker of the immune cell is an
antibody or ligand that binds to an immune cell surface marker;
preferably, the immune cell surface markers include: CD3, CD16,
CD28.
41. The multi-functional immunoconjugate of claim 40, wherein the
molecule that targets the surface marker of the immune cell is an
antibody that binds to a T cell surface marker, which forms a
T-cell-engaging bifunctional antibody with the following antibody:
an antibody specifically recognizing EGFRvIII expressed or EGFR
overexpressed by tumor cells, wherein the antibody comprises a
light chain variable region and a heavy chain variable region, and
CDR1 of the light chain variable region has an amino acid sequence
selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47,
SEQ ID NO: 55; CDR2 of the light chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 42,
SEQ ID NO: 53; CDR3 of the light chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 43,
SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57; CDR1 of
the heavy chain variable region has the amino acid sequence of SEQ
ID NO: 44; CDR2 of the heavy chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 45,
SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52; CDR3 of the heavy
chain variable region has an amino acid sequence selected from a
group consisting of SEQ ID NO: 46, SEQ ID NO: 50.
42. The multi-functional immunoconjugate of claim 41, wherein the
antibody that binds to the immune cell surface marker is an
anti-CD3 antibody.
43. The multi-functional immunoconjugate of claim 37, wherein the
multi-functional immunoconjugate is a fusion polypeptide, and
further comprises a linker peptide between the following antibody
and the functional molecule linked thereto: an antibody
specifically recognizing EGFRvIII expressed or EGFR overexpressed
by tumor cells, wherein the antibody comprises a light chain
variable region and a heavy chain variable region, and CDR1 of the
light chain variable region has an amino acid sequence selected
from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO:
55; CDR2 of the light chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID
NO: 53; CDR3 of the light chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID
NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57; CDR1 of the
heavy chain variable region has the amino acid sequence of SEQ ID
NO: 44; CDR2 of the heavy chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID
NO: 49, SEQ ID NO: 51, SEQ ID NO: 52; CDR3 of the heavy chain
variable region has an amino acid sequence selected from a group
consisting of SEQ ID NO: 46, SEQ ID NO: 50.
44. A chimeric antigen receptor expressed on the surface of an
immune cell comprising an antibody, wherein the chimeric antigen
receptor comprises: the antibody, a transmembrane region and an
intracellular signal region, which are sequentially linked; wherein
the antibody specifically recognizing EGFRvIII expressed or EGFR
overexpressed by tumor cells, wherein the antibody comprises a
light chain variable region and a heavy chain variable region, and
CDR1 of the light chain variable region has an amino acid sequence
selected from a group consisting of SEQ ID NO: 41, SEQ ID NO: 47,
SEQ ID NO: 55; CDR2 of the light chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 42,
SEQ ID NO: 53; CDR3 of the light chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 43,
SEQ ID NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57; CDR1 of
the heavy chain variable region has the amino acid sequence of SEQ
ID NO: 44; CDR2 of the heavy chain variable region has an amino
acid sequence selected from a group consisting of SEQ ID NO: 45,
SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52; CDR3 of the heavy
chain variable region has an amino acid sequence selected from a
group consisting of SEQ ID NO: 46, SEQ ID NO: 50.
45. The chimeric antigen receptor of claim 44, wherein the
intracellular signal region is selected from a group consisting of
intracellular signal region sequences of CD3.zeta.,
Fc.epsilon.RI.gamma., CD27, CD28, CD137 and CD134, or a combination
thereof.
46. The chimeric antigen receptor of claim 45, wherein the
transmembrane region comprises a transmembrane region of CD8 or
CD28.
47. The chimeric antigen receptor of claim 45, wherein the immune
cells include T lymphocyte, NK cell or NKT cell.
48. The chimeric antigen receptor of claim 45, wherein the chimeric
antigen receptor comprises an antibody, transmembrane region and
intracellular signal region which are sequentially linked as
follows: Antibody, CD8 and CD3.zeta.; Antibody, CD8, CD137 and
CD3.zeta.; Antibody, a transmembrane region of a CD28 molecule, an
intracellular signal region of a CD28 molecule, and CD3.zeta.; or
Antibody, a transmembrane region of a CD28 molecule, an
intracellular signal region of a CD28 molecule, CD137 and
CD3.zeta.; Wherein the antibody is an antibody specifically
recognizing EGFRvIII expressed or EGFR overexpressed by tumor
cells, wherein the antibody comprises a light chain variable region
and a heavy chain variable region, and CDR1 of the light chain
variable region has an amino acid sequence selected from a group
consisting of SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55; CDR2 of
the light chain variable region has an amino acid sequence selected
from a group consisting of SEQ ID NO: 42, SEQ ID NO: 53; CDR3 of
the light chain variable region has an amino acid sequence selected
from a group consisting of SEQ ID NO: 43, SEQ ID NO: 48, SEQ ID NO:
54, SEQ ID NO: 56, SEQ ID NO: 57; CDR1 of the heavy chain variable
region has the amino acid sequence of SEQ ID NO: 44; CDR2 of the
heavy chain variable region has an amino acid sequence selected
from a group consisting of SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO:
51, SEQ ID NO: 52; CDR3 of the heavy chain variable region has an
amino acid sequence selected from a group consisting of SEQ ID NO:
46, SEQ ID NO: 50.
49. The chimeric antigen receptor of claim 45, wherein the antibody
is a single chain antibody or a domain antibody.
50. The chimeric antigen receptor of claim 45, wherein the chimeric
antigen receptor comprises: SEQ ID NO: 36 or the amino acid
sequence shown in positions 285-601; or SEQ ID NO: 37 or the amino
acid sequence shown in positions 285-702; or SEQ ID NO: 38 or the
amino acid sequence shown in positions 285-744; or SEQ ID NO: 39 or
the amino acid sequence shown in positions 285-749; or SEQ ID NO:
40 or the amino acid sequence shown in positions 285-791.
51. A genetically modified immune cell, wherein it is transduced
with a nucleic acid encoding the chimeric antigen receptor of claim
44, or an expression vector comprising the nucleic acid or a virus
comprising the vector; or it expresses the chimeric antigen
receptor of claim 8 at its surface.
52. The immune cell of claim 51, wherein it further carries an
exogenous encoding sequence for cytokine; preferably, the cytokine
includes: IL-12, IL-15 or IL-21
53. The immune cell of claim 51, wherein it further expresses
another chimeric antigen receptor that does not contain CD3.zeta.,
but contains the intracellular signaling domain of CD28, the
intracellular signaling domain of CD137, or a combination of
both.
54. The immune cell of claim 51, wherein it further expresses a
chemokine receptor; preferably, the chemokine receptor includes
CCR2.
55. The immune cell of claim 51, wherein it further expresses siRNA
that can reduce PD-1 expression or a protein that can block
PD-L1.
56. The immune cell of claim 51, wherein it further expresses a
safety switch; preferably, the safety switch includes iCaspase-9,
Truancated EGFR or RQR8.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of immunology,
and in particular, the present invention relates to tumor-specific
anti-EGFR antibodies and uses thereof.
BACKGROUND
[0002] EGFR is overexpressed or mutated in many tumors, and it is
undoubtedly a very important scientific issue on how to selectively
recognize these over-expressed or mutated EGFR. Until now,
antibodies against EGFR287-302 epitope are believed to achieve the
purpose of recognizing EGFR, EGFRvIII and de4 EGFR overexpressed on
the surface of tumors, instead of EGFR in normal cells.
Unfortunately, antibodies against this epitope still have side
effects such as rashes in clinical trials
(http://meetinglibrary.asco.org/content/115945-132), suggesting
that targeting this epitope may identify EGFR in normal cells (such
as keratinocytes).
[0003] Therefore, it is very urgent to screen anti-EGFR antibodies
with higher tumor-specificity. Highly tumor-specific antibodies,
whether for tumor imaging diagnosis, individual diagnosis or tumor
targeting therapy, have a very large potential value.
SUMMARY OF THE INVENTION
[0004] The object of the present invention is to provide
tumor-specific anti-EGFR antibodies and uses thereof.
[0005] In a first aspect of the present invention, an antibody
specifically recognizing EGFRvIII expressed or EGFR overexpressed
by tumor cells is provided, wherein the antibody comprises a light
chain variable region and a heavy chain variable region,
[0006] CDR1 of the light chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 41, SEQ ID
NO: 47, SEQ ID NO: 55;
[0007] CDR2 of the light chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 42, SEQ ID
NO: 53;
[0008] CDR3 of the light chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 43, SEQ ID
NO: 48, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57;
[0009] CDR1 of the heavy chain variable region has the amino acid
sequence of SEQ ID NO: 44;
[0010] CDR2 of the heavy chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 45, SEQ ID
NO: 49, SEQ ID NO: 51, SEQ ID NO: 52;
[0011] CDR3 of the heavy chain variable region has an amino acid
sequence selected from a group consisting of SEQ ID NO: 46, SEQ ID
NO: 50.
[0012] In a preferred embodiment, the antibody includes:
[0013] antibody (a), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 43, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 45, CDR3 of SEQ ID NO: 46,
[0014] antibody (b), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 47, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 48, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 49, CDR3 of SEQ ID NO: 50,
[0015] antibody (c), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 48, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 51, CDR3 of SEQ ID NO: 50,
[0016] antibody (d), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 43, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 52, CDR3 of SEQ ID NO: 50,
[0017] antibody (e), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 43, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 45, CDR3 of SEQ ID NO: 50,
[0018] antibody (f), wherein light chain variable region thereof
has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 53, CDR3 of SEQ ID
NO: 54, or the heavy chain variable region thereof has CDR1 of SEQ
ID NO: 44, CDR2 of SEQ ID NO: 51, CDR3 of SEQ ID NO: 50,
[0019] antibody (g), wherein the light chain variable region has
CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of SEQ ID NO:
54, or the heavy chain variable region thereof has CDR1 of SEQ ID
NO: 44, CDR2 of SEQ ID NO: 51, CDR3 of SEQ ID NO: 50,
[0020] antibody (h), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 55, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 56, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 45, CDR3 of SEQ ID NO: 50,
[0021] antibody (i), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 53, CDR3 of
SEQ ID NO: 56, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 52, CDR3 of SEQ ID NO: 50,
[0022] antibody (j), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 56, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 52, CDR3 of SEQ ID NO: 50,
[0023] antibody (k), wherein the light chain variable region
thereof has CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, CDR3 of
SEQ ID NO: 57, or the heavy chain variable region thereof has CDR1
of SEQ ID NO: 44, CDR2 of SEQ ID NO: 52, CDR3 of SEQ ID NO: 50;
or
[0024] antibody (l) which recognizes the same antigenic determinant
as that recognized by the antibody according to any one of (a) to
(k).
[0025] In another preferred embodiment, the antibody specifically
recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells
can be: single chain antibody (scFV), monoclonal antibody, domain
antibody, Fab fragment, Fd fragment, Fv fragment, F (ab').sub.2
fragment and a derivative thereof, or other forms of antibody;
preferably single chain antibody.
[0026] In another preferred embodiment, the antibody specifically
recognizing EGFRvIII expressed or EGFR overexpressed by tumor cells
is humanized, fully humanized, chimeric or murine.
[0027] In another preferred embodiment, the amino acid sequence of
the heavy chain variable region of the antibody is shown in
positions 124 to 239 of SEQ ID NO: 13; or the amino acid sequence
of the light chain variable region of the antibody is shown in
positions 1-108 of SEQ ID NO: 13;
[0028] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 59;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 59;
[0029] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 61;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 61;
[0030] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 63;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 63;
[0031] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 65;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 65;
[0032] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 67;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 67;
[0033] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 69;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 69;
[0034] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 71;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 71;
[0035] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 73;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 73;
[0036] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 75;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 75; or
[0037] The amino acid sequence of the heavy chain variable region
of the antibody is shown in positions 124 to 239 of SEQ ID NO: 77;
or the amino acid sequence of the light chain variable region of
the antibody is shown in positions 1-108 of SEQ ID NO: 77.
[0038] In another preferred embodiment, the antibody is antibody
(a); more preferably, the amino acid sequence of the heavy chain
variable region of the antibody is shown in positions 124 to 239 of
SEQ ID NO: 13; or the amino acid sequence of the light chain
variable region of the antibody is shown in positions 1-108 of SEQ
ID NO: 13.
[0039] In another aspect of the invention, a nucleic acid encoding
the antibody as described above is provided.
[0040] In another aspect of the present invention, an expression
vector is provided, comprising the nucleic acid. In another
preferred embodiment, the expression vector is a PH/DHFR
vector.
[0041] In another aspect of the present invention, a host cell is
provided, comprising the expression vector or having the nucleic
acid integrated into its genome. In another preferred embodiment,
the host cell is a eukaryotic host cell or prokaryotic host cell;
preferably a eukaryotic host cell, more preferably Chinese hamster
ovary cell (CHO).
[0042] In another aspect of the invention, the use of any one of
the above described antibodies is provided for the manufacture of a
targeting drug, antibody drug conjugate, or multi-functional
antibody specifically targeting tumor cells expressing EGFRvIIIed
or over-expressing EGFR; or an agent for diagnosis of tumors that
expressed EGFRvIII or overexpress EGFR; or is used to prepare a
chimeric antigen receptor modified immune cell; preferably, the
immune cell includes: T lymphocyte, NK cell or NKT lymphocyte.
[0043] In another aspect of the present invention, a
multi-functional immunoconjugate is provided, comprising: any one
of the above described antibodies; and a functional molecule linked
thereto (including covalently linked, conjugated, attached,
adsorbed); the functional molecule is selected from a group
consisting of a molecule that targets a tumor surface marker, a
tumor-suppressing molecule, a molecule that targets a surface
marker of an immune cell, or a detectable label.
[0044] In a preferred embodiment, the molecule that targets the
tumor surface marker is an antibody or ligand that binds to a tumor
surface marker; or the tumor-suppressing molecule is an anti-tumor
cytokine or an anti-tumor toxin; preferably, the cytokines include
but are not limited to: IL-12, IL-15, IFN-beta, TNF-alpha.
[0045] In another preferred embodiment, in the multi-functional
immunoconjugate, the detectable label includes a fluorescent label
and a chromogenic label.
[0046] In another preferred embodiment, in the multi-functional
immunoconjugate, the molecule targeting the surface marker of the
immune cell is an antibody or ligand that binds to an immune cell
surface marker; preferably, the immune cell surface markers
include, but are not limited to: CD3, CD16, CD28.
[0047] In another preferred embodiment, in the multi-functional
immunoconjugate, the molecule that targets the surface marker of
the immune cell is an antibody that binds to a T cell surface
marker, which can form a T-cell-engaging bifunctional antibody with
any one of the above described antibody (bispecific T cell engager,
BiTE).
[0048] In another preferred embodiment, in the multi-functional
immunoconjugate, the antibody that binds to the immune cell surface
marker is an anti-CD3 antibody. In another preferred embodiment,
the anti-CD3 antibody is a single chain antibody (scFV), a
monoclonal antibody, a Fab fragment, an Fd fragment, an Fv
fragment, an F(ab').sub.2 fragment and a derivative thereof,
antibody; preferably single chain antibody. In another preferred
embodiment, the anti-CD3 antibody is humanized, fully human,
chimeric or murine.
[0049] In another preferred embodiment, the multi-functional
immunoconjugate is a fusion polypeptide, and further comprises a
linker peptide (linker) between any one of the above described
antibodies and the functional molecule linked thereto.
[0050] In another preferred embodiment, the linker peptide has the
sequence (GlyGlyGlyGlySer)n, wherein n is an integer from 1 to 5;
more preferably, n=3.
[0051] In another preferred embodiment, the multi-functional
immunoconjugate is administered in a form of polypeptide or in the
manner of gene administration.
[0052] In another aspect of the invention, a nucleic acid encoding
the multi-functional immunoconjugate is provided.
[0053] In another aspect of the invention, the use of any one of
the above described multi-functional immunoconjugate is provided,
for the preparation of an antineoplastic agent or an agent for
diagnosis of tumors that express EGFRvIII or overexpress EGFR; or
for the preparation of chimeric antigen receptor modified immune
cells. Preferably, the immune cells include T lymphocyte, NK cell
or NKT lymphocyte.
[0054] In another aspect of the present invention, a chimeric
antigen receptor comprising any one of the above described
antibodies is provided, and the chimeric antigen receptor is
expressed on the surface of an immune cell and comprises: any one
of the above described antibodies, a transmembrane region and an
intracellular signal region, which are sequentially linked; and the
intracellular signal region is selected from a group consisting of
intracellular signal region sequences of CD3.zeta.,
Fc.epsilon.RI.gamma., CD27, CD28, CD137 and CD134, or a combination
thereof.
[0055] In a preferred embodiment, the transmembrane region
comprises a transmembrane region of CD8 or CD28.
[0056] In another preferred embodiment, the immune cells include T
lymphocyte, NK cell or NKT cell.
[0057] In another preferred embodiment, the chimeric antigen
receptor comprises the following sequentially linked antibody,
transmembrane region and intracellular signal region:
[0058] Any one of the above described antibodies, CD8 and
CD3.zeta.;
[0059] Any one of the above described antibodies, CD8, CD137 and
CD3.zeta.;
[0060] Any one of the above described antibodies, a transmembrane
region of a CD28 molecule, an intracellular signal region of a CD28
molecule, and CD3.zeta.; or
[0061] Any one of the above described antibodies, a transmembrane
region of a CD28 molecule, an intracellular signal region of a CD28
molecule, CD137 and CD3.zeta..
[0062] In another preferred embodiment, the antibody is a single
chain antibody or a domain antibody.
[0063] In another preferred embodiment, the chimeric antigen
receptor comprises:
[0064] SEQ ID NO: 36 or the amino acid sequence shown in positions
285-601; or
[0065] SEQ ID NO: 37 or the amino acid sequence shown in positions
285-702; or
[0066] SEQ ID NO: 38 or the amino acid sequence shown in positions
285-744; or
[0067] SEQ ID NO: 39 or the amino acid sequence shown in positions
285-749; or
[0068] SEQ ID NO: 40 or the amino acid sequence shown in positions
285-791.
[0069] In another aspect of the invention, a nucleic acid encoding
any one of the above described chimeric antigen receptors is
provided. In another preferred embodiment, the nucleic acid
encoding the chimeric antigen receptor comprises:
[0070] SEQ ID NO: 31 or the nucleotide sequence shown in positions
966-1916; or
[0071] SEQ ID NO: 32 or the nucleotide sequence shown in positions
966-2219; or
[0072] SEQ ID NO: 33 or the nucleotide sequence shown in positions
966-2345; or
[0073] SEQ ID NO: 34 or the nucleotide sequence shown in positions
966-2360; or
[0074] SEQ ID NO: 35 or the nucleotide sequences shown in positions
966-2486.
[0075] In another aspect of the present invention, an expression
vector comprising the above described nucleic acid is provided.
[0076] In another preferred embodiment, the expression vector is
derived from lentiviral plasmid pWPT (or pWPT-eGFP).
[0077] In another aspect of the present invention, a virus
comprising the above described vector is provided.
[0078] Use of any one of the above described chimeric antigen
receptors or an encoding nucleic acid thereof, or an expression
vector or virus comprising the nucleic acid is provided, for the
preparation of genetically modified immune cells that target tumor
cells that express EGFRvIII or overexpress EGFR.
[0079] In another aspect of the present invention, a genetically
modified immune cell is provided, which is transduced with the
nucleic acid, or the expression vector or the virus; or expresses
the chimeric antigen receptor at its surface.
[0080] In a preferred embodiment, the immune cell further carries
an exogenous encoding sequence for cytokine; preferably, the
cytokine includes: IL-12, IL-15 or IL-21.
[0081] In another preferred embodiment, the immune cell further
expresses another chimeric antigen receptor that does not contain
CD3.zeta., but contains the intracellular signaling domain of CD28,
the intracellular signaling domain of CD137, or a combination of
both.
[0082] In another preferred embodiment, the immune cell further
expresses a chemokine receptor; preferably, the chemokine receptor
includes CCR2.
[0083] In another preferred embodiment, the immune cell further
expresses siRNA that can reduce PD-1 expression or a protein that
can block PD-L1.
[0084] In another preferred embodiment, the immune cell further
expresses a safety switch; preferably, the safety switch includes
iCaspase-9, Truancated EGFR or RQR8.
[0085] In another aspect of the invention, the use of said
genetically modified immune cells is provided for the preparation
of a tumor-inhibiting drug, and said tumor is the tumor that
expresses EGFRvIII or overexpresses EGFR.
[0086] In another aspect of the invention, a pharmaceutical
composition (including medicament or diagnostic reagent) is
provided, comprising:
[0087] any one of the above described antibodies or a nucleic acid
encoding the antibody; or
[0088] any one of the above described immunoconjugates or a nucleic
acid encoding the conjugate; or
[0089] any one of the above described chimeric antigen receptors or
a nucleic acid encoding the chimeric antigen receptor; or
[0090] any one of the above described genetically modified immune
cells.
[0091] Other aspects of the invention will be apparent to a person
skilled in the art in view of the disclosure herein.
DESCRIPTION OF DRAWINGS
[0092] FIG. 1. Antibodies 7B3 and Y022 can specifically bind to
antigens EGFRvIII and N1N2-806 (phage ELISA assay).
[0093] FIG. 2. Binding curve of antibody 7B3 vs antigen
EGFRvIII.
[0094] FIG. 3. Binding curve of antibody Y022 vs antigen
EGFRvIII.
[0095] FIG. 4. Electrophorogram of purification of three scFv-Fc
fusion antibodies.
[0096] FIG. 5. Detection of single-chain antibodies scFv-Y022-Fc,
scFv-806-Fc and scFv-C225-Fc for their ability to bind to cell
surface EGFR by FACS.
[0097] FIG. 6. Structure diagram of pH-Y022/CD3 expression
vector.
[0098] FIG. 7. SDS-PAGE detection of single-chain bifunctional
antibodies Y022/CD3, 806/CD3 and C225/CD3.
[0099] FIG. 8. Detection of Y022/CD3 single chain bifunctional
antibody for its antigen binding specificity by FACS.
[0100] FIG. 9. Cytotoxicity plots of single-chain bifunctional
antibodies.
[0101] FIG. 10. Schematic illustration of the ligation order of
various parts of the chimeric antigen receptor.
MODES FOR CARRYING OUT THE INVENTION
[0102] After intensive research and screening, the present
inventors obtained an antibody that specifically recognizes
EGFRvIII or over-expressed EGFR in tumor cells and scarcely
recognizes EGFR in normal cells. The antibody of the present
invention can be used to prepare various targeting anti-tumor drugs
and drugs for diagnosis of tumors.
[0103] Anti-EGFR Antibody
[0104] The present inventors further conducted screening and amino
acid mutations based on the humanized antibodies obtained in the
previous stage, and found an anti-EGFR antibody capable of
targeting EGFR of tumor cells with higher specificity, which
selectively binds to a tumor overexpressing EGFR or EGFRvIII, while
does not bind to EGFR on normal cells.
[0105] Antibodies of the invention may be intact immunoglobulin
molecules or antigen-binding fragments, including but not limited
to Fab fragments, Fd fragments, Fv fragments, F (ab').sub.2
fragments, complementarity determining region (CDR) fragments,
single-chain antibody (scFv), domain antibody, bivalent single
chain antibody, single chain phage antibody, bispecific diabody,
triple chain antibody, quadruple chain antibody.
[0106] The antigen-binding properties of an antibody can be
described by three specific regions located in variable regions of
the heavy and light chains, termed complementarity determining
regions (CDRs), which divide the variable regions into four
framework regions (FR), and the amino acid sequences of four FRs
are relatively conservative, not directly involved in binding
reaction. These CDRs form a loop structure, in which j-folds formed
by the FRs are located close to each other in space and the antigen
binding site of the antibody is constituted by CDRs on the heavy
chain and CDRs on the corresponding light chain. It is possible to
determine which amino acids make up FR or CDR regions by comparing
the amino acid sequences of the same type of antibody. The CDR
regions are sequences of immunologically interesting proteins and
the CDR regions of the antibodies of the invention are brand new.
The antibody may comprise two, three, four, five, or all six of the
CDR regions disclosed herein.
[0107] Another aspect of the invention includes functional variants
of the antibodies described herein. If the variant is capable of
competing with the parental antibody for specific binding to SEQ ID
NO: 1 and its ability to recognize EGFRvIII or overexpressed EGFR
in tumor cells is close to that of the specific antibodies provided
in Examples of the present invention. The functional variants may
have conservative sequence modifications, including nucleotide and
amino acid substitutions, additions and deletions. These
modifications can be introduced by standard techniques known in the
art, such as directed mutagenesis and random PCR-mediated
mutagenesis, and can include both natural and non-natural
nucleotides and amino acids. Preferably, modification of the
sequence occurs on a region outside the CDR region of the
antibody.
[0108] Immunoconjugate
[0109] In the present invention, a multifunctional immunoconjugate
is also provided, comprising the antibodies described herein and
further comprising at least one functional molecule of other type.
The functional molecule is selected from, but not limited to, a
molecule that targets a tumor surface marker, a tumor-suppressing
molecule, a molecule that targets a surface marker of an immune
cell, or a detectable label. The antibody and the functional
molecule may form a conjugate by covalent attachment, coupling,
attachment, cross-linking, or the like.
[0110] As a preferred mode, the immunoconjugate may comprise an
antibody of the invention and at least one molecule that targets a
tumor surface marker or a tumor-suppressing molecule. The
tumor-suppressing molecule may be anti-tumor cytokines or
anti-tumor toxins. Preferably, the cytokines include but are not
limited to IL-12, IL-15, IFN-beta, TNF-alpha. The molecules that
target tumor surface markers, for example, can act synergistically
with the antibodies of the invention to more precisely target tumor
cells.
[0111] As a preferred mode, the immunoconjugate may comprise an
antibody of the present invention and a detectable label. Such
detectable labels include, but are not limited to, fluorescent
labels, chromogenic labels such as enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, radioactive materials, positron-emitting metals and
non-radioactive paramagnetic metal ion. More than one marker can
also be included. The label used to label the antibody for the
purpose of detection and/or analysis and/or diagnosis depends on
the used particular detection/analysis/diagnosis technique and/or
method, eg, immunohistochemical staining (tissue) samples, flow
cytometry, and the like. Suitable labels for
detection/analysis/diagnosis techniques and/or methods known in the
art are well known to those skilled in the art.
[0112] As a preferred mode, the immunoconjugate may comprise an
antibody of the invention as well as a molecule that targets a
surface marker of an immune cell. The molecule that targets surface
markers of immune cells can recognize immune cells and carry the
antibodies of the invention to the immune cells, so that the
antibodies of the invention can target the immune cells to the
tumor cells and thus trigger immunocyte for specifically killing
tumor.
[0113] As a means of chemically generating an immunoconjugate by
conjugation, either directly or indirectly (eg, by a linker), the
immunoconjugate can be produced as a fusion protein comprising an
antibody of the invention and other suitable proteins. The fusion
protein can be produced by a method known in the art, for example
recombinantly produced by constructing and subsequently expressing
the nucleic acid molecule which comprises the nucleotide sequence
encoding the antibody in frame with a nucleotide sequence encoding
a suitable label.
[0114] In another aspect of the invention, a nucleic acid molecule
encoding at least one antibody of the invention, a functional
variant, or an immunoconjugate thereof is provided. Once obtaining
the relevant sequence, the recombination method can be used to
obtain the relevant sequence in large quantities. This is usually
done by cloning it into a vector, transferring it to a cell, and
then isolating the relevant sequence from the proliferating host
cells by conventional methods.
[0115] The present invention also relates to vectors comprising the
appropriate DNA sequences described above as well as appropriate
promoters or control sequences. These vectors can be used to
transform an appropriate host cell to enable expression of the
protein. The host cell may be a prokaryotic cell, such as a
bacterial cell; or a lower eukaryotic cell, such as a yeast cell;
or a higher eukaryotic cell, such as a mammalian cell.
[0116] Chimeric Antigen Receptor and Genetically Modified Immune
Cell
[0117] In the present invention, a chimeric antigen receptor
expressed on the surface of an immune effector cell (immune cell)
is provided, wherein the chimeric antigen receptor comprises
sequentially linked: extracellular binding region, transmembrane
region and intracellular signal region, and the extracellular
binding region comprises the antibody of the invention. By
expressing the chimeric antigen receptor on the surface of immune
effector cells, immune effector cells can have a highly specific
cytotoxic effect on tumor cells that express EGFRvIII or
overexpress EGFR.
[0118] As used herein, "immune cells" and "immune effector cells"
are used interchangeably and include: T lymphocytes, NK cells or
NKT cells, and the like.
[0119] As a preferred embodiment of the present invention, the
antibody contained in the chimeric antigen receptor is a single
chain antibody, which is connected to CD8 or the transmembrane
region of CD28 through the hinge region of CD8, and the
transmembrane region is immediately followed by the intracellular
signal region.
[0120] The invention also includes nucleic acids encoding the
chimeric antigen receptors. The present invention also relates to
variants of the above described polynucleotides, which encode a
polypeptide, or a fragment, analog and derivative of the
polypeptide having the same amino acid sequence as the present
invention.
[0121] The transmembrane region of the chimeric antigen receptor
may be selected from the transmembrane region of a protein such as
CD8 or CD28. The human CD8 protein is a heterodimer composed of two
chains, .alpha..beta. or .gamma..delta.. In one embodiment of the
invention, the transmembrane region is selected from the
transmembrane region of CD8a or CD28. In addition, the CD8a hinge
is a flexible region so that CD8 or CD28 and the transmembrane
region as well as the hinge region are used to connect the target
recognition domain scFv of the chimeric antigen receptor CAR to the
intracellular signal region.
[0122] The intracellular signal region may be selected from a group
consisting of intracellular signal region of CD3 .zeta.,
Fc.epsilon.RI.gamma., CD28, CD137, CD134 protein, and combinations
thereof. The CD3 molecule consists of five subunits, in which
CD3.zeta. subunit (also known as CD3 zeta, abbreviated as Z)
contains 3 ITAM motifs that are important signal transduction
regions in TCR-CD3 complex. CD3.delta.Z is a truncated CD3.zeta.
sequence without ITAM motif and is generally constructed in the
present invention as a negative control. Fc.epsilon.RI.gamma. is
mainly distributed on the surface of mast cells and basophils,
which contains an ITAM motif, which is similar to CD3.zeta. in
structure, distribution and function. In addition, as mentioned
above, CD28, CD137 and CD134 are co-stimulatory signaling
molecules. The co-stimulatory effect of their intracellular
signaling segments upon binding to the respective ligands results
in the continued proliferation of immune effector cells, primarily
T lymphocytes, and increase in the level of cytokines such as IL-2
and IFN-.gamma. secreted by immune effector cells, and the survival
period and anti-tumor effect of CAR immune effector cells in vivo
are increased.
[0123] The chimeric antigen receptor of the present invention can
be sequentially linked as follows:
[0124] The antibody of the invention, CD8 and CD3.zeta.;
[0125] The antibody of the invention, CD8, CD137 and CD3.zeta.;
[0126] The antibody of the invention, the transmembrane region of
CD28 molecule, the intracellular signal region of CD28 molecule and
CD3 .zeta.; or
[0127] The antibodies of the invention, the transmembrane region of
CD28 molecule, the intracellular signal region of CD28 molecule,
CD137 and CD3.zeta..
[0128] And combinations thereof, wherein CD28a in the relevant
chimeric antigen receptor protein represents the transmembrane
region of CD28 molecule and CD28b represents the intracellular
signal region of CD28 molecule. The various chimeric antigen
receptors described above are collectively referred to as scFv
(EGFR)-CAR.
[0129] The present invention also provides a vector comprising the
above-mentioned nucleic acid encoding a chimeric antigen receptor
protein expressed on the surface of an immune effector cell. In a
specific embodiment, the vector used in the present invention is a
lentiviral plasmid vector pWPT-eGFP. This plasmid belongs to the
third generation of self-inactivating lentiviral vector system. The
system has three plasmids, packaging plasmid psPAX2 encoding
protein Gag/Pol, encoding Rev protein; envelope plasmid PMD2.G
encoding VSV-G protein; and empty vector pWPT-eGFP, which can be
used for recombinant introduction of a nucleic acid sequence of
interest, i.e., a nucleic acid encoding CAR. In the empty vector
pWPT-eGFP, the expression of enhanced green fluorescent protein
(eGFP) is regulated by elongation factor-1.alpha. (EF-1.alpha.)
promoter. While in the recombinant expression vector
pWPT-eGFP-F2A-CAR containing the nucleic acid sequence encoding
CAR, co-expression of eGFP and CAR is achieved by ribosomal
skipping sequence 2A (abbreviated as F2A) from food-and-mouth
disease virus (FMDV).
[0130] The invention also includes viruses comprising the vectors
described above. The viruses of the invention include packaged
infectious viruses as well as viruses to be packaged that contain
the necessary components for packaging into infectious viruses.
Other viruses known in the art that can be used to transduce
exogenous genes into immune effector cells and their corresponding
plasmid vectors are also useful in the present invention.
[0131] The present invention further includes a genetically
modified T lymphocyte, which is transduced with a nucleic acid of
the present invention or transduced with the above-mentioned
recombinant plasmid containing the nucleic acid of the present
invention or a viral system containing the plasmid. Conventional
nucleic acid transduction methods in the art, including non-viral
and viral transduction methods, can be used in the present
invention. Non-viral transduction methods include electroporation
and transposon methods. Recently, nucleofector nuclear transfection
instrument developed by Amaxa can directly introduce foreign genes
into nucleus to achieve highly efficient transduction of target
genes. In addition, compared with conventional electroporation, the
transduction efficiency of transposon system based on Sleeping
Beauty system or PiggyBac transposon was significantly improved.
The combination of nucleofector transfection instrument and SB
Sleeping Beauty transposon system has been reported [Davies JK., et
al. Combining CD19 redirection and alloanergization to generate
tumor-specific human T cells for allogeneic cell therapy of B-cell
malignancies. Cancer Res, 2010, 70(10): OF1-10.], and high
transduction efficiency and site-directed integration of target
genes can be achieved by this method. In one embodiment of the
invention, the transduction method of a T lymphocyte modified by a
chimeric antigen receptor gene is a transduction method based on a
virus such as a retrovirus or a lentivirus. The method has the
advantages of high transduction efficiency and stable expression of
exogenous gene, and the time for in vitro culturing T lymphocytes
to clinical level can be shorten. The transduced nucleic acid is
expressed on the surface of the transgenic T lymphocytes by
transcription, translation. In vitro cytotoxicity assay performed
on various cultured tumor cells demonstrated that the immune
effector cells of the present invention have highly specific tumor
cell killing effects (also known as cytotoxicity). Therefore, the
nucleic acid encoding a chimeric antigen receptor protein of the
present invention, a plasmid comprising the nucleic acid, a virus
comprising the plasmid, and a transgenic immune effector cells
transfected with the nucleic acid, plasmid or virus described above
can be effectively used in tumor immunotherapy.
[0132] The immune cells of the present invention may also carry
exogenous encoding sequences for cytokines, including but not
limited to IL-12, IL-15 or IL-21. These cytokines have
immunomodulatory or antitumor activity, enhance the function of
effector T cells and activated NK cells, or directly exert
anti-tumor effects. Therefore, those skilled in the art will
understand that the use of these cytokines will help the immune
cells to function better.
[0133] In addition to the chimeric antigen receptor described
above, the immune cells of the present invention may also express
another chimeric antigen receptor, which does not contain CD3, but
contains intracellular signaling domain of CD28 and intracellular
signal domain of CD137, or a combination of both.
[0134] The immune cells of the present invention may also express
chemokine receptors; the chemokine receptors include, but are not
limited to, CCR2. A skilled person will understand that the CCR2
chemokine receptor can competitively bind CCR2 in the body and is
beneficial for blocking the metastasis of the tumor.
[0135] The immune cells of the present invention may also express
siRNAs that can reduce PD-1 expression or PD-L1-blocking proteins.
A skilled person will understand that competitive blocking of the
interaction between PD-L1 and its receptor PD-1 will facilitate the
recovery of anti-tumor T-cell responses, thereby inhibiting tumor
growth.
[0136] The immune cells of the present invention may also express a
safety switch; preferably, the safety switch includes iCaspase-9,
Truancated EGFR or RQR8.
[0137] Pharmaceutical Composition
[0138] The antibodies, immunoconjugates comprising the antibodies,
and genetically modified immune cells of the present invention can
be used in the preparation of a pharmaceutical composition or
diagnostic reagent. In addition to an effective amount of the
antibody, immunological conjugate, or immune cell, the composition
may further comprise a pharmaceutically acceptable carrier. The
term "pharmaceutically acceptable" means that when the molecular
entities and compositions are properly administered to animals or
humans, they do not cause adverse, allergic or other untoward
reactions.
[0139] Specific examples of some of the substances which may be
used as pharmaceutically acceptable carriers or components thereof
are sugars, such as lactose, dextrose and sucrose; starches, such
as corn starch and potato starch; cellulose and its derivatives,
such as carboxymethylcellulose sodium, ethylcellulose and
methylcellulose; gum tragacanth; 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; polyhydric alcohols such
as propylene glycol, glycerin, sorbitol, mannitol and polyethylene
glycol; alginic acid; emulsifiers such as Tween.RTM.; wetting
agents such as sodium lauryl sulfate; coloring agents; flavoring
agents; tablets, stabilizers; antioxidants; preservatives;
pyrogen-free water; isotonic saline solutions; and phosphate
buffers and the like.
[0140] The composition of the present invention can be prepared
into various dosage forms as needed, and the dosage to be
administered to a patient can be determined by a physician
according to factors, such as type, age, body weight, and general
disease condition of a patient, mode of administration, and the
like. For example, injection or other treatment may be used.
[0141] The present invention is further described below with
reference to specific embodiments. It should be understood that
these examples are only for illustrating the present invention and
are not intended to limit the scope of the present invention.
Experimental procedures in the following examples where no specific
conditions are indicated are generally carried out in accordance
with the conditions described in customary conditions such as those
compiled by J. Sambrook et al., Molecular Cloning Experiments
Guide, Third Edition, Science Press, 2002, or according to the
manufacturer Suggested conditions.
Example 1. Construction of Affinity Mature Library of Single Chain
Antibody 7B3
[0142] The single chain antibody 7B3 is a humanized antibody
fragment that specifically recognizes a cryptic epitope
(.sup.287CGADSYEMEEDGVRKC.sup.302 (SEQ ID NO: 1)) formed from the
amino acid sequence of positions 287-302 of EGFR exposed in tumor
cells. The nucleotide sequences of VL and VH genes were obtained
from the sequences SEQ ID NO: 14 and SEQ ID NO: 13 as shown in
patent application 201210094008.x and linked in the order of
VL7B3-linker-VH7B3.
[0143] Nucleotide sequence 717 base airs SEQ ID NO: 2 of single
chain antibody 7B3:
TABLE-US-00001 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
AGCAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCGTTCAG
TACGCCCAGTTCCCATATACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCAGCTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCCTGGGACGCGGCTTCCGCTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0144] The amino acid sequence of the single chain antibody 7B3
(239 amino acids, SEQ ID NO: 3; underlined area was 7B3 VL CDR1,
CDR2, CDR3, 7B3 VH CDR1, CDR2, CDR3, respectively):
TABLE-US-00002 DIQMTQSPSSLSASVGDRVTITCHASQDINSNIGWLQQKP
GKAFKGLIYHGKNLEDGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCVQYAQFPYTFGQGTKVEIKRGGGGSGGGGSGG
GGSDVQLVESGGGLVQPGGSLRLSCAVSGYSITSDYAWNW
IRQAPGKGLEWLGYISYRGRTSYNPSLKSRISITRDNSKN
TFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0145] For enhancing the ability of 7B3 single-chain antibody to
bind EGFR, some amino acids in CDR3 of light chain and CDR3 of
heavy chain were randomly mutated and corresponding mature
libraries were constructed.
[0146] 1. Construction of 7B3 Light Chain CDR3 Affinity Mature
Library
[0147] By sequence alignment and analysis of 7B3 single-chain
antibody, part of the amino acids in the third CDR region of 7B3
light chain were selected and randomized mutations were introduced
by primers to construct a light-chain affinity mature library.
[0148] To prepare a DNA fragment encoding the 7B3 mutant library,
two DNA fragments were respectively obtained by PCR using plasmid
pCantab 5E-7B3 (inserting 7B3 into sfiI/NotI site of pCantab
5E-7B3) as a template, followed by splicing through bypass PCR
method. Specifically, the following procedure was used: for
synthesizing genes, PCR reactions were performed in a volume of 50
.mu.l each using plasmid pCantab 5E-7B3 as a template with a final
concentration of 0.2 .mu.M for each primer and 5 .mu.l of
10.times.KOD Plus buffer, 4 .mu.l dNTPs (dATP, dCTP, dGTP and dTTP,
2 mM each), 2 .mu.l 25 mM MgSO.sub.4 and 1 U KOD Plus (from Takara)
were added and the PCR procedure was started in a thermal cycler
after making up the volume with water. The reaction was firstly
heated to 94.degree. C. for 5 minutes and then incubated for 25
cycles of 94.degree. C. for 30 seconds, 56.degree. C. for 30
seconds and 68.degree. C. for 30 seconds, and finally, at
68.degree. C. for 10 minutes. The first fragment was amplified
using primers pC7B3fw (SEQ ID NO: 4,
ATAACAGGCCCAGCCGGCCATGGATATTCAGATGACCCAGAG) and LR3re (SEQ ID NO:
5, CACTTTGGTGCCCTGGCCAAATGTMNNTGGGNNMNNMNNMNNCTGMNNGCAATA
ATAGGTCGCAAAATC) and the second fragment was amplified using primer
LR3f2fw (SEQ ID NO: 6, ACATTTGGCCAGGGCACCAAAG) and pC7B3re (SEQ ID
NO: 7, ATAAATGCGGCC GCGCTGCTCACGGTCAC).
[0149] Expected PCR products were identified by analytical agarose
gel electrophoresis and purified from samples by Wizard SV Gel and
PCR Clean-up Kit (available from Promega). The two fragments were
added in equimolar ratio to a second round of bridge PCR as a
template and the reaction system still used KOD Plus system
mentioned above. The reaction was firstly heated to 94.degree. C.
for 5 minutes and then incubated for 10 cycles, each cycling
reaction conditions were 94.degree. C. for 30 seconds, 60.degree.
C. for 30 seconds and 68.degree. C. for 30 seconds, and finally, at
68.degree. C. for 10 minutes. Subsequently, primers pC7B3fw and
pC7B3re were directly added to the reaction system at a final
concentration of 0.2 .mu.M, and the PCR program was started. The
reaction was firstly heated to 94.degree. C. for 5 minutes and then
incubated for 25 cycles of 94.degree. C. for 30 seconds, 56.degree.
C. for 30 seconds and 68.degree. C. for 30 seconds, and finally, at
68.degree. C. for 10 minutes. The expected PCR products were
separated by preparative agarose gel electrophoresis and purified
by Wizard SV Gel and PCR Clean-up kits according to the
manufacturer's instructions.
[0150] In the library, complete DNA fragments contained sfiI and
Notl restriction enzyme recognition sites at each end, and was
digested by restriction endonuclease sfiI/NotI (purchased from New
England Biolabs) for restriction digestion and inserted into
phagemid vector pCANTAB 5E digested by the same two enzymes.
Ligation products were isolated and desalted using Wizard SV Gel
and PCR Clean-up Kit for electrotransformation. For
electrotransformation, a home-made competent E. coli ER2738
(available from New England Biolabs) was used with electroporation
cuvette and electroporation instrument Gene Pulser II (from
Bio-Rad). A library containing 1.9.times.10.sup.9 mutants was
finally confirmed.
[0151] 2. Construction of 7B3 Heavy Chain CDR3 Affinity Mature
Library
[0152] By sequence alignment and analysis of 7B3 single-chain
antibody, part of the amino acids in the third CDR region of 7B3
heavy chain were selected and randomized mutations were introduced
by primers to construct a heavy-chain affinity mature library.
[0153] To prepare a DNA fragment encoding the 7B3 mutant library,
two DNA fragments were respectively obtained by PCR using plasmid
pCantab 5E-7B3 as a template, followed by splicing through bypass
PCR method. Specifically, the following procedure was used: for
synthesizing genes, PCR reactions were performed in a volume of 50
.mu.l each using plasmid pCantab 5E-7B3 as a template with a final
concentration of 0.2 .mu.M for each primer and 5 .mu.l of
10.times.KOD Plus buffer, 4 .mu.l dNTPs (dATP, dCTP, dGTP and dTTP,
2 mM each), 2 .mu.l 25 mM MgSO.sub.4 and 1 U KOD Plus were added
and the PCR procedure was started in a thermal cycler after making
up the volume with water. The reaction was firstly heated to
94.degree. C. for 5 minutes and then incubated for 25 cycles of
94.degree. C. for 30 seconds, 56.degree. C. for 30 seconds and
68.degree. C. for 30 seconds, and finally, at 68.degree. C. for 10
minutes. The first fragment was amplified using primers HR3f1fw
(SEQ ID NO: 8, TCGCAATTCCTTTAGTTGTTCC) and HR3f1re (SEQ ID NO: 9,
CAGGGTGCCCTGGCCCCAGTAANNMNNMNNMNNMNNMNNGCGCGCGCAATAATAC AC) and the
second fragment was amplified using primer HR3f2fw (SEQ ID NO: 10,
TACTGGGGCCAGGGCACCCTG) HR3f2re (SEQ ID NO: 11,
GGAATAGGTGTATCACCGTACTCAG).
[0154] Expected PCR products were identified by analytical agarose
gel electrophoresis and purified from samples by Wizard SV Gel and
PCR Clean-up Kit. The two fragments were added in equimolar ratio
to a second round of bridge PCR as a template and the reaction
system still used KOD Plus system mentioned above. The reaction was
firstly heated to 94.degree. C. for 5 minutes and then incubated
for 10 cycles, each cycling reaction conditions were 94.degree. C.
for 30 seconds, 60.degree. C. for 30 seconds and 68.degree. C. for
30 seconds, and finally, at 68.degree. C. for 10 minutes.
Subsequently, primers HR3f1fw and HR3f2re were directly added to
the reaction system at a final concentration of 0.2 .mu.M, and the
PCR program was started. The reaction was firstly heated to
94.degree. C. for 5 minutes and then incubated for 25 cycles of
94.degree. C. for 30 seconds, 56.degree. C. for 30 seconds and
68.degree. C. for 30 seconds, and finally, at 68.degree. C. for 10
minutes. The expected PCR products were separated by preparative
agarose gel electrophoresis and purified by Wizard SV Gel and PCR
Clean-up kits according to the manufacturer's instructions.
[0155] In the library, complete DNA fragments contained sfiI and
Notl restriction enzyme recognition sites at each end, and was
digested by restriction endonuclease sfiI/NotI for restriction
digestion and inserted into phagemid vector pCANTAB 5E digested by
the same two enzymes. Ligation products were isolated and desalted
using Wizard SV Gel and PCR Clean-up Kit for electrotransformation.
For electrotransformation, a home-made competent E. coli ER2738 was
used with electroporation cuvette and electroporation instrument
Gene Pulser II. A library containing 6.0.times.10.sup.9 mutants was
finally confirmed.
Example 2. Screening Against EGFRvIII by Using 7B3 Affinity
Maturation Library
[0156] To obtain 7B3 mutants with higher affinity, four rounds of
screening were performed using light chain and heavy chain mutant
libraries, respectively, as follows: a corresponding phage library
was obtained from the above library through infection of helper
phage M13KO7. The phage library was incubated with the
biotin-labeled antigen EGFRvIII (purchased from Shanghai raygene
biotechnology Co., LTD) for 2 hours at room temperature and then
incubated with 2% (w/v) BSA (bovine serum albumin, purchased from
Shanghai Bioengineering)-blocked streptavidin magnetic beads MyOne
Cl (from Invitrogen) at room temperature for 30 minutes. The beads
were then washed with PBST (containing 0.1% Tween-20) buffer to
remove phage not specifically bound or with weaker binding
capacity. Strongly binding phages were eluted from magnetic beads
with glycine-HCl (pH 2.2), neutralized with Tris neutralizing
solution (pH 9.1), and then used to infect E. coli ER2738 in the
mid-logarithmic growth phase for the next round of screening.
[0157] In the above described four rounds of screening, the amounts
of magnetic beads were 50 .mu.l, 25 .mu.l, 10 .mu.l and 10 .mu.l,
the concentrations of biotin-labeled antigen EGFRvIII were 10 nM, 1
nM, 0.5 nM and 0.1 nM, respectively, and PBST was used for washing
for 10, 10, 15 and 20 times. From the second round of screening,
50-, 500-, and 1000-fold excess of unlabeled antigen EGFRvIII,
respectively, was added as a competitor prior to elution to remove
mutants with weaker binding capacity.
[0158] For the production of phage displaying 7B3 single chain
antibody mutants on the surface, the strain in glycerol obtained in
Example 1 was inoculated into 400 ml of 2YT/ampicillin medium to
bring the cell density to OD.sub.600=0.1, and at 37.degree. C. and
200 rpm, cultured with shaking until the cell density reached
OD.sub.600=0.5. 10.sup.12 pfu of M13KO7 helper phage was used in
infection and incubated at 30.degree. C. and 50 rpm for 30 minutes.
After adding 50 mg/l kanamycin and shaking at 37.degree. C. and 200
rpm for 30 minutes, the pellet was separated by centrifugation (15
minutes, 1600.times.g, 4.degree. C.) and resuspended in 400 ml
2YT/ampicillin/Kanamycin medium and cultured for 16 hours at
37.degree. C. with shaking at 200 rpm. Finally, the pellet was
separated by centrifugation (5000 rpm, 4.degree. C. for 20 minutes)
and discarded. The supernatant was filtered through a 0.45 .mu.m
filter and 1/4 volume of 20% (w/v) PEG 8000, 2.5 M NaCl solution
was added and incubated in an ice bath for 1 hour for precipitating
phage pellets. The pellet was then centrifuged (20 min,
8000.times.g, 4.degree. C.) and the supernatant was discarded. The
phage was resuspended in 25 ml of prechilled PBS (137 mM NaCl, 2.7
mM KCl, 8 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4) and
centrifuged (5 minutes, 20000.times.g, 4.degree. C.). 1/4 volume of
20% (w/v) PEG 8000, 2.5 M NaCl solution was added to the
supernatant and incubated in an ice bath for 30 minutes for
precipitating the phage particles again. The pellet was obtained by
centrifugation (30 min at 20000.times.g at 4.degree. C.),
resuspended in 2 ml of prechilled PBS again, kept on ice for 30 min
and centrifuged (30 min, 17000.times.g, 4.degree. C.). The
supernatant was mixed with 4% (w/v) BSA in PBS at 1:1, placed on a
rotary mixer and incubated for 30 minutes at room temperature, and
then used directly in screening.
Example 3. Identification of 7B3 Mutants Specifically Binding to
EGFRvIII
[0159] After four rounds of screening against EGFRvIII antigen, 96
clones were randomly selected from the clones obtained in the
fourth round of screening and analyzed for their combination with
antigens EGFRvIII and N1N2-806 (purchased from Shanghai raygene
biotechnology Co., LTD) using single phage ELISA (enzyme-linked
immunosorbent assay), where N1N2-806 is a fusion protein of N1N2
domain of M13 phage PIII protein and amino acids at positions
287-302 of EGFR. For this purpose, each single colony was
inoculated into 300 .mu.l of 2YT/ampicillin medium (containing 2%
glucose) in a 96-well deep-well plate and cultured with shaking at
37.degree. C. and 250 rpm for 16 hours. 20 .mu.l of culture was
innoculated into 500 .mu.l of 2YT/ampicillin medium (containing
0.1% glucose) and shaken at 37.degree. C. and 250 rpm for 1.5
hours. To prepare the helper phage solution, 75 .mu.l of M13KO7
(titer of 3.times.10.sup.12 pfu/ml) was taken and mixed into 15 ml
of 2YT medium and added into a plate at 50 .mu.l/well. Incubation
was performed at 37.degree. C. and 150 rpm for 30 minutes, and then
50 .mu.l/well of prepared kanamycin solution (180 .mu.l of 50 mg/ml
kanamycin taken and added into 15 ml of 2YT medium) was added and
cultured at 37.degree. C. and 250 rpm for 16 hours with shaking.
Finally, the cells were precipitated by centrifugation (30 minutes
at 5000.times.g, 4.degree. C.) and the supernatant was transferred
to a new 96-well deep-well plate.
[0160] To perform single-phage ELISA, 100 ng/well of antigen
EGFRvIII, N1N2-806 and negative control proteins BSA and N1N2
(purchased from Shanghai raygene biotechnology Co., LTD) were used
on 96-well MediSorp ELISA plate (purchased from Nunc) at 50
.mu.l/well, and coated overnight at 4.degree. C. Each well was
blocked with PBST containing 2% BSA (w/v). The wells were then
washed with PBST for three times. Then, each phage solution
prepared above was added to each well of the plate at 100
.mu.l/well. After incubation for 2 hours at 37.degree. C., it was
washed for three times with PBST. To detect bound phage, anti-M13
antibody superoxide dismutase conjugate (purchased from GE
Healthcare) was diluted at 1:5000 in PBST and 100 .mu.l was added
to each well. After incubating at 37.degree. C. for 1 hour, the
wells were rinsed for three times with PBST and then rinsed for
three times with PBS. Finally, 50 .mu.l of TMB substrate was added
into the wells and developed for 10 minutes at room temperature,
followed by addition of 50 .mu.l of 2M H.sub.2SO.sub.4 per well to
stop the color reaction. Extinction values were measured at 450 nm
with an enzyme-linked immunosorbent (Bio-Rad).
[0161] Clones with stronger signal of binding antigen instead of
BSA in ELISA were selected and used in subsequent evaluation and
sequencing analysis. The antibodies obtained from the light chain
affinity maturation library were then further combined with the
antibodies obtained from Example 2 in the heavy chain affinity
maturation library on light chain variable region sequence and
heavy chain variable region sequence and the resulting antibodies
are also capable of specifically binding antigens EGFRvIII and
N1N2-806, instead of the control proteins BSA and N1N2.
[0162] From the crystal structure of the antibody and antigenic
determinant, the binding of combined antibodies obtained from 7B3
light and heavy chain mutations and EGFR287-302 was structurally
analyzed, and finally, some amino acid positions were selected for
further mutation for higher affinity and stability. All of the
altered amino acid positions include S31 in the light chain CDR1
region, V89, A92, Q93, F94 and Y96 in the light chain CDR3 region,
S182 in the heavy chain CDR2 region, L222, R224, G225, F226 and
R227 in the heavy chain CDR3 region. Based on the sequences of
combined antibodies after mutagenesis of the light and heavy
chains, the mutation sites were further introduced to obtain the
antibody Y022. Compared with the parental antibody 7B3, Y022
contains 12 amino acid mutation sites (S31V, V89N, A92E, Q93N,
F941, Y96L, S182Q, L222M, R224K, G225N, F226W, and R227D). As shown
in FIG. 1, in a single phage ELISA assay, Y022 was able to
specifically bind to antigens EGFRvIII and N1N2-806 without binding
to control proteins BSA and N1N2.
[0163] Nucleotide sequence of single chain antibody Y022 (717
bases; SEQ ID NO: 12):
TABLE-US-00003 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
GTGAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCAATCAG
TATGAAAATATCCCACTGACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCCAGTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCATGGGTAAGAATTGGGATTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0164] Amino acid sequence of single chain antibody Y022 (239 amino
acids; SEQ ID NO: 13):
TABLE-US-00004 DIQMTQSPSSLSASVGDRVTITCHASQDIN
VNIGWLQQKPGKAFKGLIYHGKNLEDGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCNQ
YENIPLTFGQGTKVEIKRGGGGSGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGR TQYNPSLKSRISITRDNSKNTFFLQLNSLR
AEDTAVYYCARMGKNWDYWGQGTLVTVSS
[0165] Wherein the light chain is in positions 1-108 and the light
chain CDR1 sequence: HASQDINVNIG (SEQ ID NO: 41), CDR2 sequence:
HGKNLED (SEQ ID NO: 42), CDR3 sequence: NQYENIPLT (SEQ ID NO:
43).
[0166] Wherein the heavy chain is in positions 124-239 and the
heavy chain CDR1 sequence: GYSITSDYAWN (SEQ ID NO: 44), CDR2
sequence: YISYRGRTQYNPSLKS (SEQ ID NO: 45), CDR3 sequence: MGKNWDY
(SEQ ID NO: 46).
[0167] Since it was screened in the mutant library constructed
previously and subjected to site-directed mutagenesis, the
nucleotide sequence of Y022 was contained in pCantab 5E, named as
pCantab 5E-Y022 plasmid.
[0168] The inventors also obtained 10 additional antibody clones
with significantly improved affinity and stability using the same
method as for the production of antibody Y022, namely M14, M15,
M25, M26, S7, S8, S17, S22, S23 and S29. Compared with the parent
antibody 7B3, all single-chain antibodies contained amino acid
mutation sites as shown in Table 1.
TABLE-US-00005 TABLE 1 Antibody amino acid mutation site Y022 S31V,
V89N, A92E, Q93N, F94I, Y96L, S182Q, L222M, R224K, G225N, F226W,
R227D M14 V89N, A92E, Q93N, F94N, Y96I, S182N M15 S31V, V89N, A92E,
Q93N, F94N, Y96I M25 S31V, V89N, A92E, Q93N, F94I, Y96L, S182R M26
S31V, V89N, A92E, Q93N, F94I, Y96L, S182Q S7 S31V, K53T, V89N,
A92E, Q93N, F94N, Y96I S8 S31V, A44S, V89N, A92E, Q93N, F94N, Y96I
S17 S31T, V89N, A92E, Q93N, F94N, Y96L, S182Q S22 S31V, K53T, V89N,
A92E, Q93N, F94N, Y96L, S182R S23 S31V, A44S, V89N, A92E, Q93N,
F94N, Y96L, S182R S29 S31V, V89N, A92E, Q93N, Y96L, S182R
[0169] Nucleotide sequence of single chain antibody M14 (717 bases;
SEQ ID NO: 58):
TABLE-US-00006 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
AGCAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCAATCAG
TATGAAAATAACCCAATTACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCAACTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCCTGGGACGCGGCTTCCGCTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0170] Amino acid sequence of single chain antibody M14 (239 amino
acids; SEQ ID NO: 59):
TABLE-US-00007 DIQMTQSPSSLSASVGDRVTITCHASQDIN
SNIGWLQQKPGKAFKGLIYHGKNLEDGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCNQ
YENNPITFGQGTKVEIKRGGGGSGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGR TNYNPSLKSRISITRDNSKNTFFLQLNSLR
AEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0171] Amino acid sequences of M14 light chain CDR1(HASQDINSNIG),
CDR2(HGKNLED), CDR3(NQYENNPIT) and heavy chain CDR1 (GYSITSDYAWN),
CDR2 (YISYRGRTNYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NO: 47, 42, 48,
44, 49, 50, respectively.
[0172] Nucleotide sequence of single chain antibody M15 (717 bases;
SEQ ID NO: 60):
TABLE-US-00008 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
GTGAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCAATCAG
TATGAAAATAACCCAATTACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCAGCTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCCTGGGACGCGGCTTCCGCTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0173] Amino acid sequence of single chain antibody M15 (239 amino
acids; SEQ ID NO: 61):
TABLE-US-00009 DIQMTQSPSSLSASVGDRVTITCHASQDIN
VNIGWLQQKPGKAFKGLIYHGKNLEDGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCNQ
YENNPITFGQGTKVEIKRGGGGSGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGR TSYNPSLKSRISITRDNSKNTFFLQLNSLR
AEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0174] Amino acid sequences of M15 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLED), CDR3 (NQYENNPIT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 48, 44, 51, 50, respectively.
[0175] Nucleotide sequence of single chain antibody M25 (717 bases;
SEQ ID NO: 62):
TABLE-US-00010 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
GTGAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCAATCAG
TATGAAAATATCCCACTGACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCCGCTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCCTGGGACGCGGCTTCCGCTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0176] Amino acid sequence of single chain antibody M25 (239 amino
acids: SEQ ID NO: 63):
TABLE-US-00011 DIQMTQSPSSLSASVGDRVTITCHASQDIN
VNIGWLQQKPGKAFKGLIYHGKNLEDGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCNQ
YENIPLTFGQGTKVEIKRGGGGSGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGR TRYNPSLKSRISITRDNSKNTFFLQLNSLR
AEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0177] Amino acid sequences of M25 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLED), CDR3 (NQYENIPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 43, 44, 52, 50, respectively.
[0178] Nucleotide sequence of single chain antibody M26 (717 bases;
SEQ ID NO: 64):
TABLE-US-00012 GATATTCAGATGACCCAGAGCCCGAGCAGC
CTGAGCGCGAGCGTGGGCGACCGTGTGACC ATTACCTGCCATGCGAGCCAGGATATTAAC
GTGAACATTGGCTGGCTGCAGCAGAAACCG GGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGC CGTTTTAGCGGCAGCGGCAGCGGCACCGAT
TTTACCCTGACCATTAGCAGCCTGCAGCCG GAAGATTTTGCGACCTATTATTGCAATCAG
TATGAAAATATCCCACTGACATTTGGCCAG GGCACCAAAGTGGAAATTAAACGTGGTGGA
GGCGGTTCAGGCGGAGGTGGCTCTGGCGGT GGCGGATCGGATGTGCAGCTGGTGGAAAGC
GGCGGCGGCCTGGTGCAGCCGGGCGGCAGC CTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGG ATTCGTCAGGCGCCGGGCAAAGGCCTGGAA
TGGCTGGGCTATATTAGCTATCGTGGCCGC ACCCAGTATAACCCGAGCCTGAAAAGCCGT
ATTAGCATTACCCGTGATAACAGCAAAAAC ACCTTTTTCCTGCAGCTGAACAGCCTGCGT
GCGGAAGATACCGCGGTGTATTATTGCGCG CGCCTGGGACGCGGCTTCCGCTACTGGGGC
CAGGGCACCCTGGTGACCGTGAGCAGC
[0179] Amino acid sequence of single chain antibody M26 (239 amino
acids; SEQ ID NO: 65):
TABLE-US-00013 DIQMTQSPSSLSASVGDRVTITCHASQDIN
VNIGWLQQKPGKAFKGLIYHGKNLEDGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCNQ
YENIPLTFGQGTKVEIKRGGGGSGGGGSGG GGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGR TQYNPSLKSRISITRDNSKNTFFLQLNSLR
AEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0180] Amino acid sequences of M26 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLED), CDR3 (NQYENIPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTQYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 43, 44, 45, 50, respectively.
[0181] Nucleotide sequence of single chain antibody S7 (717 bases;
SEQ ID NO: 66):
TABLE-US-00014 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCG
ACCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACGTGAACAT
TGGCTGGCTGCAGCAGAAACCGGGCAAAGCGTTTAAAGGCCTGATTTAT
CATGGCACCAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCG
GCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGA
TTTTGCGACCTATTATTGCAATCAGTATGAAAATAACCCAATTACATTT
GGCCAGGGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCG
GAGGTGGCTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGG
CGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTG
AGCGGCTATAGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGG
CGCCGGGCAAAGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCG
CACCAGCTATAACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGAT
AACAGCAAAAACACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAG
ATACCGCGGTGTATTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTG
GGGCCAGGGCACCCTGGTGACCGTGAGCAGC
[0182] Amino acid sequence of single chain antibody S7 (239 amino
acids; SEQ ID NO: 67):
TABLE-US-00015 DIQMTQSPSSLSASVGDRVTITCHASQDINVNIGWLQQKPGKAFKGLIY
HGTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENNPITF
GQGTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAV
SGYSITSDYAWNWIRQAPGKGLEWLGYISYRGRTSYNPSLKSRISITRD
NSKNTFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0183] Amino acid sequences of S7 light chain CDR1 (HASQDINVNIG),
CDR2 (HGTNLED), CDR3 (NQYENNPIT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 53, 54, 44, 51, 50, respectively.
[0184] Nucleotide sequence of single chain antibody S8 (717 bases;
SEQ ID NO: 68):
TABLE-US-00016 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCG
ACCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACGTGAACAT
TGGCTGGCTGCAGCAGAAACCGGGCAAAAGCTTTAAAGGCCTGATTTAT
CATGGCAAAAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCG
GCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGA
TTTTGCGACCTATTATTGCAATCAGTATGAAAATAACCCAATTACATTT
GGCCAGGGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCG
GAGGTGGCTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGG
CGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTG
AGCGGCTATAGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGG
CGCCGGGCAAAGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCG
CACCAGCTATAACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGAT
AACAGCAAAAACACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAG
ATACCGCGGTGTATTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTG
GGGCCAGGGCACCCTGGTGACCGTGAGCAGC
[0185] Amino acid sequence of single chain antibody S8 (239 amino
acids; SEQ ID NO: 69):
TABLE-US-00017 DIQMTQSPSSLSASVGDRVTITCHASQDINVNIGWLQQKPGKSFKGLIYH
GKNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENNPITFGQ
GTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGRTSYNPSLKSRISITRDNSKN
TFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0186] Amino acid sequences of S8 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLED), CDR3 (NQYENNPIT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 54, 44, 51, 50, respectively.
[0187] Nucleotide sequence of single chain antibody S17 (717 bases;
SEQ ID NO: 70):
TABLE-US-00018 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGA
CCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACACCAACATTG
GCTGGCTGCAGCAGAAACCGGGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCGGCAG
CGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTG
CGACCTATTATTGCAATCAGTATGAAAATAACCCACTGACATTTGGCCAG
GGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCGGAGGTGG
CTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGGCGGCGGCC
TGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGGCGCCGGGCAA
AGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCGCACCCAGTATA
ACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGATAACAGCAAAAAC
ACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAGATACCGCGGTGTA
TTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTGGGGCCAGGGCACCC
TGGTGACCGTGAGCAGC
[0188] Amino acid sequence of single chain antibody S17 (239 amino
acids; SEQ ID NO: 71):
TABLE-US-00019 DIQMTQSPSSLSASVGDRVTITCHASQDINTNIGWLQQKPGKAFKGLIYH
GKNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENNPLTFGQ
GTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGRTQYNPSLKSRISITRDNSKN
TFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0189] Amino acid sequences of S17 light chain CDR1 (HASQDINTNIG),
CDR2 (HGKNLED), CDR3 (NQYENNPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTQYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 55, 42, 56, 44, 45, 50, respectively.
[0190] Nucleotide sequence of single chain antibody S22 (717 bases;
SEQ ID NO: 72):
TABLE-US-00020 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGA
CCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACGTGAACATTG
GCTGGCTGCAGCAGAAACCGGGCAAAGCGTTTAAAGGCCTGATTTATCAT
GGCACCAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCGGCAG
CGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTG
CGACCTATTATTGCAATCAGTATGAAAATAACCCACTGACATTTGGCCAG
GGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCGGAGGTGG
CTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGGCGGCGGCC
TGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGGCGCCGGGCAA
AGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCGCACCCGCTATA
ACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGATAACAGCAAAAAC
ACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAGATACCGCGGTGTA
TTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTGGGGCCAGGGCACCC
TGGTGACCGTGAGCAGC
[0191] Amino acid sequence of single chain antibody S22 (239 amino
acids; SEQ ID NO: 73):
TABLE-US-00021 DIQMTQSPSSLSASVGDRVTITCHASQDINVNIGWLQQKPGKAFKGLIYH
GTNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENNPLTFGQ
GTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAVSGY
SITSDYAWNWIRQAPGKGLEWLGYISYRGRTRYNPSLKSRISITRDNSKN
TFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0192] Amino acid sequences of S22 light chain CDR1 (HASQDINVNIG),
CDR2 (HGTNLED), CDR3 (NQYENNPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3(LGRGFRY) are SEQ ID
NO: 41, 53, 56, 44, 52, 50, respectively.
[0193] Nucleotide sequence of single chain antibody S23 (717 bases;
SEQ ID NO: 74):
TABLE-US-00022 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGA
CCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACGTGAACATTG
GCTGGCTGCAGCAGAAACCGGGCAAAAGCTTTAAAGGCCTGATTTATCAT
GGCAAAAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCGGCAG
CGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTG
CGACCTATTATTGCAATCAGTATGAAAATAACCCACTGACATTTGGCCAG
GGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCGGAGGTGG
CTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGGCGGCGGCC
TGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTGAGCGGCTAT
AGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGGCGCCGGGCAA
AGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCGCACCCGCTATA
ACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGATAACAGCAAAAAC
ACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAGATACCGCGGTGTA
TTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTGGGGCCAGGGCACCC
TGGTGACCGTGAGCAGC
[0194] Amino acid sequence of single chain antibody S23 (239 amino
acids; SEQ ID NO: 75):
TABLE-US-00023 DIQMTQSPSSLSASVGDRVTITCHASQDINVNIGWLQQKPGKSFKGLIY
HGKNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENNPLTF
GQGTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAV
SGYSITSDYAWNWIRQAPGKGLEWLGYISYRGRTRYNPSLKSRISITRD
NSKNTFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0195] Amino acid sequences of S23 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLEDG), CDR3 (NQYENNPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 56, 44, 52, 50, respectively.
[0196] Nucleotide sequence of single chain antibody S29717 bases;
SEQ ID NO: 76):
TABLE-US-00024 GATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCG
ACCGTGTGACCATTACCTGCCATGCGAGCCAGGATATTAACGTGAACAT
TGGCTGGCTGCAGCAGAAACCGGGCAAAGCGTTTAAAGGCCTGATTTAT
CATGGCAAAAACCTGGAAGATGGCGTGCCGAGCCGTTTTAGCGGCAGCG
GCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGA
TTTTGCGACCTATTATTGCAATCAGTATGAAAATTTCCCACTGACATTT
GGCCAGGGCACCAAAGTGGAAATTAAACGTGGTGGAGGCGGTTCAGGCG
GAGGTGGCTCTGGCGGTGGCGGATCGGATGTGCAGCTGGTGGAAAGCGG
CGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGTG
AGCGGCTATAGCATTACCAGCGATTATGCGTGGAACTGGATTCGTCAGG
CGCCGGGCAAAGGCCTGGAATGGCTGGGCTATATTAGCTATCGTGGCCG
CACCCGCTATAACCCGAGCCTGAAAAGCCGTATTAGCATTACCCGTGAT
AACAGCAAAAACACCTTTTTCCTGCAGCTGAACAGCCTGCGTGCGGAAG
ATACCGCGGTGTATTATTGCGCGCGCCTGGGACGCGGCTTCCGCTACTG
GGGCCAGGGCACCCTGGTGACCGTGAGCAGC
[0197] Amino acid sequence of single chain antibody S29 (239 amino
acids; SEQ ID NO: 77):
TABLE-US-00025 DIQMTQSPSSLSASVGDRVTITCHASQDINVNIGWLQQKPGKAFKGLIY
HGKNLEDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCNQYENFPLTF
GQGTKVEIKRGGGGSGGGGSGGGGSDVQLVESGGGLVQPGGSLRLSCAV
SGYSITSDYAWNWIRQAPGKGLEWLGYISYRGRTRYNPSLKSRISITRD
NSKNTFFLQLNSLRAEDTAVYYCARLGRGFRYWGQGTLVTVSS
[0198] Amino acid sequences of S23 light chain CDR1 (HASQDINVNIG),
CDR2 (HGKNLED), CDR3 (NQYENFPLT) and heavy chain CDR1
(GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID
NO: 41, 42, 57, 44, 52, 50, respectively.
Example 4. Expression and Purification of Antibody
[0199] Genes of each antibody were inserted into NdeI/XhoI site of
the expression vector pET22B(+), antibody proteins were
recombinantly produced in E. coli BL21 (DE3) and purified by nickel
columns using polypeptides with carboxy-terminal fused
6.times.histidine. In particular, to prepare the antibody protein,
each single colony was inoculated into 5 ml of
2.times.YT/ampicillin medium and cultured with shaking at
37.degree. C. and 220 rpm for 16 hours. 1 ml of this preculture was
used to inoculate 100 ml of 2.times. YT/ampicillin medium and
cultured with shaking at 37.degree. C. and 220 rpm until the cell
density reached OD.sub.600=0.5. After induction of foreign gene
expression by 1 mM .alpha.-D-isopropylthiogalactoside (IPTG), the
culture was shaken for 6 hours at 30.degree. C. and 220 rpm. The
cells were then precipitated by centrifugation (15 minutes at
3500.times.g, 4.degree. C.) and resuspended in 35 ml breaking
buffer (50 mM PB, 300 mM NaCl, 2 M urea, 0.5% Triton X-100, pH
8.0). After sonication, the sample was shaken at room temperature
for 30 minutes to completely lyse cell debris. Inclusion body
pellets were then collected by centrifugation (15 minutes,
10,000.times.g, 4.degree. C.) and 20 ml denaturing buffer (50 mM
PB, 300 mM NaCl, 8 M urea, 10 mM imidazole, pH 8.0) was added and
shaken for one hour at room temperature. The pellets were removed
by centrifugation (15 min, 10,000.times.g, 4.degree. C.), the
lysate was collected and the protein was purified with 5 ml HisTrap
HP purification column (commercially available from GE Healthcare).
The purity of the purified antibody protein was analyzed by SDS
polyacrylamide gel electrophoresis and the protein concentration
was determined by the BCA method.
Example 5. Binding Activity Assay of Antibody
[0200] Binding activity of an antibody to antigen EGFRvIII was
determined by concentration gradient ELISA assay. For this purpose,
the antigen EGFRvIII was diluted with 0.1 M NaHCO.sub.3 (pH 9.6)
coating solution and each well was coated with 200 ng at 50
.mu.l/well overnight at 4.degree. C. and blocked with PBST
containing 2% (w/v) BSA for 2 hours at room temperature. The plate
was then rinsed for three times with PBST. Subsequently, 100 .mu.l
of each antibody protein solution in PBST containing a series of
concentrations (initial concentration of 50 ng/well, 18 nM diluted
until to 1:81) was added to each well plate and each sample was
assayed using parallel three-well analysis. After incubation for 2
hours at 37.degree. C., plate was rinsed for three times with PBST
followed by adding 100 .mu.l/well of a 1:2000 dilution of mouse
anti-His-tag antibody (available from Santa cruz) for 1 hour at
37.degree. C. To test the bound antibody, HRP-labeled goat
anti-mouse antibody (purchased from Santa Cruz) was diluted in PBST
at a 1:15,000 dilution and 100 .mu.l per well was added and
incubated at 37.degree. C. for 1 hour. For detection, wells were
rinsed for three times with PBST followed by rinsing for three
times with PBS and finally TMB was added for development for 15
mins. The chromogenic reaction was stopped with 50 .mu.l of 2 M
H.sub.2SO.sub.4 per well and extinction value was measured at 450
nm using enzyme-linked immunoassay (Bio-Rad). The absorbance values
obtained were evaluated using Sigma Plot software and the binding
strength of an antibody was calculated. For this purpose, the
extinction value measured in each case was plotted against the
corresponding antibody concentration and the resulting curve was
fitted using the following non-linear regression.
y = a * x ( b + x ) ##EQU00001##
[0201] Wherein the binding/dissociation equilibrium identified
between the immobilized antigen and the antibody protein is:
[0202] x=concentration of antibody protein;
[0203] y=concentration of antigen/antibody complex (indirectly
measured by absorbance after color reaction);
[0204] a=total concentration of immobilized antigen;
[0205] b=dissociation constant (K.sub.D).
[0206] The binding curve obtained for antibody 7B3 in
concentration-gradient ELISA assay is exemplarily shown in FIG. 2
with K.sub.D of about 22.4 nM; the binding curve of antibody Y022
to EGFRvIII is shown in FIG. 3 with apparent K.sub.D of about 2.7
nM.
Example 6. Activity Analysis on Binding of Y022 to Cell Surface
EGFR
[0207] 1. Expression and Purification of scFv-Y022-Fc, scFv-806-Fc
and scFv-C225-Fc Fusion Antibody
[0208] According to a standard scheme, scFv-Y022 fragment was
amplified from the resulting clones using the primer pair V5-Y022-F
(SEQ ID NO: 14, ACAGTGCTAGCAGATATTCAGATGACCCAG) and V5-Y022-R (SEQ
ID NO: 15, AAGAATGCGGCCGCGCTGCTCACGGTCACCAG); ScFv-806 was
amplified using the primer pair V5-806-F (SEQ ID NO: 16,
ACAGTGCTAGCAGACATCCTGATGACCCAAT) and V5-806-R (SEQ ID NO: 17,
AAGAATGCGGCCGCTGCAGAGACAGTGACCAG) and pH-806/CD3 (see
201210094008.X) as the template; scFv-C225 fragment was cloned
using the primer pairs V5-C225-F (SEQ ID NO: 18,
ACAGTGCTAGCAGACATCTTGCTGACTCAG) and V5-C225-R (SEQ ID NO: 19,
AAGAATGCGGCCGCTGCAGAGACAGTGACCAG) and C225 (VL-linker-VH) DNA
fragment (sequence thereof was determined according to SEQ ID NO:
10 and SEQ ID NO: 12 in US20090099339A1 and obtained through
whole-genome synthesis by Shanghai raygene biotechnology Co., LTD)
as the template; and the amplified product was digested by
NheI/NotI (purchased from NEB), linked with NheI/NotI-digested
vector plasmid pCMV-V5-Fc (in the vector, the Fc fragment of human
IgG1 was fused downstream to the multiple cloning site, abbreviated
as V5-Fc, purchased from Shanghai raygene biotechnology Co., LTD)
with T4 DNA ligase (purchased from NEB) and transformed into host
strain TOP10. Clones were selected and the positive clones were
identified by PCR and confirmed by sequencing, so as to obtain
V5-scFv-Y022-Fc, V5-scFv-806-Fc and V5-scFv-C225-Fc eukaryotic
expression plasmids, respectively.
[0209] The above expression plasmids were respectively transfected
into well-growing HEK-293F cells, cultured at 37.degree. C., 5%
CO.sub.2, 125 rpm shaker for 7 days, and centrifuged at 4000 rpm
for 10 min. The precipitate was removed, the supernatant was
collected and filtered with 0.45 .mu.m membrane. The sample was
affinity-purified with protein A (from GE) affinity column to
finally obtain the purified antibody-Fc fusion proteins
scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc. Results are shown in
FIG. 4.
[0210] 2. Detection of Binding Ability of Single-Chain
scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to Cell-Surface EGFR by
FACS
[0211] The binding capacities of each of single-chain antibodies
scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to the following cell
lines were analyzed by fluorescence activated cell sorter (FACS)
(BD, FACS Calibur).
[0212] Specific methods are as follows:
[0213] 1) tumor cells in logarithmic growth phase as listed in
Table 2 were inoculated into a 6 cm dish with a inoculation cell
density of about 90%, and incubated at 37.degree. C. incubator
overnight.
[0214] 2) Cells were digested with 10 mM EDTA and cells were
collected by centrifugation at 200 g for 5 min. Cells were
resuspended in 1% phosphate buffered saline (NBS PBS) containing
calf serum at a concentration of 1.times.10.sup.6 to
1.times.10.sup.7/mL, and added in a flow tube in an amount of 100
.mu.l/tube.
[0215] 3) Cells were centrifuged at 200 g.times.5 min, and the
supernatant was discarded.
[0216] 4) Antibodies to be tested, scFv-Y022-Fc, scFv-806-Fc and
scFv-C225-Fc, were added respectively. And PBS was used as a
negative control. The final concentration of antibody was 20
.mu.g/ml, 100 .mu.l was added to each tube and placed in an ice
bath for 45 minutes.
[0217] 5) 2 ml of 1% NBS PBS was added to each tube and centrifuged
at 200 g.times.5 min for two times.
[0218] 6) Supernatant was discarded and FITC fluorescent-labeled
goat anti-human antibody (from Shanghai Kangcheng Bio-engineering
Company) at a dilution of 1:50 was added, and 100 ul was added to
each tube and placed in an ice bath for 45 minutes.
[0219] 7) 2 ml of 1% NBS PBS was added to each tube and centrifuged
at 200 g.times.5 min for two times.
[0220] 8) Supernatant was discarded, resuspended in 300 ul of 1%
NBS PBS and detected by flow cytometry.
[0221] 9) Data was analyzed by using flow cytometry data analysis
software WinMDI 2.9.
TABLE-US-00026 TABLE 2 Name of Expression tumor cell Source
Properties of cell of EGFR U87 ATCC Glioma cell line Low-expressed
EGFR U87-EGFR Shanghai Cancer EGFR-transfected U87 cell
over-expressed Institute line EGFR U87-EGFRvIII Shanghai Cancer
EGFRvIII-transfected over-expressed Institute U87 cell line
EGFRvIII A431 ATCC Vaginal epithelial cancer over-expressed EGFR
CAL 27 ATCC Tongue cancer cell line over-expressed EGFR MDA-MB-468
ATCC Breast cancer cell line over-expressed EGFR RWPE-1 ATCC
Prostate normal normally-expressed epithelial cells EGFR K2
Shanghai Cancer Human primary keratinocytes normallyr-expressed
Institute EGFR
[0222] Results are shown in FIG. 5, single chain antibody Y022 of
the present invention can, with different degrees, bind to U87-EGFR
exogenously overexpressing EGFR (Construction method can be found
in Wang H., et al., Identification of an Exon 4-Deletion Variant of
Epidermal Growth Factor Receptor with Increased
Metastasis-Promoting Capacity. Neoplasia, 2011, 13, 461-471) and
U87-EGFRvIII overexpressing EGFRvIII (construction method can be
found in WO/2011/035465), A431, CAL27, MDA-MB-468 endogenously
overexpressing EGFR, and especially strongly bind to U87-EGFRvIII
and A431 cells, but its binding ability was not as high as that of
single-chain antibody 806. Binding ability of the single-chain
antibody C225 to these cells is very strong. These single-chain
antibodies have little binding to U87 cells.
[0223] In addition, both Y022 and 806 single-chain antibodies
almost did not bind to glioma cell line U87 cells. Especially,
single chain antibody Y022 also does not bind to Normal prostate
epithelial cells RWPE-1 and human primary keratinocyte K2, whereas
single-chain antibody 806 binds to both of these normal cells with
different degrees.
[0224] These results indicate that the single chain antibody Y022
specifically binds to tumor cells overexpressing EGFR as well as
EGFRvIII, while little binding to normal EGFR-expressing cells.
Example 7. Construction of Expression Vector Containing Nucleotide
Sequence Encoding Y022/CD3 Single Chain Bifunctional Antibodies
[0225] PCR amplification was performed using the pCantab 5E-Y022
plasmid obtained in Example 3 as a template and a primer pair, the
forward primer pH7B3f2_fw (SEQ ID NO: 20,
GATATTCAGATGACCCAGAGCCCGAGCAG) and the reverse primer pH7B3f2_re
(SEQ ID NO: 21, AATAGGATCCACCACCTCCGCTGCTCACGGTCAC) to obtain DNA
fragment of Y022 scFv. Another DNA fragment containing the pH
vector signal peptide sequence was obtained by PCR using pH-7B3/CD3
plasmid (see 201210094008.X Example 3 and FIG. 2) as a template and
the forward primer pH7B3f1_fw (SEQ ID NO: 22
CCATTGACGCAAATGGGCGGTAGG) and reverse primer pH7B3f1_re (SEQ ID NO:
23, CTGCTCGGGCTCTGGGTCATCTGAATATC). The two fragments were mixed in
equimolar ratio for fragment splicing and PCR. The splicing
conditions were: denaturation: 94.degree. C. for 4 min;
denaturation: 94.degree. C. for 40 s; annealing: 60.degree. C. for
40 s; extension: 68.degree. C. for 140 s for 5 cycles, and then the
total extension of 68.degree. C., 10 min. And then DNA polymerase
and forward primer pH7B3f1_fw and reverse primer pH7B3f2_re were
supplemented, 30 cycles of amplification were performed, and
amplification conditions were: 94.degree. C., 4 min; denaturation:
94.degree. C., 40 s; annealing: 60.degree. C., 40 s; 68.degree. C.
for 140 s for 30 cycles and then total extension 68.degree. C. for
10 min.
[0226] The amplified sequence was digested with the restriction
endonuclease NheI/BamHI and double-digested according to the
reaction conditions recommended by the enzyme supplier (New England
Biolabs, NEB). The expression vector pH (see 201210094008.X Example
3 and FIG. 2) was also similarly digested with the restriction
enzyme NheI/BamHI. The double-digested Y022 scFv fragment and pH
vector fragment were then ligated with T4 DNA ligase following the
reaction conditions recommended by the enzyme supplier (NEB). The
nucleotide sequence encoding for Y022 single chain antibody
polypeptide thus obtained was cloned into a vector, and was
transcribed together with the nucleotide sequence already contained
in the vector encoding CD3 single-chain antibody polypeptide into
an mRNA, which finally translated into Y022/CD3 single-chain
bifunctional antibody polypeptide. The new plasmid was named as
pH-Y022/CD3, and the detailed structure was shown in FIG. 6.
Example 8. Expression and Purification of Single Chain Bifunctional
Antibody Y022/CD3, pH-806/CD3 and pH-C225/CD3
[0227] The expression vectors pH-Y022/CD3, pH-806/CD3 and
pH-C225/CD3 (see 201210094008.X) were transfected into Chinese
hamster ovary (CHO) cells according to the procedure of FreeStyle
MAX Reagent Transfection Reagent (from Invitrogen). And then the
stable clones were screened according to OptiCHO.TM. protein
expression kit (from Invitrogen). Stable clones of CHO cells
transfected with each of the above expression vectors were cultured
in shake flasks at 37.degree. C. for 7 days at 130 rpm, and the
used medium was CD OptiCHO (from Gibco). The culture supernatant
was obtained by centrifugation and then stored at -20.degree.
C.
[0228] Protein purification was performed using a histidine
affinity column (His Trap HP column, available from GE Healthcare)
according to the manufacturer's method steps. Specifically, the
column was equilibrated with buffer A (20 mM sodium phosphate pH
7.4, 0.4 M NaCl) and then the cell culture supernatant (500 mL of
the supernatant) was added to the column (1 mL) with a flow rate of
3 ml/min after dialysis against PBS. The column was then washed
with 5 volumes of buffer A and 10 volumes of buffer A containing 50
mM imidazole to remove the impurity protein. The bound protein of
interest was eluted with the same buffer A supplemented with 250 mM
imidazole. All purification steps were performed at 4.degree.
C.
[0229] Purified single-chain bifunctional antibodies were detected
by reducing SDS-PAGE. As shown in FIG. 7, the molecular weights of
these antibody molecules were all around 60 kD, which corresponded
to the molecular weight of the single-chain bifunctional antibody
calculated from the amino acid sequences.
Example 9. Analysis of Antigen Binding Specificity of Single Chain
Bifunctional Antibody Such as Y022/CD3
[0230] The binding capacities of single chain bifunctional antibody
Y022/CD3 to EGFR were analyzed by fluorescence activated cell
sorter (FACS) (BD, FACS Calibur).
[0231] Specific methods are as follows:
[0232] 1. Tumor cells in logarithmic growth phase as listed in
Table 2 were inoculated into a 6 cm dish with inoculation cell
density of about 90%, and incubated at 37.degree. C. incubator
overnight.
[0233] 2. Cells were digested with 10 mM EDTA and cells were
collected by centrifugation at 200 g for 5 min. Cells were
resuspended in 1% phosphate buffered saline (NBS PBS) containing
calf serum at a concentration of 1.times.10.sup.6 to
1.times.10.sup.7/mL, and added in a flow tube in an amount of 100
.mu.l/tube.
[0234] 3. Cells were centrifuged at 200 g.times.5 min, and the
supernatant was discarded.
[0235] 4. Antibody Y022/CD3 to be tested was added. And irrelevant
antibody NGR/CD3 was used as a negative control. The final
concentration of antibody was 5 .mu.g/ml, 100 .mu.l was added to
each tube and placed in an ice bath for 45 minutes.
[0236] 5. 2 ml of 1% NBS PBS was added to each tube and centrifuged
at 200 g.times.5 min for two times.
[0237] 6. Supernatant was discarded and mouse anti-his tag antibody
(from Shanghai Genomics Technology Co., Ltd.) at a dilution of 1:50
was added, and 100 ul was added to each tube and placed in an ice
bath for 45 minutes.
[0238] 7. 2 ml of 1% NBS PBS was added to each tube and centrifuged
at 200 g.times.5 min for two times.
[0239] 8. Supernatant was discarded and FITC fluorescent-labeled
goat anti-mouse antibody (from Shanghai Kangcheng Bio-engineering
Company) at a dilution of 1:50 was added, and 100 ul was added to
each tube and placed in an ice bath for 45 minutes.
[0240] 9. 2 ml of 1% NBS PBS was added to each tube and centrifuged
at 200 g.times.5 min for two times.
[0241] 10. Supernatant was discarded, resuspended in 300 ul of 1%
NBS PBS and detected by flow cytometry.
[0242] 11. Data was analyzed by using flow cytom6etry data analysis
software WinMDI 2.9.
[0243] Results are shown in FIG. 8, the bifunctional antibody
Y022/CD3 of the present invention can bind to U87-EGFR,
U87-EGFRvIII and A431 cells, however, hardly bind to U87 and human
keratinous epithelial cells. These results indicate that Y022/CD3
can specifically bind to tumor cells expressing mutant human EGFR
and overexpressing EGFR, but not to tissues that normally express
EGFR.
[0244] In addition, Y022/CD3 can also bind to human peripheral
blood mononuclear cells (PBMCs) or Jurkat cells (human peripheral
blood leukemia T cells, CD3 positive) as shown in the figure,
suggesting that the bifunctional antibody of the present invention
can specifically bind to CD3 antigen of T cell surface.
[0245] The expression plasmids were constructed according to the
methods mentioned in Examples 7 and 8, respectively (Y022 in
Examples 7 and 8 were replaced with other mutated forms of
antibodies), and M14/CD3, M15/CD3, M25/CD3, M26/CD3, S7/CD3,
S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3 were expressed and
purified. According to the method of this example, the binding
abilities of these antibodies to U87-EGFRvIII overexpressing
EGFRvIII and to CAL27 cells endogenously overexpressing EGFR were
determined respectively. The above antibodies were able to bind
both of these cells, and their mean fluorescence intensity (MFI)
values are shown in Table 13.
TABLE-US-00027 TABLE 13 Antibody U87MG-EGFRvIII CAL 27 PBS 1 3.11
M14 36.52 28.39 M15 37.86 29.43 M25 36.52 24.14 M26 41.42 24.58 S7
42.17 27.88 S8 38.54 29.96 S17 31.62 25.03 S22 31.34 24.58 S23 32.2
29.69 S29 34.6 25.71
Example 10. Biological Activity Analysis of Single-Chain
Bifunctional Antibody, Such as Y022/CD3--Cytotoxicity to Various
Tumor Cells
[0246] Peripheral blood mononuclear cells (PBMCs) were isolated
from healthy human-donated blood following standard procedures
using Ficoll (from Biochrom) density gradient centrifugation. After
centrifugation, the cells were washed with phosphate buffered
saline (PBS) at a concentration of 0.1 M and then resuspended in
RPMI 1640 complete medium (Gibco) and the cell concentration was
adjusted to 5.times.10.sup.5/mL. PBMCs served as effector cells in
cytotoxicity experiments. Different tumor cells act as target
cells. The target cell concentration was adjusted to
5.times.10.sup.4/mL with RPMI 1640 complete medium. The same volume
of target cells and effector cells were mixed such that the
effector cell: target cell (E:T) ratio was 10:1.
[0247] The mixed cell suspension was added to a 96-well plate in a
volume of 75 .mu.L/well. Then 25 .mu.L of the following reagent
serially diluted ten times from 1000 ng/mL to 0.1 ng/mL was added
to each well:
[0248] (1) Y022/CD3 Single chain bifunctional antibody (BiTe);
[0249] (2) RPMI 1640 complete medium (background);
[0250] (3) NGR/CD3 Single chain bifunctional antibody (negative
control, NGR was a neovascular targeting peptide that has no
cross-binding site with EGFR, and prepared according to a
conventional method)
[0251] After incubation for 40 hours in a 37.degree. C., 5%
CO.sub.2 incubator, the cytotoxicity of the antibody was tested
using CytoTox 96R Non-Radioactive Cytotoxicity Assay kit (from
Promega) according to the manufacturer's instructions.
[0252] The CytoTox 96@ Non-Radioactive Cytotoxicity Assay is a
colorimetric based assay that can replace 51Cr release assay.
CytoTox 96@ Assay measures lactate dehydrogenase (LDH)
quantitatively. LDH is a stable cytosolic enzyme that is released
upon lysis of cells and is released in the same way as radioactive
51Cr is released. The supernatant with released LDH medium can be
detected by a 30-minute coupled enzyme reaction in which LDH
converts a tetrazolium salt (INT) to a red formazan. The amount of
red product produced is proportional to the number of lysed
cells.
[0253] Five EGFR-associated tumor cells as listed in Table 3 below
were used to analyze T cell tumors killing ability mediated by the
bifunctional antibody Y022/CD3 of the present invention and the
NGR/CD3 single-chain bifunctional antibody that is not associated
with EGFR as a control, respectively.
[0254] Tumor cell killing rate (i.e., cytotoxicity %) was
calculated based on the following formula provided in the manual of
CytoTox 96@ non-radioactive cytotoxicity assay G1780:
cytotoxicity %=[(experiment-effector cell spontaneously-target cell
spontaneously)/(target cell maxium-target cell
spontaneously)].times.100
[0255] wherein:
[0256] "Experiment" refers to LDH release produced in the
experimental wells, in which the antibody/effector/target cells
were added,
[0257] "effector cell spontaneously" refers to LDH release produced
by effector cells spontaneously,
[0258] "target cell spontaneously" refers to LDH release produced
by cells that are not treated by other factors,
[0259] "target cell maxium" refers to LDH release resulting from
complete lysis of target cells after treatment with 0.8% Triton
X-100,
[0260] "target cell maxium-target cell spontaneously" refers to LDH
release resulting from complete lysis of cells after external
treatment.
TABLE-US-00028 TABLE 3 1000 ng/ml 1000 ng/ml Tumor Cytotoxicity
Cytotoxicity cell line % of Y022/CD3 % of NGR/CD3 U87 2.0 3.4
U87-EGFR 32.1 3.7 U87-EGFRvIII 66.2 6.3 A431 48.7 5.2 K2 5.1
4.5
[0261] The results of Table 3 above show that all of the tumor
cells expressing mutant EGFR and/or overexpressing EGFR, such as
U87-EGFRvIII, U87-EGFR and A431, will be specifically killed by
T-cells directed by the bifunctional antibody Y022/CD3.
[0262] Specifically, in the above tumor cell group treated with
Y022/CD3, the minimum specific cytotoxicity was 32.1% and the
maximum was 66.2%. While the cytotoxicity of Y022/CD3 to cells
expressing low levels of EGFR, U87, and human primary keratinocytes
was very low at 3.4% and 4.5%, respectively, which were
significantly lower than those to the above-mentioned mutant cells
expressing EGFR and/or overexpressing EGFR.
[0263] More specifically, cytotoxicity % of Y022/CD3 and control
antibody NGR/CD3 at various concentrations to each tumor is shown
in Tables 4-8 below.
TABLE-US-00029 TABLE 4 U87 ng/ml NGR/CD3 Y022/CD3 1000 3.4 .+-. 1.2
2.0 .+-. 1.3 100 4.8 .+-. 1.1 1.6 .+-. 3.2 10 4.3 .+-. 1.5 2.5 .+-.
2.3 1 5.2 .+-. 2.1 0.5 .+-. 1.2 0.1 5.4 .+-. 2.2 0.2 .+-. 1.7
TABLE-US-00030 TABLE 5 U87-EGFR ng/ml NGR/CD3 Y022/CD3 1000 3.7
.+-. 2.6 32.1 .+-. 3.1 100 4.9 .+-. 1.7 21.7 .+-. 4.4 10 4.3 .+-.
2.7 12.6 .+-. 3.2 1 3.3 .+-. 1.9 6.3 .+-. 2.6 0.1 0.7 .+-. 1.2 5.1
.+-. 2.0
TABLE-US-00031 TABLE 6 U87-EGFRvIII ng/ml NGR/CD3 Y022/CD3 1000 6.3
.+-. 1.3 66.2 .+-. 5.8 100 7.4 .+-. 2.4 52.5 .+-. 4.5 10 6.5 .+-.
0.8 33.6 .+-. 3.2 1 4.7 .+-. 2.1 25.3 .+-. 2.9 0.1 2.6 .+-. 1.4 6.7
.+-. 2.3
TABLE-US-00032 TABLE 7 A431 ng/ml NGR/CD3 Y022/CD3 1000 5.2 .+-.
2.9 48.7 .+-. 4.3 100 5.6 .+-. 2.7 35.3 .+-. 5.1 10 3.7 .+-. 2.4
22.7 .+-. 3.3 1 1.3 .+-. 0.6 10.8 .+-. 4.4 0.1 1.5 .+-. 1.1 4.3
.+-. 2.1
TABLE-US-00033 TABLE 8 K2 ng/ml NGR/CD3 Y022/CD3 1000 4.5 .+-. 2.2
5.1 .+-. 1.1 100 4.1 .+-. 2.8 3.2 .+-. 1.2 10 3.5 .+-. 2.4 1.7 .+-.
1.0 1 2.1 .+-. 1.8 2.7 .+-. 1.2 0.1 4.3 .+-. 2.9 2.1 .+-. 1.3
[0264] In addition, in vitro toxicity analysis was performed by the
same method on the following expressed and purified BiTe: M14/CD3,
M15/CD3, M25/CD3, M26/CD3, S7/CD3, S8/CD3, S17/CD3, S22/S29/CD3,
and the results are shown in FIG. 9.
[0265] As can be seen in FIG. 9, tumor cells expressing mutated
EGFR and/or overexpressing EGFR, such as U87-EGFRvIII, U87-EGFR,
and CAL27, can be killed by T cells directed by the bifunctional
specific antibodies M14/CD3, M15/CD3, M25/M26/CD3, S7/CD3, S8/CD3,
S17/CD3, S22/CD3, S23/CD3, S29/CD3 to different degrees. While for
U87, a cell that expresses low levels of EGFR, there is little
killing effect.
Example 11. Construction of Lentiviral Plasmid Expressing the
Chimeric Antigen Receptor Protein Encoded by the Nucleic Acid of
the Present Invention and Virus Packaging
[0266] Construction of the chimeric antigen receptor, and the
connection order of chimeric antigen receptor exemplified in the
present invention, is shown in Table 9 and FIG. 10.
TABLE-US-00034 TABLE 9 Extracellular binding region - transmembrane
region - intracellular signal region 1 - chimeric antigen
intracellular signal region 2 and receptor the like Description
Y022-.delta.Z scFv(EGFR)-CD8-CD3.delta.zeta Negative control Y022-Z
scFv(EGFR)-CD8-CD3 zeta 1.sup.st generation Y022-BBZ
scFv(EGFR)-CD8-CD137-CD3 zeta 2.sup.nd generation Y022-28Z
scFv(EGFR)-CD28a-CD28b-CD3 zeta 2.sup.nd generation Y022-28BBZ
scFv(EGFR)-CD28a-CD28b-CD137- 3.sup.rd CD3 zeta generation Note:
CD28a represents the transmembrane region of CD28 molecule and
CD28b represents the intracellular signaling region of CD28
molecule.
[0267] 1. Amplification of Nucleic Acid Fragments
[0268] (1) Amplification of scFv Sequences
[0269] Y022 scFv was obtained by PCR using pCantab 5E-Y022 plasmid
as a template with a forward primer (SEQ ID NO: 24, comprising part
of the sequence of CD8 signal peptide) and a reverse primer (SEQ ID
NO: 25, comprising part of the sequence of CD8 hinge).
TABLE-US-00035 SEQ ID NO: 24
(TGCTCCACGCCGCCAGGCCGGATATTCAGATGACCCAG) SEQ ID NO: 25
(CGCGGCGCTGGCGTCGTGGTGCTGCTCACGGTCAC)
[0270] (2) Nucleic Acid Sequences of Other Parts of the Chimeric
Antigen Receptor
[0271] The nucleic acid sequences of other parts of the
anti-EGFRvIII chimeric antigen receptor protein except for Y022
scFv were respectively obtained by PCR using the sequences SEQ ID
NO: 26, 27, 28, 29 and 30 disclosed in Patent Application No.
201310164725.X as templates. Specifically, the eGFP-F2A-CD8sp
sequence was obtained by PCR amplification using SEQ ID NO: 27
plasmid contained in Patent Application No. 201310164725.X as a
template and primer pairs (SEQ ID NOs: 26 and 27). CD8-CD36 zeta
(6Z) was obtained by PCR amplification using SEQ ID NO: 26 plasmid
in the patent application CN201310164725.X as a template and primer
pairs (SEQ ID NOs: 28 and 29). The CD8-CD3 zeta (Z), CD8-CD137-CD3
zeta (BBZ), CD28.alpha.-CD28b-CD3 zeta (28Z) and
CD28a-CD28b-CD137-CD3 zeta (28BBZ) were obtained by PCR
amplification respectively using SEQ ID NO: 27, SEQ ID NO: 28, SEQ
ID NO: 29 and SEQ ID NO: 30 in the patent application
CN201310164725.X as templates and primer pairs (SEQ ID NO: 28,
30).
TABLE-US-00036 SEQ ID NO: 26 (TGCAGTAGTCGCCGTGAAC) SEQ ID NO: 27
(CGGCCTGGCGGCGTGGAGCA) SEQ ID NO: 28 (ACCACGACGCCAGCGCCGCGACCAC)
SEQ ID NO: 29 (GAGGTCGACCTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCACTCT)
SEQ ID NO: 30 (GAGGTCGACCTAGCGAGGGGGCAGGGCCTGCATGTGAAG)
[0272] 2. Splicing of Nucleic Acid Fragments
[0273] eGFP-F2A-CD8sp nucleic acid fragment obtained as described
above and equimolar Y022 scFv nucleic acid fragment and equimolar
CD8-CD36 zeta (6Z) or CD8-CD3 zeta (Z) or CD8-CD137-CD3 zeta (BBZ)
or CD28a-CD28b-CD3 zeta (28Z) or CD28a-CD28b-CD137-CD3 zeta (28BBZ)
nucleic acid fragments were subjected to three-segment splicing and
PCR as shown in FIG. 9 under the following conditions:
Pre-denaturation: 94.degree. C. for 4 min; denaturation: 94.degree.
C. for 40 s; annealing: 60.degree. C. for 40 s; extension:
68.degree. C. for 140 s for 5 cycles and then total extension
68.degree. C. for 10 min. DNA polymerase and forward primer (SEQ ID
NO: 24) and reverse primer (reverse primer corresponding to
CD8-CD36 zeta was SEQ ID NO: 29, and other is SEQ ID NO: 30) were
supplemented, and then PCR was performed for 30 cycles with the
amplification conditions: Pre-denaturation: 94.degree. C. for 4
min; denaturation: 94.degree. C. for 40 s; annealing: 60.degree. C.
for 40 s; extension: 68.degree. C. for 140 s for 30 cycles and then
total extension 68.degree. C. for 10 min. The amplified fragments
were referred to as (Table 2):
[0274] eGFP-F2A-Y022 scFv-6Z (SEQ ID NO: 31),
[0275] eGFP-F2A-Y022 scFv-Z (SEQ ID NO: 32),
[0276] eGFP-F2A-Y022 scFv-BBZ (SEQ ID NO: 33),
[0277] eGFP-F2A-Y022 scFv-28Z (SEQ ID NO: 34),
[0278] eGFP-F2A-Y022 scFv-28BBZ (SEQ ID NO: 35).
[0279] 3. Construction of Lentiviral Plasmid Vector
[0280] By way of example, the vector system used for the lentiviral
plasmid vectors constructed below belongs to self-inactivating
lentiviral vector system of the third generation, which has three
plasmids, namely, packaging plasmid psPAX2 encoding protein
Gag/Pol, encoding Rev protein (from addgene); envelope plasmid
PMD2.G encoding VSV-G protein (from addgene); and the recombinant
expression vector encoding the gene of interest CAR based on empty
vector pWPT-eGFP (from addgene).
[0281] In the empty vector pWPT-eGFP, the expression of enhanced
green fluorescent protein (eGFP) is regulated by elongation
factor-1.alpha. (EF-1.alpha.) promoter. After inserting the
constructs constructed as described in this example into an empty
vector, a recombinant expression vector encoding CAR of the target
gene was formed, wherein co-expression of eGFP and target gene CAR
is achieved ribosomal skipping sequence from food and mouth disease
virus (FMDV, F2A). F2A is a core sequence of 2A (or "self-cleaving
polypeptide 2A") from foot-and-mouth disease virus possessing the
"self-shearing" function of 2A that enables upstream and downstream
gene co-expression. 2A provides an effective and viable strategy
for constructing polycistronic vectors for gene therapy due to its
high shearing efficiency, high upstream and downstream gene
expression balance and short sequence itself. Especially in
immunotherapy based on chimeric antigen receptor gene modified T
lymphocytes, this sequence is frequently used to achieve the
co-expression of the target gene with GFP or eGFP. The expression
of CAR can be indirectly detected by detecting GFP or eGFP.
[0282] In this example, a lentiviral expression vector
co-expressing eGFP and specific CAR linked by F2A was constructed,
collectively referred to as pWPT-eGFP-F2A-CAR. The target gene
eGFP-F2A-CAR (see 2 in Example 7, the component after F2A is
abbreviated as CAR) obtained in the above step 2 was
double-digested by MluI and SalI restriction enzymes and ligated
into the same double digested pWPT vector to construct a lentiviral
vector expressing each chimeric antigen receptor. The constructed
vector was identified by MluI and SalI digestion and sequenced
correctly, which was ready for lentivirus packaging. As mentioned
above, eGFP-F2A-CAR was transcribed into one mRNA but eventually
translated into two peptide chains of eGFP and anti-EGFRvIII
chimeric antigen receptors, where the anti-EGFRvIII chimeric
antigen receptor will be localized under the guidance of CD8a
signal peptide on the cell membrane.
[0283] The vectors containing the desired CARs are as follows (the
components following F2A may be abbreviated as CAR):
[0284] pWPT-eGFP-F2A-Y022 scFv-6Z;
[0285] pWPT-eGFP-F2A-Y022 scFv-Z;
[0286] pWPT-eGFP-F2A-Y022 scFv-BBZ;
[0287] pWPT-eGFP-F2A-Y022 scFv-28Z;
[0288] pWPT-eGFP-F2A-Y022 scFv-28BBZ.
[0289] 5 eGFP-F2A-CAR polypeptide sequences were respectively
obtained through the above construction, which are named as:
[0290] eGFP-F2A-Y022 scFv-6Z (SEQ ID NO: 36);
[0291] eGFP-F2A-Y022 scFv-Z (SEQ ID NO: 37);
[0292] eGFP-F2A-Y022 scFv-BBZ (SEQ ID NO: 38);
[0293] eGFP-F2A-Y022 scFv-28Z (SEQ ID NO: 39);
[0294] eGFP-F2A-Y022 scFv-28BBZ (SEQ ID NO: 40).
[0295] 4. Plasmid-Transfected 293T Packaging Lentivirus
[0296] HEK-293T cells (ATCC: CRL-11268) cultured at passage 6 to
passage 10 were seeded at a density of 6.times.10.sup.6 in 10 cm
dishes and cultured overnight at 37.degree. C. in 5% CO.sub.2 for
transfection. The medium was DMEM (available from PAA) containing
10% fetal bovine serum (purchased from PAA).
[0297] Transfection steps are as follows:
[0298] 4.1 Preparation of liquid A: dissolving 10 .mu.g of mock
control or 10 .mu.g of each of the desired gene plasmids
pWPT-eGFP-F2A-CAR with 7.5 .mu.g of packaging plasmid PAX2: and 3
.mu.g of envelope plasmid pMD2.G into 800 .mu.L of serum-free DMEM
medium and mixing well.
[0299] 4.2 Preparation of liquid B: dissolving 60 .mu.g PEI
(polyethylenimine, purchased from Polysciences) in 800 .mu.L
serum-free DMEM medium, mixing gently and incubating at room
temperature for 5 min.
[0300] 4.3 Formation of transfection complex: adding liquid A into
liquid B and gently mixing, vortexing or gently mixing immediately
after addition, incubating at room temperature for 20 min.
[0301] 4.4 Adding 1.6 ml of the transfection complex into HEK-293T
cells dropwise, and after 4-5 h, changing to DMEM with 2% FBS for
transfected 293T cells.
[0302] In the next day after transfection, the transfection
efficiency (that is, the proportion of green fluorescent cells) was
observed: .about. 80% of the positive transfection efficiency
represents the successful transfection experiments. After 72 h of
transfection, the virus was collected by filtration using a 0.45
.mu.m filter (available from Millipore Corporation) and centrifuged
at 28,000 rpm using a Beckman Optima L-100XP ultracentrifuge for 2
hours at 4.degree. C. The supernatant was discarded and the
resulting pellet was centrifuged at 1/10.about. 1/50 stock solution
of AIM-V (purchased from Invitrogen) and resuspend at 100
.mu.L/tube in -80.degree. C. for virus titration or infection of T
lymphocytes.
[0303] 5. Determination of Lentiviral Titers Packaged with Mock or
eGFP-F2A-CAR
[0304] On the first day, 293T cells were inoculated at
1.times.10.sup.5/mL in 96-well culture plates, 100 .mu.L/well, and
cultured at 37.degree. C., 5% CO.sub.2, and the culture medium was
DMEM containing 10% fetal bovine serum. On the next day, 50
.mu.L/well of culture supernatant was discarded, 50 .mu.L/well of
fresh medium was supplemented, and polybrene at final concentration
of 6 .mu.g/mL was contained. The culture was incubated for 30 min
at 37.degree. C. with 5% CO.sub.2. 10 .mu.L/well of virus stock or
1 .mu.L/well of virus concentrate was added (3-fold diluted, 6
gradients, two replicate wells) and incubated at 37.degree. C. in
5% CO.sub.2. 48 h after infection, eGFP was detected by flow
cytometry, cells with 5 to 20% of the positive rate are appropriate
to calculate the titer (U/mL)=positive rate.times.dilution
times.times.100.times.10.sup.4. The titers of virus comprising the
above-mentioned mock empty vector control and each eGFP-F2A-CAR
packaged in the PEI transfection method were both about 0.5 to
1.times.10.sup.7U/mL, and the detected virus titer after
concentration was about 0.5.about. 1.times.10.sup.8U/mL.
Example 12. T Cells Infected by Recombinant Lentivirus
[0305] Human peripheral blood mononuclear cells were obtained from
healthy human peripheral blood by density gradient centrifugation
(supplied by Shanghai Blood Center) and added in AIM-V lymphocyte
medium (purchased from Invitrogen) at a density of about
2.times.10.sup.6/mL and added. The magnetic beads coated with
anti-CD3 and CD28 antibodies (Invitrogen) were added in a 1:1 ratio
of cells to magnetic beads, and recombinant human IL-2 (purchased
from Shanghai Huaxin Biotechnology Co., Ltd.) at a final
concentration of 300 U/mL was added for stimulation and culture for
48 h. And then T cells were infected with the above recombinant
lentivirus (MOI.apprxeq.15). Infected T cells were detected by flow
cytometry on day 8 of culture for the expression of different
chimeric antigen receptors. Since eGFP was co-expressed with CAR,
the detected eGFP-positive cells were positive cells expressing
chimeric antigen receptors. Using uninfected T lymphocytes as a
negative control, the positive rates of virus-infected T cells
expressing different chimeric antigen receptors are shown in Table
10. The positive rate results show that a certain positive rate of
CAR T cells can be obtained by lentivirus infection.
TABLE-US-00037 TABLE 10 T cells transfected eGFP positive rate by
following CARs of CAR T cells Y022-.delta.Z(Mock) 66% Y022-Z 58%
Y022-BBZ 53% Y022-28Z 54% Y022-28BBZ 52%
[0306] T cells were infected with viruses that had different
chimeric antigen receptors packaged, respectively, and then
subcultured at a cell density of 5.times.10.sup.5/ml quaque die
alterna, counted, and supplemented with IL-2 (final concentration
of 300 U/ml). On the 11th day of culture, about 100.about.1000
times of amplification was obtained, indicating that the T cells
expressing different chimeric antigen receptors can be expanded in
a certain amount in vitro, which ensures subsequent in vitro
toxicity tests and in vivo experiments.
Example 13. In Vitro Antitumor Activity of CAR-Y022
[0307] In vitro toxicity experiments used the following
materials:
[0308] The target cells were U87, U87-EGFR, U87-EGFRvIII, A431, CAL
27, MDA-MB-468, RWPE-1 cells and human primary keratinocyte K2 as
shown in Table 5, respectively. Effector cells were T lymphocytes
(CAR T cells) cultured for 12 days in vitro, which were detected
chimeric antigen receptor positive by FACS.
[0309] Effective target ratios were 3:1, 1:1 and 1:3, respectively.
The number of target cells was 10000/well, and each group had 5
replicate wells. Detection time was 18 h.
[0310] Each experimental group and each control group are listed as
follows:
[0311] Each experimental group: each target cell+CAR T lymphocytes
expressing different chimeric antigen receptors;
[0312] Control group 1: target cells with maxium LDH release;
[0313] Control group 2: target cells with spontaneous LDH
release;
[0314] Control group 3: effector cells with spontaneous LDH
release.
[0315] Detection method: CytoTox 96.RTM. Non-Radioactive
Cytotoxicity Assay (Promega) is used, which is a colorimetric based
assay that can replace 51Cr release assay. CytoTox 96.RTM. Assay
measures lactate dehydrogenase (LDH) quantitatively. LDH is a
stable cytosolic enzyme that is released upon lysis of cells and is
released in the same way as radioactive 51Cr is released. The
supernatant with released LDH medium can be detected by a 30-minute
coupled enzyme reaction in which LDH converts a tetrazolium salt
(INT) to a red formazan. The amount of red product produced is
proportional to the number of lysed cells. Details can be found in
instructions of CytoTox 96 non-radioactive cytotoxicity detection
kit.
[0316] Cytotoxicity is calculated as:
Cytotoxicity %=[(experiment group-control group 2-control group
3)/(control group 1-control group 2)].times.100
[0317] Specifically, as shown in Table 11 and Table 12, compared
with 806-CAR T, Y022-28Z CAR T and Y022-28BBZ CAR T expressing
chimeric antigen receptors of the present invention at different
effector target ratios showed a significantly killing effects on
cells highly expressing EGFR and EGFRvIII and a effector target
ratio gradient dependency, that is, the higher the effector target
ratio, the stronger the cytotoxic effects.
Effector-target-ratio-dependency data further demonstrate the
specific cytotoxic effects of CAR T cells expressing chimeric
antigen receptors of the invention on cells that highly express
EGFR and its variants.
[0318] It is noteworthy that Y022-CAR T has almost no killing
effect on RWPE-1 cells that normally express EGFR and human primary
keratinocytes K2. At the effector target ratio of 3:1, Cytotoxicity
of the chimeric antigen receptor Y022-28BBZ CAR T-lymphocyte to
RWPE-1 cells and human primary keratinocytes K2 was 12% and 2%,
respectively. Cytotoxicity of Y022-28Z CAR T lymphocytes to RWPE-1
cells and human primary keratinocytes K2 was 8% and 3%. In
contrast, 806-CAR T had different degrees of cytotoxicity on both
of these cells. The cytotoxicity of 806-28BBZ CAR T lymphocytes to
RWPE-1 cells and human primary keratinocytes K2 were 25% and 22%,
respectively, and the cytotoxicity of 806-28Z CAR T lymphocytes to
RWPE-1 cells and human primary keratinocytes K2 was 15% and 13%,
respectively.
[0319] In addition, CAR T, as a negative control, transfected with
a virus containing the mock plasmid (carrying scFv-Y022-6Z) showed
very low cytotoxic effects on the above cell lines.
[0320] The above results indicate that the chimeric antigen
receptor Y022-CAR T, which is constructed from a single chain
antibody against EGFR and its variants, can selectively kill tumor
cells that highly express EGFR and its variant (EGFRvIII), while
hardly kill cells normally expressing EGFR. In addition, from the
cytotoxicity data, CAR T of the third generation (Y022-28BBZ) was
more cytotoxic to target cells than the second generation
(Y022-28Z) CART.
TABLE-US-00038 TABLE 11 Y022-28BBZ Y022-28Z mock Different effector
Different effector Different effector target ratio target ratio
target ratio Cytotoxicity % 3:1 1:1 1:3 3:1 1:1 1:3 3:1 1:1 1:3 U87
2 4 5 4 3 1 -4 -3 2 U87-EGFR 35 17 6 25 16 7 -1 5 1 U87-EGFRvIII 73
42 19 65 31 9 3 0.3 1 A431 67 37 12 45 24 5 9 7 4 CAL27 70 49 15 50
23 8 7 5 5 MDA-MB-468 57 45 14 41 23 1 6 8 7 RWPE-1 12 7 3 8 6 4 3
2 0.2 K2 11 2 0.3 3 0.6 2 -3 -1 2
TABLE-US-00039 TABLE 12 806-28BBZ 806-28Z mock Different effector
Different effector Different effector target ratio target ratio
target ratio Cytotoxicity % 3:1 1:1 1:3 3:1 1:1 1:3 3:1 1:1 1:3 U87
3 4 2 5 0.6 2 1 3 -3 U87-EGFR 68 49 18 55 36 12 3 7 4 U87-EGFRvIII
83 51 23 75 42 15 6 5 2 A431 75 48 16 56 35 12 7 7 5 CAL27 81 57 25
65 33 18 3 6 2 MDA-MB-468 62 49 32 51 32 16 6 9 7 RWPE-1 29 17 10
15 10 7 -2 -3 2 K2 27 15 2 13 9 4 0.2 1 3
[0321] All references mentioned in the present application are
incorporated herein by reference, as if each reference was
individually incorporated by reference. In addition, it should be
understood that after reading the above teachings of the present
invention, those skilled in the art can make various modifications
or changes to the present invention, and such equivalent forms also
fall within the scope of the appended claims of the present
application.
Sequence CWU 1
1
77116PRTHomo Sapiens 1Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp
Gly Val Arg Lys Cys1 5 10 152717DNAArtificial sequenceencoding
sequence of single chain antibody 7B3 2gatattcaga tgacccagag
cccgagcagc ctgagcgcga gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca
ggatattaac agcaacattg gctggctgca gcagaaaccg 120ggcaaagcgt
ttaaaggcct gatttatcat ggcaaaaacc tggaagatgg cgtgccgagc
180cgttttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag
cctgcagccg 240gaagattttg cgacctatta ttgcgttcag tacgcccagt
tcccatatac atttggccag 300ggcaccaaag tggaaattaa acgtggtgga
ggcggttcag gcggaggtgg ctctggcggt 360ggcggatcgg atgtgcagct
ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc 420ctgcgtctga
gctgcgcggt gagcggctat agcattacca gcgattatgc gtggaactgg
480attcgtcagg cgccgggcaa aggcctggaa tggctgggct atattagcta
tcgtggccgc 540accagctata acccgagcct gaaaagccgt attagcatta
cccgtgataa cagcaaaaac 600acctttttcc tgcagctgaa cagcctgcgt
gcggaagata ccgcggtgta ttattgcgcg 660cgcctgggac gcggcttccg
ctactggggc cagggcaccc tggtgaccgt gagcagc 7173239PRTArtificial
sequenceamino acid sequence of single chain antibody 7B3 3Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp
Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp Ile Asn Ser Asn 20 25
30Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile
35 40 45Tyr His Gly Lys Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala
Gln Phe Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Gly Gly Gly Gly 100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Asp Val Gln Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly
Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170
175Tyr Arg Gly Arg Thr Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser
180 185 190Ile Thr Arg Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu
Asn Ser 195 200 205Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Arg Leu Gly Arg 210 215 220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser225 230 235442DNAArtificial sequencePrimer
4ataacaggcc cagccggcca tggatattca gatgacccag ag 42569DNAArtificial
sequencePrimer(26)..(27)n is a, c, g, or t(32)..(33)n is a, c, g,
or t(35)..(36)n is a, c, g, or t(38)..(39)n is a, c, g, or
t(41)..(42)n is a, c, g, or t(47)..(48)n is a, c, g, or t
5cactttggtg ccctggccaa atgtmnntgg gnnmnnmnnm nnctgmnngc aataataggt
60cgcaaaatc 69622DNAArtificial sequencePrimer 6acatttggcc
agggcaccaa ag 22729DNAArtificial sequencePrimer 7ataaatgcgg
ccgcgctgct cacggtcac 29822DNAArtificial sequencePrimer 8tcgcaattcc
tttagttgtt cc 22957DNAArtificial sequencePrimer(23)..(24)n is a, c,
g, or t(26)..(27)n is a, c, g, or t(29)..(30)n is a, c, g, or
t(32)..(33)n is a, c, g, or t(35)..(36)n is a, c, g, or
t(38)..(39)n is a, c, g, or t 9cagggtgccc tggccccagt aannmnnmnn
mnnmnnmnng cgcgcgcaat aatacac 571021DNAArtificial sequencePrimer
10tactggggcc agggcaccct g 211125DNAArtificial sequencePrimer
11ggaataggtg tatcaccgta ctcag 2512717DNAArtificial
sequencenucelotide sequence of single chain antibody Y022
12gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga ccgtgtgacc
60attacctgcc atgcgagcca ggatattaac gtgaacattg gctggctgca gcagaaaccg
120ggcaaagcgt ttaaaggcct gatttatcat ggcaaaaacc tggaagatgg
cgtgccgagc 180cgttttagcg gcagcggcag cggcaccgat tttaccctga
ccattagcag cctgcagccg 240gaagattttg cgacctatta ttgcaatcag
tatgaaaata tcccactgac atttggccag 300ggcaccaaag tggaaattaa
acgtggtgga ggcggttcag gcggaggtgg ctctggcggt 360ggcggatcgg
atgtgcagct ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc
420ctgcgtctga gctgcgcggt gagcggctat agcattacca gcgattatgc
gtggaactgg 480attcgtcagg cgccgggcaa aggcctggaa tggctgggct
atattagcta tcgtggccgc 540acccagtata acccgagcct gaaaagccgt
attagcatta cccgtgataa cagcaaaaac 600acctttttcc tgcagctgaa
cagcctgcgt gcggaagata ccgcggtgta ttattgcgcg 660cgcatgggta
agaattggga ttactggggc cagggcaccc tggtgaccgt gagcagc
71713239PRTArtificial sequenceamino acid sequence of single chain
antibody Y022 13Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp
Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala
Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys Asn Leu Glu Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Ile Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 115 120 125Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135
140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr Gln Tyr Asn Pro Ser
Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg Asp Asn Ser Lys Asn
Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Met Gly Lys 210 215 220Asn Trp Asp Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225 230
2351430DNAArtificial sequencePrimer 14acagtgctag cagatattca
gatgacccag 301532DNAArtificial sequencePrimer 15aagaatgcgg
ccgcgctgct cacggtcacc ag 321631DNAArtificial sequencePrimer
16acagtgctag cagacatcct gatgacccaa t 311732DNAArtificial
sequencePrimer 17aagaatgcgg ccgctgcaga gacagtgacc ag
321830DNAArtificial sequencePrimer 18acagtgctag cagacatctt
gctgactcag 301932DNAArtificial sequencePrimer 19aagaatgcgg
ccgctgcaga gacagtgacc ag 322029DNAArtificial sequencePrimer
20gatattcaga tgacccagag cccgagcag 292134DNAArtificial
sequencePrimer 21aataggatcc accacctccg ctgctcacgg tcac
342224DNAArtificial sequencePrimer 22ccattgacgc aaatgggcgg tagg
242329DNAArtificial sequencePrimer 23ctgctcgggc tctgggtcat
ctgaatatc 292438DNAArtificial sequencePrimer 24tgctccacgc
cgccaggccg gatattcaga tgacccag 382535DNAArtificial sequencePrimer
25cgcggcgctg gcgtcgtggt gctgctcacg gtcac 352619DNAArtificial
sequencePrimer 26tgcagtagtc gccgtgaac 192720DNAArtificial
sequencePrimer 27cggcctggcg gcgtggagca 202825DNAArtificial
sequencePrimer 28accacgacgc cagcgccgcg accac 252948DNAArtificial
sequencePrimer 29gaggtcgacc tacgcggggg cgtctgcgct cctgctgaac
ttcactct 483039DNAArtificial sequencePrimer 30gaggtcgacc tagcgagggg
gcagggcctg catgtgaag 39311928DNAArtificial sequenceeGFP-F2A-Y022
scFv-Z nucleotide sequence 31tgcagtagtc gccgtgaacg ttctttttcg
caacgggttt gccgccagaa cacaggtgtc 60gtgacgcgga tccaggccta agcttacgcg
tcctagcgct accggtcgcc accatggtga 120gcaagggcga ggagctgttc
accggggtgg tgcccatcct ggtcgagctg gacggcgacg 180taaacggcca
caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
240tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
accctcgtga 300ccaccctgac ctacggcgtg cagtgcttca gccgctaccc
cgaccacatg aagcagcacg 360acttcttcaa gtccgccatg cccgaaggct
acgtccagga gcgcaccatc ttcttcaagg 420acgacggcaa ctacaagacc
cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 480gcatcgagct
gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
540agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
aacggcatca 600aggtgaactt caagatccgc cacaacatcg aggacggcag
cgtgcagctc gccgaccact 660accagcagaa cacccccatc ggcgacggcc
ccgtgctgct gcccgacaac cactacctga 720gcacccagtc cgccctgagc
aaagacccca acgagaagcg cgatcacatg gtcctgctgg 780agttcgtgac
cgccgccggg atcactctcg gcatggacga gctgtacaag tccggagtga
840aacagacttt gaattttgac cttctgaagt tggcaggaga cgttgagtcc
aaccctgggc 900ccatggcctt accagtgacc gccttgctcc tgccgctggc
cttgctgctc cacgccgcca 960ggccggatat tcagatgacc cagagcccga
gcagcctgag cgcgagcgtg ggcgaccgtg 1020tgaccattac ctgccatgcg
agccaggata ttaacgtgaa cattggctgg ctgcagcaga 1080aaccgggcaa
agcgtttaaa ggcctgattt atcatggcaa aaacctggaa gatggcgtgc
1140cgagccgttt tagcggcagc ggcagcggca ccgattttac cctgaccatt
agcagcctgc 1200agccggaaga ttttgcgacc tattattgca atcagtatga
aaatatccca ctgacatttg 1260gccagggcac caaagtggaa attaaacgtg
gtggaggcgg ttcaggcgga ggtggctctg 1320gcggtggcgg atcggatgtg
cagctggtgg aaagcggcgg cggcctggtg cagccgggcg 1380gcagcctgcg
tctgagctgc gcggtgagcg gctatagcat taccagcgat tatgcgtgga
1440actggattcg tcaggcgccg ggcaaaggcc tggaatggct gggctatatt
agctatcgtg 1500gccgcaccca gtataacccg agcctgaaaa gccgtattag
cattacccgt gataacagca 1560aaaacacctt tttcctgcag ctgaacagcc
tgcgtgcgga agataccgcg gtgtattatt 1620gcgcgcgcat gggtaagaat
tgggattact ggggccaggg caccctggtg accgtgagca 1680gcaccacgac
gccagcgccg cgaccaccaa caccggcgcc caccatcgcg tcgcagcccc
1740tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac
acgagggggc 1800tggacttcgc ctgtgatatc tacatctggg cgcccttggc
cgggacttgt ggggtccttc 1860tcctgtcact ggttatcacc agagtgaagt
tcagcaggag cgcagacgcc cccgcgtagg 1920tcgacctc
1928322231DNAArtificial sequenceeGFP-F2A-Y022 scFv-Z nucleotide
sequence 32tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
cacaggtgtc 60gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc
accatggtga 120gcaagggcga ggagctgttc accggggtgg tgcccatcct
ggtcgagctg gacggcgacg 180taaacggcca caagttcagc gtgtccggcg
agggcgaggg cgatgccacc tacggcaagc 240tgaccctgaa gttcatctgc
accaccggca agctgcccgt gccctggccc accctcgtga 300ccaccctgac
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
360acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
ttcttcaagg 420acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
gggcgacacc ctggtgaacc 480gcatcgagct gaagggcatc gacttcaagg
aggacggcaa catcctgggg cacaagctgg 540agtacaacta caacagccac
aacgtctata tcatggccga caagcagaag aacggcatca 600aggtgaactt
caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
660accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
cactacctga 720gcacccagtc cgccctgagc aaagacccca acgagaagcg
cgatcacatg gtcctgctgg 780agttcgtgac cgccgccggg atcactctcg
gcatggacga gctgtacaag tccggagtga 840aacagacttt gaattttgac
cttctgaagt tggcaggaga cgttgagtcc aaccctgggc 900ccatggcctt
accagtgacc gccttgctcc tgccgctggc cttgctgctc cacgccgcca
960ggccggatat tcagatgacc cagagcccga gcagcctgag cgcgagcgtg
ggcgaccgtg 1020tgaccattac ctgccatgcg agccaggata ttaacgtgaa
cattggctgg ctgcagcaga 1080aaccgggcaa agcgtttaaa ggcctgattt
atcatggcaa aaacctggaa gatggcgtgc 1140cgagccgttt tagcggcagc
ggcagcggca ccgattttac cctgaccatt agcagcctgc 1200agccggaaga
ttttgcgacc tattattgca atcagtatga aaatatccca ctgacatttg
1260gccagggcac caaagtggaa attaaacgtg gtggaggcgg ttcaggcgga
ggtggctctg 1320gcggtggcgg atcggatgtg cagctggtgg aaagcggcgg
cggcctggtg cagccgggcg 1380gcagcctgcg tctgagctgc gcggtgagcg
gctatagcat taccagcgat tatgcgtgga 1440actggattcg tcaggcgccg
ggcaaaggcc tggaatggct gggctatatt agctatcgtg 1500gccgcaccca
gtataacccg agcctgaaaa gccgtattag cattacccgt gataacagca
1560aaaacacctt tttcctgcag ctgaacagcc tgcgtgcgga agataccgcg
gtgtattatt 1620gcgcgcgcat gggtaagaat tgggattact ggggccaggg
caccctggtg accgtgagca 1680gcaccacgac gccagcgccg cgaccaccaa
caccggcgcc caccatcgcg tcgcagcccc 1740tgtccctgcg cccagaggcg
tgccggccag cggcgggggg cgcagtgcac acgagggggc 1800tggacttcgc
ctgtgatatc tacatctggg cgcccttggc cgggacttgt ggggtccttc
1860tcctgtcact ggttatcacc agagtgaagt tcagcaggag cgcagacgcc
cccgcgtacc 1920agcagggcca gaaccagctc tataacgagc tcaatctagg
acgaagagag gagtacgatg 1980ttttggacaa gagacgtggc cgggaccctg
agatgggggg aaagccgcag agaaggaaga 2040accctcagga aggcctgtac
aatgaactgc agaaagataa gatggcggag gcctacagtg 2100agattgggat
gaaaggcgag cgccggaggg gcaaggggca cgatggcctt taccagggtc
2160tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg
ccccctcgct 2220aggtcgacct c 2231332357DNAArtificial
sequenceeGFP-F2A-Y022 scFv-BBZ nucleotide sequence 33tgcagtagtc
gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc 60gtgacgcgga
tccaggccta agcttacgcg tcctagcgct accggtcgcc accatggtga
120gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
gacggcgacg 180taaacggcca caagttcagc gtgtccggcg agggcgaggg
cgatgccacc tacggcaagc 240tgaccctgaa gttcatctgc accaccggca
agctgcccgt gccctggccc accctcgtga 300ccaccctgac ctacggcgtg
cagtgcttca gccgctaccc cgaccacatg aagcagcacg 360acttcttcaa
gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
420acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
ctggtgaacc 480gcatcgagct gaagggcatc gacttcaagg aggacggcaa
catcctgggg cacaagctgg 540agtacaacta caacagccac aacgtctata
tcatggccga caagcagaag aacggcatca 600aggtgaactt caagatccgc
cacaacatcg aggacggcag cgtgcagctc gccgaccact 660accagcagaa
cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
720gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
gtcctgctgg 780agttcgtgac cgccgccggg atcactctcg gcatggacga
gctgtacaag tccggagtga 840aacagacttt gaattttgac cttctgaagt
tggcaggaga cgttgagtcc aaccctgggc 900ccatggcctt accagtgacc
gccttgctcc tgccgctggc cttgctgctc cacgccgcca 960ggccggatat
tcagatgacc cagagcccga gcagcctgag cgcgagcgtg ggcgaccgtg
1020tgaccattac ctgccatgcg agccaggata ttaacgtgaa cattggctgg
ctgcagcaga 1080aaccgggcaa agcgtttaaa ggcctgattt atcatggcaa
aaacctggaa gatggcgtgc 1140cgagccgttt tagcggcagc ggcagcggca
ccgattttac cctgaccatt agcagcctgc 1200agccggaaga ttttgcgacc
tattattgca atcagtatga aaatatccca ctgacatttg 1260gccagggcac
caaagtggaa attaaacgtg gtggaggcgg ttcaggcgga ggtggctctg
1320gcggtggcgg atcggatgtg cagctggtgg aaagcggcgg cggcctggtg
cagccgggcg 1380gcagcctgcg tctgagctgc gcggtgagcg gctatagcat
taccagcgat tatgcgtgga 1440actggattcg tcaggcgccg ggcaaaggcc
tggaatggct gggctatatt agctatcgtg 1500gccgcaccca gtataacccg
agcctgaaaa gccgtattag cattacccgt gataacagca 1560aaaacacctt
tttcctgcag ctgaacagcc tgcgtgcgga agataccgcg gtgtattatt
1620gcgcgcgcat gggtaagaat tgggattact ggggccaggg caccctggtg
accgtgagca 1680gcaccacgac gccagcgccg cgaccaccaa caccggcgcc
caccatcgcg tcgcagcccc 1740tgtccctgcg cccagaggcg tgccggccag
cggcgggggg cgcagtgcac acgagggggc 1800tggacttcgc ctgtgatatc
tacatctggg cgcccttggc cgggacttgt ggggtccttc 1860tcctgtcact
ggttatcacc aaacggggca gaaagaaact cctgtatata ttcaaacaac
1920catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc
cgatttccag 1980aagaagaaga aggaggatgt gaactgagag tgaagttcag
caggagcgca gacgcccccg 2040cgtaccagca gggccagaac cagctctata
acgagctcaa tctaggacga agagaggagt 2100acgatgtttt ggacaagaga
cgtggccggg accctgagat ggggggaaag ccgcagagaa 2160ggaagaaccc
tcaggaaggc ctgtacaatg aactgcagaa agataagatg gcggaggcct
2220acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat
ggcctttacc 2280agggtctcag tacagccacc aaggacacct acgacgccct
tcacatgcag gccctgcccc 2340ctcgctaggt cgacctc
2357342372DNAArtificial sequenceeGFP-F2A-Y022 scFv-28Z nucleotide
sequence 34tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
cacaggtgtc 60gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc
accatggtga 120gcaagggcga ggagctgttc accggggtgg tgcccatcct
ggtcgagctg gacggcgacg 180taaacggcca caagttcagc gtgtccggcg
agggcgaggg cgatgccacc tacggcaagc 240tgaccctgaa gttcatctgc
accaccggca agctgcccgt gccctggccc accctcgtga 300ccaccctgac
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
360acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
ttcttcaagg 420acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
gggcgacacc ctggtgaacc 480gcatcgagct gaagggcatc gacttcaagg
aggacggcaa catcctgggg cacaagctgg 540agtacaacta caacagccac
aacgtctata tcatggccga caagcagaag aacggcatca 600aggtgaactt
caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
660accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
cactacctga 720gcacccagtc cgccctgagc aaagacccca acgagaagcg
cgatcacatg gtcctgctgg 780agttcgtgac cgccgccggg atcactctcg
gcatggacga gctgtacaag
tccggagtga 840aacagacttt gaattttgac cttctgaagt tggcaggaga
cgttgagtcc aaccctgggc 900ccatggcctt accagtgacc gccttgctcc
tgccgctggc cttgctgctc cacgccgcca 960ggccggatat tcagatgacc
cagagcccga gcagcctgag cgcgagcgtg ggcgaccgtg 1020tgaccattac
ctgccatgcg agccaggata ttaacgtgaa cattggctgg ctgcagcaga
1080aaccgggcaa agcgtttaaa ggcctgattt atcatggcaa aaacctggaa
gatggcgtgc 1140cgagccgttt tagcggcagc ggcagcggca ccgattttac
cctgaccatt agcagcctgc 1200agccggaaga ttttgcgacc tattattgca
atcagtatga aaatatccca ctgacatttg 1260gccagggcac caaagtggaa
attaaacgtg gtggaggcgg ttcaggcgga ggtggctctg 1320gcggtggcgg
atcggatgtg cagctggtgg aaagcggcgg cggcctggtg cagccgggcg
1380gcagcctgcg tctgagctgc gcggtgagcg gctatagcat taccagcgat
tatgcgtgga 1440actggattcg tcaggcgccg ggcaaaggcc tggaatggct
gggctatatt agctatcgtg 1500gccgcaccca gtataacccg agcctgaaaa
gccgtattag cattacccgt gataacagca 1560aaaacacctt tttcctgcag
ctgaacagcc tgcgtgcgga agataccgcg gtgtattatt 1620gcgcgcgcat
gggtaagaat tgggattact ggggccaggg caccctggtg accgtgagca
1680gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg
tcgcagcccc 1740tgtccctgcg cccagaggcg tgccggccag cggcgggggg
cgcagtgcac acgagggggc 1800tggacttcgc ctgtgatttt tgggtgctgg
tggtggttgg tggagtcctg gcttgctata 1860gcttgctagt aacagtggcc
tttattattt tctgggtgag gagtaagagg agcaggctcc 1920tgcacagtga
ctacatgaac atgactcccc gccgccccgg gccaacccgc aagcattacc
1980agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag
ttcagcagga 2040gcgcagacgc ccccgcgtac cagcagggcc agaaccagct
ctataacgag ctcaatctag 2100gacgaagaga ggagtacgat gttttggaca
agagacgtgg ccgggaccct gagatggggg 2160gaaagccgca gagaaggaag
aaccctcagg aaggcctgta caatgaactg cagaaagata 2220agatggcgga
ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc
2280acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac
gcccttcaca 2340tgcaggccct gccccctcgc taggtcgacc tc
2372352498DNAArtificial sequenceeGFP-F2A-Y022 scFv-28BBZ nucleotide
sequence 35tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
cacaggtgtc 60gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc
accatggtga 120gcaagggcga ggagctgttc accggggtgg tgcccatcct
ggtcgagctg gacggcgacg 180taaacggcca caagttcagc gtgtccggcg
agggcgaggg cgatgccacc tacggcaagc 240tgaccctgaa gttcatctgc
accaccggca agctgcccgt gccctggccc accctcgtga 300ccaccctgac
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
360acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
ttcttcaagg 420acgacggcaa ctacaagacc cgcgccgagg tgaagttcga
gggcgacacc ctggtgaacc 480gcatcgagct gaagggcatc gacttcaagg
aggacggcaa catcctgggg cacaagctgg 540agtacaacta caacagccac
aacgtctata tcatggccga caagcagaag aacggcatca 600aggtgaactt
caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
660accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
cactacctga 720gcacccagtc cgccctgagc aaagacccca acgagaagcg
cgatcacatg gtcctgctgg 780agttcgtgac cgccgccggg atcactctcg
gcatggacga gctgtacaag tccggagtga 840aacagacttt gaattttgac
cttctgaagt tggcaggaga cgttgagtcc aaccctgggc 900ccatggcctt
accagtgacc gccttgctcc tgccgctggc cttgctgctc cacgccgcca
960ggccggatat tcagatgacc cagagcccga gcagcctgag cgcgagcgtg
ggcgaccgtg 1020tgaccattac ctgccatgcg agccaggata ttaacgtgaa
cattggctgg ctgcagcaga 1080aaccgggcaa agcgtttaaa ggcctgattt
atcatggcaa aaacctggaa gatggcgtgc 1140cgagccgttt tagcggcagc
ggcagcggca ccgattttac cctgaccatt agcagcctgc 1200agccggaaga
ttttgcgacc tattattgca atcagtatga aaatatccca ctgacatttg
1260gccagggcac caaagtggaa attaaacgtg gtggaggcgg ttcaggcgga
ggtggctctg 1320gcggtggcgg atcggatgtg cagctggtgg aaagcggcgg
cggcctggtg cagccgggcg 1380gcagcctgcg tctgagctgc gcggtgagcg
gctatagcat taccagcgat tatgcgtgga 1440actggattcg tcaggcgccg
ggcaaaggcc tggaatggct gggctatatt agctatcgtg 1500gccgcaccca
gtataacccg agcctgaaaa gccgtattag cattacccgt gataacagca
1560aaaacacctt tttcctgcag ctgaacagcc tgcgtgcgga agataccgcg
gtgtattatt 1620gcgcgcgcat gggtaagaat tgggattact ggggccaggg
caccctggtg accgtgagca 1680gcaccacgac gccagcgccg cgaccaccaa
caccggcgcc caccatcgcg tcgcagcccc 1740tgtccctgcg cccagaggcg
tgccggccag cggcgggggg cgcagtgcac acgagggggc 1800tggacttcgc
ctgtgatttt tgggtgctgg tggtggttgg tggagtcctg gcttgctata
1860gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg
agcaggctcc 1920tgcacagtga ctacatgaac atgactcccc gccgccccgg
gccaacccgc aagcattacc 1980agccctatgc cccaccacgc gacttcgcag
cctatcgctc caaacggggc agaaagaaac 2040tcctgtatat attcaaacaa
ccatttatga gaccagtaca aactactcaa gaggaagatg 2100gctgtagctg
ccgatttcca gaagaagaag aaggaggatg tgaactgaga gtgaagttca
2160gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat
aacgagctca 2220atctaggacg aagagaggag tacgatgttt tggacaagag
acgtggccgg gaccctgaga 2280tggggggaaa gccgcagaga aggaagaacc
ctcaggaagg cctgtacaat gaactgcaga 2340aagataagat ggcggaggcc
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca 2400aggggcacga
tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc
2460ttcacatgca ggccctgccc cctcgctagg tcgacctc
249836601PRTArtificial sequenceeGFP-F2A-Y022 scFv-Z amino acid
sequence 36Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150
155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr
Leu Gly Met Asp Glu Leu Tyr Lys Ser225 230 235 240Gly Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255Val Glu
Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265
270Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met
275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 290 295 300Ile Thr Cys His Ala Ser Gln Asp Ile Asn Val Asn
Ile Gly Trp Leu305 310 315 320Gln Gln Lys Pro Gly Lys Ala Phe Lys
Gly Leu Ile Tyr His Gly Lys 325 330 335Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360 365Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Ile Pro Leu Thr Phe Gly Gln 370 375 380Gly
Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly385 390
395 400Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly
Gly 405 410 415Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Val Ser 420 425 430Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp Ile Arg Gln Ala 435 440 445Pro Gly Lys Gly Leu Glu Trp Leu Gly
Tyr Ile Ser Tyr Arg Gly Arg 450 455 460Thr Gln Tyr Asn Pro Ser Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp465 470 475 480Asn Ser Lys Asn
Thr Phe Phe Leu Gln Leu Asn Ser Leu Arg Ala Glu 485 490 495Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Met Gly Lys Asn Trp Asp Tyr 500 505
510Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala
515 520 525Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser 530 535 540Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr545 550 555 560Arg Gly Leu Asp Phe Ala Cys Asp Ile
Tyr Ile Trp Ala Pro Leu Ala 565 570 575Gly Thr Cys Gly Val Leu Leu
Leu Ser Leu Val Ile Thr Arg Val Lys 580 585 590Phe Ser Arg Ser Ala
Asp Ala Pro Ala 595 60037702PRTArtificial sequenceeGFP-F2A-Y022
scFv-Z amino acid sequence 37Met Val Ser Lys Gly Glu Glu Leu Phe
Thr Gly Val Val Pro Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn
Gly His Lys Phe Ser Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr
Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu
Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60Leu Thr Tyr Gly Val
Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr
Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105
110Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
Glu Tyr 130 135 140Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp
Lys Gln Lys Asn145 150 155 160Gly Ile Lys Val Asn Phe Lys Ile Arg
His Asn Ile Glu Asp Gly Ser 165 170 175Val Gln Leu Ala Asp His Tyr
Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190Pro Val Leu Leu Pro
Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp
Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220Val
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Ser225 230
235 240Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly
Asp 245 250 255Val Glu Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr
Ala Leu Leu 260 265 270Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg
Pro Asp Ile Gln Met 275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly Asp Arg Val Thr 290 295 300Ile Thr Cys His Ala Ser Gln
Asp Ile Asn Val Asn Ile Gly Trp Leu305 310 315 320Gln Gln Lys Pro
Gly Lys Ala Phe Lys Gly Leu Ile Tyr His Gly Lys 325 330 335Asn Leu
Glu Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345
350Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
355 360 365Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Ile Pro Leu Thr Phe
Gly Gln 370 375 380Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
Ser Gly Gly Gly385 390 395 400Gly Ser Gly Gly Gly Gly Ser Asp Val
Gln Leu Val Glu Ser Gly Gly 405 410 415Gly Leu Val Gln Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Val Ser 420 425 430Gly Tyr Ser Ile Thr
Ser Asp Tyr Ala Trp Asn Trp Ile Arg Gln Ala 435 440 445Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser Tyr Arg Gly Arg 450 455 460Thr
Gln Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp465 470
475 480Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser Leu Arg Ala
Glu 485 490 495Asp Thr Ala Val Tyr Tyr Cys Ala Arg Met Gly Lys Asn
Trp Asp Tyr 500 505 510Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Thr Thr Thr Pro Ala 515 520 525Pro Arg Pro Pro Thr Pro Ala Pro Thr
Ile Ala Ser Gln Pro Leu Ser 530 535 540Leu Arg Pro Glu Ala Cys Arg
Pro Ala Ala Gly Gly Ala Val His Thr545 550 555 560Arg Gly Leu Asp
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 565 570 575Gly Thr
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Arg Val Lys 580 585
590Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
595 600 605Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu 610 615 620Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro Gln Arg625 630 635 640Arg Lys Asn Pro Gln Glu Gly Leu Tyr
Asn Glu Leu Gln Lys Asp Lys 645 650 655Met Ala Glu Ala Tyr Ser Glu
Ile Gly Met Lys Gly Glu Arg Arg Arg 660 665 670Gly Lys Gly His Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys 675 680 685Asp Thr Tyr
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 690 695
70038744PRTArtificial sequenceeGFP-F2A-Y022 scFv-BBZ amino acid
sequence 38Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150
155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr
Leu Gly Met Asp Glu Leu Tyr Lys Ser225 230 235 240Gly Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255Val Glu
Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265
270Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met
275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 290 295 300Ile Thr Cys His Ala Ser Gln Asp Ile Asn Val Asn
Ile Gly Trp Leu305 310 315 320Gln Gln Lys Pro Gly Lys Ala Phe Lys
Gly Leu Ile Tyr His Gly Lys 325 330 335Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360 365Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Ile Pro Leu Thr Phe Gly Gln 370 375 380Gly
Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly385 390
395 400Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly
Gly 405 410 415Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Val Ser 420 425 430Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp Ile Arg Gln Ala 435 440 445Pro Gly Lys Gly Leu Glu Trp Leu Gly
Tyr Ile Ser Tyr Arg Gly Arg 450 455
460Thr Gln Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg
Asp465 470 475 480Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser
Leu Arg Ala Glu 485 490 495Asp Thr Ala Val Tyr Tyr Cys Ala Arg Met
Gly Lys Asn Trp Asp Tyr 500 505 510Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser Thr Thr Thr Pro Ala 515 520 525Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 530 535 540Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr545 550 555 560Arg
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 565 570
575Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly
580 585 590Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
Pro Val 595 600 605Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
Phe Pro Glu Glu 610 615 620Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
Phe Ser Arg Ser Ala Asp625 630 635 640Ala Pro Ala Tyr Gln Gln Gly
Gln Asn Gln Leu Tyr Asn Glu Leu Asn 645 650 655Leu Gly Arg Arg Glu
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 660 665 670Asp Pro Glu
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 675 680 685Gly
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 690 695
700Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp
Gly705 710 715 720Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu 725 730 735His Met Gln Ala Leu Pro Pro Arg
74039749PRTArtificial sequenceeGFP-F2A-Y022 scFv-28Z amino acid
sequence 39Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150
155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr
Leu Gly Met Asp Glu Leu Tyr Lys Ser225 230 235 240Gly Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255Val Glu
Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265
270Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met
275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 290 295 300Ile Thr Cys His Ala Ser Gln Asp Ile Asn Val Asn
Ile Gly Trp Leu305 310 315 320Gln Gln Lys Pro Gly Lys Ala Phe Lys
Gly Leu Ile Tyr His Gly Lys 325 330 335Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360 365Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Ile Pro Leu Thr Phe Gly Gln 370 375 380Gly
Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly385 390
395 400Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly
Gly 405 410 415Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Val Ser 420 425 430Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp Ile Arg Gln Ala 435 440 445Pro Gly Lys Gly Leu Glu Trp Leu Gly
Tyr Ile Ser Tyr Arg Gly Arg 450 455 460Thr Gln Tyr Asn Pro Ser Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp465 470 475 480Asn Ser Lys Asn
Thr Phe Phe Leu Gln Leu Asn Ser Leu Arg Ala Glu 485 490 495Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Met Gly Lys Asn Trp Asp Tyr 500 505
510Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala
515 520 525Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser 530 535 540Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr545 550 555 560Arg Gly Leu Asp Phe Ala Cys Asp Phe
Trp Val Leu Val Val Val Gly 565 570 575Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala Phe Ile Ile 580 585 590Phe Trp Val Arg Ser
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 595 600 605Asn Met Thr
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 610 615 620Tyr
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe625 630
635 640Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
Leu 645 650 655Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp 660 665 670Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
Lys Pro Gln Arg Arg 675 680 685Lys Asn Pro Gln Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met 690 695 700Ala Glu Ala Tyr Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly705 710 715 720Lys Gly His Asp
Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 725 730 735Thr Tyr
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 740
74540791PRTArtificial sequenceeGFP-F2A-Y022 scFv-28BBZ amino acid
sequence 40Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu1 5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
Tyr Pro Asp His Met Lys65 70 75 80Gln His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150
155 160Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly
Ser 165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr
Leu Gly Met Asp Glu Leu Tyr Lys Ser225 230 235 240Gly Val Lys Gln
Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp 245 250 255Val Glu
Ser Asn Pro Gly Pro Met Ala Leu Pro Val Thr Ala Leu Leu 260 265
270Leu Pro Leu Ala Leu Leu Leu His Ala Ala Arg Pro Asp Ile Gln Met
275 280 285Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg
Val Thr 290 295 300Ile Thr Cys His Ala Ser Gln Asp Ile Asn Val Asn
Ile Gly Trp Leu305 310 315 320Gln Gln Lys Pro Gly Lys Ala Phe Lys
Gly Leu Ile Tyr His Gly Lys 325 330 335Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly 340 345 350Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 355 360 365Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Ile Pro Leu Thr Phe Gly Gln 370 375 380Gly
Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly385 390
395 400Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val Glu Ser Gly
Gly 405 410 415Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
Ala Val Ser 420 425 430Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp Ile Arg Gln Ala 435 440 445Pro Gly Lys Gly Leu Glu Trp Leu Gly
Tyr Ile Ser Tyr Arg Gly Arg 450 455 460Thr Gln Tyr Asn Pro Ser Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp465 470 475 480Asn Ser Lys Asn
Thr Phe Phe Leu Gln Leu Asn Ser Leu Arg Ala Glu 485 490 495Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Met Gly Lys Asn Trp Asp Tyr 500 505
510Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala
515 520 525Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
Leu Ser 530 535 540Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
Ala Val His Thr545 550 555 560Arg Gly Leu Asp Phe Ala Cys Asp Phe
Trp Val Leu Val Val Val Gly 565 570 575Gly Val Leu Ala Cys Tyr Ser
Leu Leu Val Thr Val Ala Phe Ile Ile 580 585 590Phe Trp Val Arg Ser
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 595 600 605Asn Met Thr
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 610 615 620Tyr
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg625 630
635 640Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
Gln 645 650 655Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
Glu Glu Glu 660 665 670Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
Arg Ser Ala Asp Ala 675 680 685Pro Ala Tyr Gln Gln Gly Gln Asn Gln
Leu Tyr Asn Glu Leu Asn Leu 690 695 700Gly Arg Arg Glu Glu Tyr Asp
Val Leu Asp Lys Arg Arg Gly Arg Asp705 710 715 720Pro Glu Met Gly
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 725 730 735Leu Tyr
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 740 745
750Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
755 760 765Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His 770 775 780Met Gln Ala Leu Pro Pro Arg785
7904111PRTArtificial sequenceY022 light chain CDR1 41His Ala Ser
Gln Asp Ile Asn Val Asn Ile Gly1 5 10427PRTArtificial sequenceY022
light chainCDR2 42His Gly Lys Asn Leu Glu Asp1 5439PRTArtificial
sequenceY022 light chain CDR3 43Asn Gln Tyr Glu Asn Ile Pro Leu
Thr1 54411PRTArtificial sequenceY022 heavy chain CDR1 44Gly Tyr Ser
Ile Thr Ser Asp Tyr Ala Trp Asn1 5 104516PRTArtificial sequenceY022
heavy chain CDR2 45Tyr Ile Ser Tyr Arg Gly Arg Thr Gln Tyr Asn Pro
Ser Leu Lys Ser1 5 10 15467PRTArtificial sequenceY022 heavy chain
CDR3 46Met Gly Lys Asn Trp Asp Tyr1 54711PRTArtificial sequenceM14
light chain CDR1 amino acid sequence 47His Ala Ser Gln Asp Ile Asn
Ser Asn Ile Gly1 5 10489PRTArtificial sequenceM14 light chain CDR3
amino acid sequence 48Asn Gln Tyr Glu Asn Asn Pro Ile Thr1
54916PRTArtificial sequenceM14 heavy chain CDR2 49Tyr Ile Ser Tyr
Arg Gly Arg Thr Asn Tyr Asn Pro Ser Leu Lys Ser1 5 10
15507PRTArtificial sequenceM14 heavy chain CDR3 50Leu Gly Arg Gly
Phe Arg Tyr1 55116PRTArtificial sequenceM15 heavy chain CDR2 51Tyr
Ile Ser Tyr Arg Gly Arg Thr Ser Tyr Asn Pro Ser Leu Lys Ser1 5 10
155216PRTArtificial sequenceM25 heavy chain CDR2 52Tyr Ile Ser Tyr
Arg Gly Arg Thr Arg Tyr Asn Pro Ser Leu Lys Ser1 5 10
15537PRTArtificial sequenceS7 light chain CDR2 53His Gly Thr Asn
Leu Glu Asp1 5549PRTArtificial sequenceS7 light chain CDR3 54Asn
Gln Tyr Glu Asn Asn Pro Ile Thr1 55511PRTArtificial sequenceS17
light chain CDR1 55His Ala Ser Gln Asp Ile Asn Thr Asn Ile Gly1 5
10569PRTArtificial sequenceS17 light chain CDR3 56Asn Gln Tyr Glu
Asn Asn Pro Leu Thr1 5579PRTArtificial sequenceS23 light chain CDR3
57Asn Gln Tyr Glu Asn Phe Pro Leu Thr1 558717DNAArtificial
sequencenucleotide sequence of single chain antibody M14
58gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga ccgtgtgacc
60attacctgcc atgcgagcca ggatattaac agcaacattg gctggctgca gcagaaaccg
120ggcaaagcgt ttaaaggcct gatttatcat ggcaaaaacc tggaagatgg
cgtgccgagc 180cgttttagcg gcagcggcag cggcaccgat tttaccctga
ccattagcag cctgcagccg 240gaagattttg cgacctatta ttgcaatcag
tatgaaaata acccaattac atttggccag 300ggcaccaaag tggaaattaa
acgtggtgga ggcggttcag gcggaggtgg ctctggcggt 360ggcggatcgg
atgtgcagct ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc
420ctgcgtctga gctgcgcggt gagcggctat agcattacca gcgattatgc
gtggaactgg 480attcgtcagg cgccgggcaa aggcctggaa tggctgggct
atattagcta tcgtggccgc 540accaactata acccgagcct gaaaagccgt
attagcatta cccgtgataa cagcaaaaac 600acctttttcc tgcagctgaa
cagcctgcgt gcggaagata ccgcggtgta ttattgcgcg 660cgcctgggac
gcggcttccg ctactggggc cagggcaccc tggtgaccgt gagcagc
71759239PRTArtificial sequenceamino acid sequence of single chain
antibody M14 59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp
Ile Asn Ser Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala
Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys Asn Leu Glu Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Asn Pro Ile 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 115 120 125Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135
140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr Asn Tyr Asn Pro Ser
Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg Asp Asn Ser
Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215 220Gly Phe
Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225 230
23560717DNAArtificial sequencenucleotide sequence of single chain
antibody M15 60gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata acccaattac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540accagctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71761239PRTArtificial sequenceamino acid
sequence of single chain antibody M15 61Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Asn Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23562717DNAArtificial sequencenucleotide sequence of single
chain antibody M25 62gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata tcccactgac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540acccgctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71763239PRTArtificial sequenceamino acid
sequence of single chain antibody M25 63Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Ile Pro Leu 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23564717DNAArtificial sequencenucleotide sequence of single
chain antibody M26 64gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata tcccactgac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540acccagtata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71765239PRTArtificial sequenceamino acid
sequence of single chain antibody M26 65Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Ile Pro Leu 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Gln Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23566717DNAArtificial sequencenucleotide sequence of single
chain antibody S7 66gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaccaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata acccaattac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540accagctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71767239PRTArtificial sequenceamino acid
sequence of single chain antibody S7 67Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Thr
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Asn Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23568717DNAArtificial sequencenucleotide sequence of single
chain antibody S8 68gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaaagct ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata acccaattac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540accagctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71769239PRTArtificial sequenceamino acid
sequence of single chain antibody S8 69Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Asn Pro Ile 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Ser Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23570717DNAArtificial sequencenucleotide sequence of single
chain antibody S17 70gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac accaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata acccactgac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540acccagtata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71771239PRTArtificial sequenceamino acid
sequence of single chain antibody S17 71Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Thr Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Asn Pro Leu 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Gln Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 23572717DNAArtificial sequencenucleotide sequence of single
chain antibody S22 72gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaccaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaata acccactgac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540acccgctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71773239PRTArtificial sequenceamino acid
sequence of single chain antibody S22 73Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile
Asn Val Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ala Phe
Lys Gly Leu Ile 35 40 45Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys
Asn Gln Tyr Glu Asn Asn Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 115 120 125Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135 140Cys
Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp145 150
155 160Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Gly Tyr Ile
Ser 165 170 175Tyr Arg Gly Arg Thr Arg Tyr Asn Pro Ser Leu Lys Ser
Arg Ile Ser 180 185 190Ile Thr Arg Asp Asn Ser Lys Asn Thr Phe Phe
Leu Gln Leu Asn Ser 195 200 205Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg Leu Gly Arg 210 215 220Gly Phe Arg Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser225 230 23574717DNAArtificial
sequencenucleotide sequence of single chain antibody S23
74gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga ccgtgtgacc
60attacctgcc atgcgagcca ggatattaac gtgaacattg gctggctgca gcagaaaccg
120ggcaaaagct ttaaaggcct gatttatcat ggcaaaaacc tggaagatgg
cgtgccgagc 180cgttttagcg gcagcggcag cggcaccgat tttaccctga
ccattagcag cctgcagccg 240gaagattttg cgacctatta ttgcaatcag
tatgaaaata acccactgac atttggccag 300ggcaccaaag tggaaattaa
acgtggtgga ggcggttcag gcggaggtgg ctctggcggt 360ggcggatcgg
atgtgcagct ggtggaaagc ggcggcggcc tggtgcagcc gggcggcagc
420ctgcgtctga gctgcgcggt gagcggctat agcattacca gcgattatgc
gtggaactgg 480attcgtcagg cgccgggcaa aggcctggaa tggctgggct
atattagcta tcgtggccgc 540acccgctata acccgagcct gaaaagccgt
attagcatta cccgtgataa cagcaaaaac 600acctttttcc tgcagctgaa
cagcctgcgt gcggaagata ccgcggtgta ttattgcgcg 660cgcctgggac
gcggcttccg ctactggggc cagggcaccc tggtgaccgt gagcagc
71775239PRTArtificial sequenceamino acid sequence of single chain
antibody S23 75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys His Ala Ser Gln Asp
Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser
Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys Asn Leu Glu Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr
Cys Asn Gln Tyr Glu Asn Asn Pro Leu 85 90 95Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Gly Gly Gly Gly 100 105 110Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Leu Val 115 120 125Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 130 135
140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn
Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu
Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr Arg Tyr Asn Pro Ser
Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg Asp Asn Ser Lys Asn
Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215 220Gly Phe Arg Tyr
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225 230
23576717DNAArtificial sequencenucleotide sequence of single chain
antibody S29 76gatattcaga tgacccagag cccgagcagc ctgagcgcga
gcgtgggcga ccgtgtgacc 60attacctgcc atgcgagcca ggatattaac gtgaacattg
gctggctgca gcagaaaccg 120ggcaaagcgt ttaaaggcct gatttatcat
ggcaaaaacc tggaagatgg cgtgccgagc 180cgttttagcg gcagcggcag
cggcaccgat tttaccctga ccattagcag cctgcagccg 240gaagattttg
cgacctatta ttgcaatcag tatgaaaatt tcccactgac atttggccag
300ggcaccaaag tggaaattaa acgtggtgga ggcggttcag gcggaggtgg
ctctggcggt 360ggcggatcgg atgtgcagct ggtggaaagc ggcggcggcc
tggtgcagcc gggcggcagc 420ctgcgtctga gctgcgcggt gagcggctat
agcattacca gcgattatgc gtggaactgg 480attcgtcagg cgccgggcaa
aggcctggaa tggctgggct atattagcta tcgtggccgc 540acccgctata
acccgagcct gaaaagccgt attagcatta cccgtgataa cagcaaaaac
600acctttttcc tgcagctgaa cagcctgcgt gcggaagata ccgcggtgta
ttattgcgcg 660cgcctgggac gcggcttccg ctactggggc cagggcaccc
tggtgaccgt gagcagc 71777239PRTArtificial sequenceamino acid
sequence of single chain antibody S29 77Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr
Cys His Ala Ser Gln Asp Ile Asn Val Asn 20 25 30Ile Gly Trp Leu Gln
Gln Lys Pro Gly Lys Ala Phe Lys Gly Leu Ile 35 40 45Tyr His Gly Lys
Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu
Asp Phe Ala Thr Tyr Tyr Cys Asn Gln Tyr Glu Asn Phe Pro Leu 85 90
95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln
Leu Val 115 120 125Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser
Leu Arg Leu Ser 130 135 140Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser
Asp Tyr Ala Trp Asn Trp145 150 155 160Ile Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Leu Gly Tyr Ile Ser 165 170 175Tyr Arg Gly Arg Thr
Arg Tyr Asn Pro Ser Leu Lys Ser Arg Ile Ser 180 185 190Ile Thr Arg
Asp Asn Ser Lys Asn Thr Phe Phe Leu Gln Leu Asn Ser 195 200 205Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Gly Arg 210 215
220Gly Phe Arg Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser225
230 235
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References